- Open Access
Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues
© I. Holzapfel Publishers 2010
- Received: 30 June 2010
- Accepted: 26 July 2010
- Published: 25 October 2010
The Retraction Note to this article has been published in European Journal of Medical Research 2011 16:II
The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved protein . Ubiquitylated proteins were degraded by the 26S protea-some in an ATP-depended manner. The degradation of ubiquitylated proteins were controlled by isopeptidase cleavage. A well characterised system of ubiquitylation and deubiquitylation is the calmodulin system in vitro . Detection of ubiquityl-calmodulin conjugtates in vivo have not been shown so far. In this article we discuss the detection of ubiquitin calmodulin conjugates in vivo by incubation with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues. Proteins with a molecular weight of ubiquityl-calmodulin conjugates could be detected in all organs tested. Incubation with ubiquitylprotein-isopeptidase showed clearly a decrease of ubiquitin calmodulin conjugates in vivo with an origination of unbounded ubiquitin. These results suggest that only few ubiquitin calmodulin conjugates exist in rabbit tissues.
- protein degradation
A: absorption, optical density; BSA: bovine serum albumin; DTE: dithioerythriol; CaM: calmodulin; APFII: DEAE fraction II; SDS-Page: sodium dodecyl sulfate polyacrylamide electrophoresis; TCA: trichloracetic acid; w/v: weight in g per volume; uCam: ubiquityl-calmodulin (has two meanings:a. general name for all conjugates of calmodulin with ubiquitin: b. if specified designates the monoconjugate); uCam-Syn F1: uCam synthetase protein factor 1; uCaM-Syn F2: uCaM synthetase protein factor2.
ATP-dependent-26-S-protease (26S-proteasome, EC 3.4.99. -)
ATP-ubiquitin-dependent proteolytic pathway
(ubiquitin protein ligase + ATP-dependent-26-S-protease)
Multicatalytic endopeptidase complex (20S-proteasome, EC 184.108.40.206)
Ubiquitin-calmodulin ligase (ubiquityl-calmodulin synthetase, EC 220.127.116.11.);
Ubiquitin-calmodulin hydrolase, (ubiquityl-calmodulin isopeptidase, EC 3.4.99.-);
Ubiquitin-protein ligase, (E1, E2, E3; EC 18.104.22.168.); Ubiquitin thiolesterase (ubiquitin carboxyl-terminal esterase, EC 22.214.171.124).
Two forms of ubiquitin function have been described: (a) catabolic and (b) non-catabolic.
In the (a) catabolic pathway, protein ubiquitylation, which involves the covalent binding of multiple ubiquitin molecules with a specific ATP-dependent ligase system on substrates following ATP-dependent protein breakdown via 26S proteasome.
Examples of the catabolic protein ubiquitylation are:
Unassembled mutant type I collagen pro-alpha1 (I) chains , a-casein , growth hormone receptor  p53 , cyclin , nuclear oncoprotein , MHC class I heavy chains  and RNA-polymerase II .
The ubiquitin/proteasome system is a major pathway of selective protein degradation in eukaryotic cells. Ubiquitin-mediated degradation of proteins plays important roles in the control of numerous processes, including cell-cycle  cell division , stress response , extracellular modulators like NFκB [13–15], morphogenesis of neurons , modulation of cell receptors , ion channels  and DNA-repair [18, 19].
(b) non-catabolic protein ubiquitylation whitout terminating in degradation by the 26S proteasome. Examples of the non-catabolic protein ubiquitylation are:
The pathway for the protein breakdown contains an ubiquitin-protein-conjugating system, a protease and an isopeptidase. The ubiquitin-conjugating system is made of three different enzymatic components. E1 is the ubiquitin activating enzyme, E2 is the ubiquitin-conjugating enzyme and E3 is the ubiquitin-protein ligase. Ubiquitin is first of all adenylated by the conjugating enzyme (E1) and then transferred to a thiol group for covalent linkage. This is followed by a transesterfication to the conjugating enzyme (E2) which can either transfer ubiquitin directly to a target protein or together with the ubiquitin-protein ligase (E3).
Of major biological relevance is the dependence of this reaction on μM Ca2++ concentrations [23, 24] making calmodulin the first protein where ubiquitylation is regulated by second messenger. UCaM-synthetase has been detected in most tissues of the rabbit  and also in the simple eukaryote, yeast (S. cervisiae)  and leads to a calmodulin molecule multiubiquitylated (up to u3/4CaM and u5CaM) at a single lysine residue [25, 27]. UCaM-synthetase can be separated into two essentially inactive protein components  which have recently been purified [29, 30]. The first one (uCaM-Syn F1, 224 kDa) binds to ubiquitin-Sepharose and is the ubiquitin activating enzyme (E1). The second component (uCaM-Syn F2, 623 kDa) binds to calmodulin-Sepharose and bestows specificity to the reaction . Although the biological function of calmodulin ubiquitylation is not exactly known it has been suggested that this covalent modification suppresses the biological activity of cal modulin in the activation of phosphorylase kinase . In this connection the hypothesis emerged that ubiquitylation of calmodulin may be a physiologically reversible process similar to protein phosphorylation and dephosphorylation [2, 26] possibly catalyzed by a highly specific ubiquitylprotein-isopeptidase as described in detail in this paper. Andersen et al [31, 32] first reported the splitting of an N...-ubiquityl-protein bound in a natural ubiquitin conjugate. This isopeptidase was capable of splitting the protein A24 (histone 2A-ubiquitin conjugate) into histone 2A and ubiquitin. Matsui et al.  identified the reaction products in detailed analysis as intact ubiquitin and histone 2A.
This isopeptidase was shown to have a molecular mass of 38 kDa and to be present in the cytoplasm of most eukaryotes . Since no other natural ubiquitin conjugates were available putative isopeptidase activity in other work has been measured with more or less nonspecific substrates such as artificial ubiquitin-protein conjugates, amides or esters [34–37]. A first group of enzymes hydrolyzing small molecules conjugated to ubiquitin (e.g. residual peptides attached to ubiquitin) has been termed "ubiquitin carboxyl-terminal esterases/hydrolases" (EC 126.96.36.199) and appear not to have the ability to hydrolyze endogenous 12© I-ubiquityl-protein conjugates [37, 38]. These hydrolases usually have molecular masses in the range of 30 kDa [35, 37, 39–41]. A typical enzyme of this group is the ubiquitin carboxyl-terminal esterase L1 (UCH-L1) and ubiquitin carboxyl-terminal esterase L3 (UCH-L3) . This enzyme is found in nerve cells throughout the brain. Ubiquitin carboxyl-terminal esterase L1 is probably involved in the cell machinery that breaks down unwanted proteins. Although the exact function of ubiquitin carboxyl-terminal esterase L1 is not fully understood, it appears to have two enzyme activities. One activity, called hydrolase, removes and recycles ubiquitin molecules from degraded proteins. This recycling step is important to sustain the degradation process. The other enzyme activity, known as ligase, links together ubiquitin molecules for use in tagging proteins for disposal. An association between M. Alzheimer  and M. Parkinson , but also the Huntington  disease are discussed.
A second group of isopeptidases (ubiquityl-protein hydrolases) capable of splitting larger ubiquityl-protein conjugates has been estimated to be in a molecular mass range of 100-200 kDa . A third type of isopeptidase which cleaves free homooligomers and homopolymers of ubiquitin i.e. diubiquitin and multiubiquitin chains (ubiquityl-ubiquitin hydrolase) has been reported as a monomer with a native molecular mass of 100 kDa  An ATP-dependent C-terminal hydrolase  and ATP-dependent isopeptidase [47, 48] have also been reported which may be associated with the 26S proteasome [46, 48]. Recently enzymes containing both C-terminal hydrolase and isopeptidase activities have been reported [49, 50]. At the moment the biological function of these isopeptidases is unclear.
Against this background we tested the hypothesis that the incubation of rabbit muscle tissues with ubiquitylprotein-isopeptidase could detect ubiquityl-calmodulin conjugates in vivo.
Ubiquitin, Anti-Sheep IgG Peroxidase Conjugate Product No. A-3415 was purchased from Sigma (Munich). Nembutal® was obtained from Sanofi (Hannover). Leupeptine, iodacetamide and dithioerytol (DTE) were obtained from Biomol (Hamburg). For chromatographic application fractogel EMD-DEAE 650 (s) and TSK HW-65 (s) Merck (Darmstadt) were used. Problott‚-membrane (pit size of 0,025 μm) was purchased from Applied Biosystems (Weiterstadt). RPN ECL western blotting detection reagents and the hyperfilm were purchased by Amersham (Braunschweig). The western-blot equipment was obtained from Sartorius (Göttingen). Coomassie Brillant Blue R-250 (= Serva Blue R), methanol and BSA were obtained from Serva (Heidelberg). The molecular weight standards for SDS-PAGE bovine serum albumin (BSA) 66 kDa, ovalbumin 45 kDa, glyceraldehyde 3-phosphate dehydrogenase 36 kDa, carbonic anhydratase 29.2 kDa, trypsinogen 25 kDa, trypsin inhibitor 20.1 kDa and lactalbumin 14.2 kDa were obtained from Sigma (Munich).
The ubiquitin- antibody employed in this work was set up and featured by Gehrke and Jennissen . The affinity purification of ubiquitin-antibody was carried out by an unpublished method from G. Botzet and H.P. Jennissen on ubiquitin-sepharose.
All chemicals were of the highest available or analytical grade. Water was deionized, distilled and then passed through a Milli-Q-system (Millipore, Witten) before use.
Reticulocyte-rich blood (ca. 85%) was generated in rabbits (3-4 kg) by the phenylhydrazine method [51, 52]. In this procedure, 2.5% (w/v) phenylhydrazine in 0.9% NaCl, pH 7.4, is injected s.c. into the back of rabbits. On days 1-4 an amount of 0.13 ml/kg was injected. On day 8 rabbits were given a lethal overdose of 250 mg Evipan and bled for harvesting of reticulocyte by incision of the jugulars. Coagulation was inhibited by addition of 1 M sodium citrate, pH 7.4, to a final concentration of 10 mM into polyethylene beakers coated with silicone spray. All further work was performed with beakers and tubes siliconized in this way. The reticulocytes were washed twice at 5°C in a 10-fold volume of buffer containing 10 mM potassium phosphate, 0.15 M NaCl, pH 7.4
Preparation of reticulocytes without ATP depletion
To prevent reticulocytes from ATP depletion the procedure of red blood cell preparation described by Lew and Garcia-Sancho  was modified as follows.
Freshly drawn blood (anticoagulated by sodium citrate, pH 7.4 at final concentration of 10 mM) was centrifuged for 20 min. at 2200 g at room temperature and the pelleted cells were resuspended in 10 volumes of buffer supplemented with glucose and containing 145 mM NaCl, 5 mM KCl, 10 mM NaOH-neutralized HEPES, 10 mM glucose, 0.15 mM MgCl2, 0.1 mM EGTA, (pH 7.4). The wash was repeated three times, supernatant, buffy coat and topmost cell layer were removed after each centrifugation. An amount of 150 ml of resulted cell pellet was incubated for 60 min. at 37°C in 2 volumes of the same buffer supplemented with 1 mM Ca2+ and centrifuged again under the same conditions. Reticulocytes were then lysed with 2 volumes of lyses buffer containing 10 mM iodacetamide, 20 mM EGTA, and pH 7.0. Lysate was centrifuged at 100000 g for 90 min. at 4°C and supernatant was used for further preparations.
Rabbits were given a lethal dose of Nembutal® (see above) and immediately exsanguinated by decapitation and suspending from the hind legs. The excised tissues are: heart and white muscle (Musculus psoas minor). The tissues were extensively washed with water to remove residual blood. The excised tissue was cut into small pieces and immediately frozen in liquid nitrogen. The frozen tissue was then transferred to a -80°C freezer and stored. For the described experiments the tissues of five New Zealand rabbits (CHBBch) were pooled. The preparation of tissue extracts is based on the previously described procedure . According to this method 6 g of frozen (-80°C) rabbit tissue was homogenized in 3 mM K2HPO4/KH2PO4, 1 mM DTE, 5 μg/ml leupeptine, 15 mM iodacetamide, 5 mM EDTA, 5 mM EGTA, 5 × 10-5 M PMSF, pH 7.4 (buffer B) in a Bühler homogenizer (E.Bühler, Bodelshausen 10 ml vessel, diameter of the knife 1 cm) at full speed for 1 min at 4°C. This homogenate was centrifuged at 40 000 × g for 30 min in a Beckmann L-7/80 ultracentrifuge. The supernatant was given in a gauze filled measuring cylinder.
Cleaning of the chromatography glass column with chromosulfuric acid
The glass column (8.2 cm × 1.2 cm × 0.5 cm Merck, Darmstadt) was heated for one hour in chromosulfuric acid and then washed up with Millipore water as long as no chromosulfuric acid was externally visible. The glass column was finally treated in boiled Millipore water for one hour.
Lysate (250 mg) was applied (4 ml/h) by a peristaltic pump (Perpex, Guldner) to the column (8.2 cm × 1.2 cm × 0.5 cm Merck, Darmstadt) with 400 μl packed fractogel EMD-DEAE 650 (s). The fractogel cartridges were protected by a pre-column containing 250 μl packed underivatized fractogel TSK-HW 65 (S). The column was eluted in a single step with 4 ml/h 500 mM KCl, 20 mM Tris/HCl, 5 μg/ml leupeptine, 5 mM iodacetamide, pH 7.2. The elution pool was dialyzed on a rotating glass frame for 4 h against 6 l of 20 mM Tris/HCl, 1 mM DTE, 5 mM iodacetamide, pH 7.6. The eluate, the wash-fraction and the pass-through-fraction.
Ubiquitin was coupled to Sepharose 4B by the divinylsulfone method [51, 54]. Sepharose was first activated by divinylsulfone (= DVS-Sepharose) as described in . Ubiquitin coupling solution was made by adding 540 mg ubiquitin to 45 ml 0.2 M NaHCO2, pH 9.5. This solution was dialyzed in SpectraPor (cutoff 6-8 kDa) dialysis bags against 0.2 M NaHCO2, pH 9.5 for 3 hours with one buffer change after 1 hour. After dialysis the solution (45 ml) had a protein concentration of 6.3 mg/ml. The 45 ml of this ubiquitin coupling solution were added to 45 g "wet weight"  DVS-Sepharose 4B and incubated first for 6 hours at room temperature and then for another 21 hours at 5°C. After this time the gel was sucked dry on a small Büchner funnel and 45 ml stopping buffer containing 0.2 M NaHCO3 with 40 mg/ml glycine pH 9.5 was added. This stopping mixture was incubated for 4 hours at room temperature. The gel was then extensively washed with 20 mM Tris/HCl, pH 7.0 followed by washing at room temperature with 5-10 vol. each of H2O, 1% SDS, H2O, BSA
(5 mg/ml in H2O), H2O, 1 M NaCl, H2O, respectively. It was then stored at 5°C in H2O with 0.02% NaN3. The degree of substitution was measured by the depletion procedure (decrease of free ubiquitin in the bulk) employing the stainless steel grid method . The coupling efficiency was ca. 95% (not shown). For regeneration after several cycles of use ubiquitin-Sepharose was washed at room temperature with 5-10 vol. each of H2O, 1% SDS, H2O, BSA (5 mg/ml in H2O), H2O, 1 M NaCl, H2O respectively and then stored at 5°C in H2O with 0.02% NaN2.
For the preparative isolation of affinity-enriched isopeptidase activity fractogel-APF II was applied to ubiquitin-Sepharose (5.9 mg Ubiquitin/ml packed gel, 5 cm i.D. × 2.8 cm gel height, flow rate 25 ml/h, fraction vol. 2.5 ml) as described by . The gel was equilibrated with 50 mM Tris/HCl, 5 mM ATP, 10 mM MgCl2, pH 7.5 (buffer G). To a sample of fractogel-APF II (175 mg) 5 mM ATP, 10 mM MgCl2 (final concentration) were added and the mixture was applied to the column and then washed with 3 volumes of buffer G. The column was eluted with 5 volumes 50 mM Tris/HCl, 0,1 mM EDTA, 10 mM DTE, pH 9.0 (Buffer H). The eluate was pooled, concentrated in Centricon 30 tubes (Amicon, Witten) in a preparative Beckmann centrifuge (Rotor JA 20, 4000 × g, 4-5°C) and simultaneously dialyzed against 50 mM Tris/HCl, 0.5 mM DTE, 5 μg/ml leupeptine, pH 7.2 (buffer I)
Ubiquitin calmodulin conjugates
Reaction mixtures for the synthesis of preparative amounts of ubiquityl-calmodulin conjugates contained in a final volume of 10 ml: 50 mM Tris/HCl, 1 mM DTE, 5 mM MgCl2, 1 mM ATP, 10 mM phosphocreatine, 0.1 mM CaCl2, 0.1 mg/ml creatine kinase, 3.0 mg/ml ubiquitin, 0.5 mg/ml 125I-BH-calmodulin and 3.6 mg/ml. The reaction was stopped after 4 h at 37°C by adding 30 ml of 20 mM sodium
β-glycerophosphate, 1 mM CaCl2, pH 7.0 (buffer H) and heating to 96°C for 10 min. Subsequently the mixture was placed on ice for 5 min. Denatured proteins were spun down (20000 × g, 15 min, 4°C) and the supernatant (39 ml, 1.9 mg/ml) was applied at room temperature to a fluphenazine column (1.5 cm i.d. × 2.8 cm gel height; 5 ml packed gel) equilibrated with buffer H (flow rate 33 ml/h, fraction vol.). The column was washed with 10 ml buffer H followed by 80 - 100 ml 20 mM sodium β-glycerophosphate, 1 mM CaCl2, 300 mM NaCl, pH 7.0. In this way the unabsorbed ubiquitin was separated from the adsorbed conjugates and unconjugated free calmodulin. The ubiquityl-calmodulin conjugates and free calmodulin were eluted from the column with 4050 ml 20 mM sodium β-glycerophosphate, 10 mM EGTA, 500 mM NaCl, pH 7.0. This "EGTA-eluate" from the FP-Sepharose column was concentrated by TCA precipitation (5%), the resulting pellet was neutralized with 1 M sodium phosphate and resuspended in 2-4 ml 20 mM sodium phosphate, 1 M NaCl, pH 7.0 (buffer J).
The concentrated EGTA-eluate from the FP-Sepharose (1.8 ml; 3.5 mg/ml) was directly applied to a column of Chelating Sepharose Fast Flow (1.5 cm i.d. × 2.8 cm gel height, flow rate 14 ml/h, fraction vol. 5 ml) charged with Cu2+  and equilibrated with buffer J ("column procedure"). After application of the sample the column was washed with 20 ml buffer J (elution of free calmodulin). Elution of the ubiquityl-calmodulin conjugates was facilitated stepwise by a series of acetate buffers of different volumes adjusted to the different pH values as follows: 40 ml 0.1 M sodium acetate, 1 M NaCl, pH 6.0; 30 ml 0.1 M sodium acetate, 1 M NaCl, pH 5.5; 110 ml 0.1 M sodium acetate, 1 M NaCl, pH 5.0; 60 ml 0.1 M sodium acetate, 1 M NaCl, pH 4.75; 20 ml 0.1 M sodium acetate, 1 M NaCl, pH 4.5; 30 ml 0.1 M sodium acetate, 1 M NaCl, pH 3.2. The separation of ubiquityl-calmodulin conjugates from free calmodulin was monitored by 15% SDS-PAGE. Fractions containing ubiquityl-calmodulin devoid of free calmodulin were pooled, concentrated and dialyzed on Centricon 10 tubes against 10 mM sodium β-glycerophosphate, 0.1 mM CaCl2, pH 7.0. In the case that the different conjugate fractions were not separated all conjugates were eluted at pH 3.0 in one step. The conjugate yield in this procedure was ca. 1 mg.
Primary ubiquitin antibody
The ubiquitin antibody was prepared and characterized by Gehrke and Jennissen . The ubiquitin antibody was in subsequent purified by affinity chromatography on the ubiquitin-sepharose (unpublished data). The ubiquitin antibody was diluted 1:1000 in 10 mM Tris/HCl, 150 mM NaCl, 30 mg/ml BSA, pH 7.0 (buffer B).
SDS-PAGE was performed with 15% gels according to . 200 μg protein per lane were applicated. The molecular weight standards for SDS-PAGE and the ubiquitin-calmodulin standards are given under Materials.
The protein transfer of Problott®-membrane based on a method of Towbin et al. . The method was modified and optimized by M. Dietsch and H.P. Jennissen (unpublished data). Two graphite plates (Sartorius, Göttingen) were chosen as electrodes in a semidry-blotting system .
Blot-arrangement anode: 3 layers of Whatman®-filter paper (Nr. 3, Whatman®, Madestone, UK) in 300 mM Tris/HCl, 20% methanol, pH 10.4. 2 layers of What-man® filter paper in 25 mM Tris/HCl, 20% methanol, pH 10.4. 1 layer Problott®-membrane, 1 layer SDSPAGE. Blot-arrangement cathode: 2 layers of Whatman® filter paper in 25 mM Tris/HCl 20% methanol, pH 9.4. The blot was weighted with 2.5 kg and blotted with 40 mA for 31/2 h. After the transfer the Problott®-membrane was washed four times in 10 mM Tris, 150 mM NaCl, pH 7.0 (buffer A) and then air dried. The molecular standards (Serva Blue R) were stained with 0.1% amidoschwarz, 40% methanol and 1% acetic acid for 20-30 sec. Amidoschwarz was eliminated from the Problott®-membrane with millipore water. The destained Problott®-membrane was dried by air and then incubated for 1 h at 120°C under vacuum in a dessicator (Swerdlow et al., 1986). After the heat processing the Problott®-membrane was incubated for 10 min in methanol and washed for 10 min in 10 mM Tris, 150 mM NaCl, pH 7.0 and finally reblocked with 10 mM Tris/HCl, 150 mM NaCl, 30 mg/ml BSA, pH 7.0 for 90 min (buffer B). Both primary and secondary antibodies were diluted 1:1000 in buffer B. After 90 min incubation with primary antibody the Problott®-membrane was washed four times for 15 min in buffer A. The Problott®-membrane was reblocked 5 min in buffer B and in a further step incubated 30 min with secondary antibody (peroxidase labelled anti sheep antibody, Sigma) following by rinsing one time each 5 and 10 min and three times 15 min in buffer A. The Problott®-membrane was incubated 1 min in 1 vol. detection reagent 1 + 1 vol. detection reagent 2 (Amersham) and placed between two layers of thin plastic foil in the presence of enhancer foils (Cronex Lightning Plus, Dupont de Nemours) and were exposed to hyper-film (18 × 24 cm Amersham) for 2-5 min.
Competitive ubiquitin western blot
In the case of ubiquitin displacement blot, unconjugated ubiquitin were added in a concentration of 5 mg/ml to the primary antibody.
Splitting of ubiquitin-conjugates with ubiquitylprotein-isopeptidase
Before incubation of the rabbit tissues with the ubiquitylprotein-isopeptidase, the probes were dialyzed against 20 mM Tris/HCl, 20 mM β-mercaptoethanol, 5 × 10-5 PMSF, pH 8.0. to eliminate iodacetamide. Iodacetamide inhibits the ubiquitylprotein-isopeptidase irreversible.
The batches, incubated with ubiquitylprotein-isopeptidase had a final concentration of 50 mM Tris/HCl, 1 mM DTE, 50 μM PMSF und 5 μg/ml leupeptine, pH 8.0. After incubation at 37°C for a given time (60 min.) in a waterbath, the reaction were irreversible inhibited by a final concentration of 5 mM iodacetamide and 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each batch was incubated with 25 μg ubiquitylprotein-isopeptidase.
Ubiquityl-calmodulin isopeptidase test
Ubiquityl-calmodulin isopeptidase test was used for experimental verification of "true" ubiquityl-protein conjugates (as well as free branched multiubiquitin chains) in examined probes and its discrimination from linear polyubiquitin chains. Reaction mixtures for ubiquitylcalmodulin isopeptidase tests contained in a total volume of 230.8 μl 50 mM Tris/HCl, 5 mM Mg acetate, 8.5 μg ubiquityl-calmodulin conjugates or 100 μg of APF II fraction from non-ATP depleted reticulocytes and 50 μg enriched ubiquityl-calmodulin isopeptidase fraction. Incubation was stopped after 60 min. by adding of TCA at final concentration of 5%. The pellets were neutralized with 60 μl laemmli sample buffer, heated to 96°C for 10 min and then subjected for SDS-PAGE. Respective volumes of 50 mM Tris/HCl, 5 mM iodacetamide were added to control probes instead of the enzyme solution.
Amino acid analysis
Purified ubiquitin, calmodulin and monoubiquitination products of calmodulin were hydrolyzed in vacuum for 24 and 48 hours in 6 N HCl, 0.1% (w/v) phenol at 110°C. Amino acid analysis (OPA method) was done on a Spherisorb O.D.S. II column (Fa. Grom, Herrenberg) as described by [25, 61].
Splitting of internal rabbit muscle ubiquitin-conjugates with trypsin
The batches (200 μg protein) incubated with trypsin had a final concentration of 20 mM Trsi/HCl, 20 mM βmercaptoethanol, 5 × 10-5 PMSF, pH 8.0. After incubation at 37°C for a given time (30 min) in a water bath, the reaction was stopped irreversible with 10% w/v TCA (final concentration 5% TCA, 20 min, 0°C). Each bath was incubated with 65 μg Trypsin.
Protein was determined after TCA precipitation (5%), washing and resolubilization to the method of (Lowry) on an AutoAnalyzer (Technicon) employing BSA as standard.
Incubation of ubiquitin calmodulin conjugates with ubiquitylprotein-isopeptidase
Amino acid analysis of the first order ubiquityl-calmodulin conjugate
First order Ubiquityl-Calmodulin Conjugate
Composition from sequence
Aminoacid analysis of ubiquitin and calmodulin released from first-order ubiquitylcalmodulin conjugate
Composition from Sequence
Composition from Sequence
Characterisation of the blotting system
Competitive western blot
Sensitivity of anti-ubiquitin antibodies against free ubiquitin
Western-blot analysis of organ tissues with ubiquitin-antibody
Detection of ubiquityl-calmodulin conjugates in reticulocyte APFII
Since, the calmodulin concentration in skeletal muscle is very high and the muscle degradation is controlled by the proteasome system [63–66] we hypothesized the existence of ubiquityl-calmodulin conjugates also in the skeletal muscle.
Western-blot analysis of heart muscle with ubiquitin-antibody
Incubation of organ tissues with ubiquitylprotein-isopeptidase
Incubation of the white muscle-extract and APFII with the ubiquitylprotein-isopeptidase
In conclusion, ubiquityl-calmodulin conjugates could only be detected in the reticulocyte APFII and in the white muscle extract (uCaM I) and APFII (uCaM I and uCaM II).
The loss of trypsin incubation experiments is the absence of facility to distinguish between the decomposition product of the "mother-protein" or the multiubiquitin chains. Ubiquitin-T arises during the splitting of ubiquitin-chains. Of note, the ubiquitin antibody detected the ubiquitin-T 10-12 fold worser than unconjugated ubiquitin (data not shown). However, the occurrence of ubiquitin-T after trypsin incubation strongly indicates the existence of ubiquitin-conjugates in the organ tissues.
Trypsin-incubation of the heart muscle
Trypsin-incubation of the white muscle
In all employed rabbit organs the ubiquitylproteinisopeptidase incubation led to a signal reduction of high molecular weight proteins and/or unconjugated ubiquitin. we identified for the first time in vivo ubiquitylcalmodulin conjugates in the investigated organ tissues. substrates of the ubiquitylprotein-isopeptidase could be identified and had a molecular weight between 30 and 40 kDa, consistent with the molecular weight of ubiquityl-calmodulin conjugates (ucaM I and uCaM II). the splitting of ubiquitin-conjugates with the ubiquitylprotein-isopeptidase is a new highly specific method for the detection of endogenous ubiquitin calmodulin conjugates in organ tissues.
Polyubiquitylated calmodulin is split into the two components: free calmodulin and free ubiquitin. Both the molecular mass and the amino acid composition (Table 1) are identical in the reaction products and the native proteins calmodulin and ubiquitin proving that only the connecting isopeptide bonds are cleaved leaving the primary structures intact. The enzyme splits the N...-ubiquityl-calmodulin bond which has been shown to be located in the n-terminal portion (amino acids 1- 107) of calmodulin . The amino acid to which calmodulin is linked is a lysine residue since methylation of calmodulin by formaldehyde followed by reduction (unpublished) abolishes ubiquitylation.
In the case of the enzyme incubation in this work the isopeptidase should have a high specificity, because only few proteins were deubiquitylated by this enzyme. In contrast to the trypsin-incubation all high molecular ubiquitin positive protein peaks were cleaved by trypsin and ubiquitin-T arises. This indicates a high specificity of the employed isopeptidase and underlines the theory of selective cleavage of ubiquityl-calmodulin conjugates in vivo.
Ubiquityl-calmodulin conjugates (uCaM I and uCaM II) would serve as "internal-standards" with the isopeptidease-incubation. The calmodulin is bound at position Lys 21 with the ubiquitin by means of a particular enzyme system . The deubiquitylation of proteins was induced by specific isopeptidases. These enzymes catalyse the cleavage of isopeptide bound of ubiquitin and the targed protein. Consequently unconjugated ubiquitin arises.
Isopeptidases have an important physiological function. They adjust disassembly of ubiquitylated proteins and consequently regulate the degradation of proteins by the 26S proteasome . This mechanism can be adjusted by the isopeptidases and/or can be made reversible so that this step in the degradation of ubiquitylated proteins is a key position.
Another possibility for the regulation of ubiquitylated proteins is E4 . E4 regulates the length of ubiquitin chains and therefore the degradation over the 26S proteasome. In the case of the non-catabole ubiquitylation the biological function is adjusted by deubiquitylation.
Furthermore, the possibility exists via the binding of ubiquitin to multiubiquitin chains to adjust the disassembly of ubiquitylated proteins. Ubiquitin possess seven lysines. It is theoretical possible that ubiquitin chains originates from every lysine in vitro. In vivo ubiquitin chains were only linked at Lys 6, Lys 11, Lys 29, Lys 48 and Lys 63. Only Lys 48 serves as a signal for disassembly via the 26S proteasome [72, 73]. Lys 63 serves as a DNA repair signal and the lysines: Lys 6, Lys 11, Lys 29 presumable for the receptor internalisation . In contrast to the ubiquitin-system, ubiquitin-like proteins such as the sentrines have their own isopeptidases. The isopeptidase Ulp1  and SENP1-SENP 7  divides exclusively sentrine-conjugates and can not cleave ubiquitinconjugates. It is improbable that ubiquitin-isopeptidases also cleave ubiquitin-like proteins. The binding of the sentrine-conjugates occurs similar to the ubiquitin-conjugates. Via a particular enzyme system, the sentrines were coupled in an ATP-depending manner to their target protein.
Cross-reactivity with ubiquitin-like proteins can be excluded, because of the high specifity of the primary antibody used in this work. However, ubiquitin-like proteins have their own, very specific isopeptidases [76–81].
Amino acid alignment of ubiquitin and ubiquitin-like proteins
Both ubiquitin and UCRP are recognized at equimolar amount of an affinity purified polyclonal ubiquitinantibody . In contrast, affinity purified polyclonal UCRP-antibody reacts considerably worse with ubiquitin than UCRP . In a numerous of different organ tissues , for example lymphoid cells, smooth and stripped muscle, in epithelial cells and neurons, the UCRP was detected and has similar function like ubiquitin. A further "ubiquitin like-protein" was described in 1997. It is a protein regarding to the group of sentrines (Sentrin-1, Sentrin-2 und Sentrin-3). They were also called SUMO-1, PIC-1, GMP-1, UBL1 und SMT3C [85–92] and are detectable in all investigated tissues . Sentrines have a molecular weight of 6, 14 and 90 kDa. Sentrine-1 has 101 amino acids and possesses a ubiquitin-like domain (amino acid 22-97) which is 18% identical and 48% homologous (Table 3) to human ubiquitin. Sentrine-2 has 95 amino acids which are 46% identical and 66% homologous (Table 3) to Sentrin-1. Sentrine-3 has 103 amino acids which are 97% (Table 3) identical to Sentrin-2. Further ubiquitin-like proteins were described in 1997 (NEDD8)  and 1998 (Apg12) . NEDD8 and Rub1 have a molecular weight of 6 kDa.
Only for the UCRP experimental data exist, that ubiquitin-antibody reacts equimolar, what is to be traced back to the high sequence homology (up to 66%). Therefore, it can not be excluded that UCRP-conjugates represent a part of the found protein peaks. The fact however, that in the analyzed organ tissues no protein peak with a molecular weight of 15 kDa was detectable, and UCRP ubiquitously in the organs occur, opens improbably the cross-reaction with our ubiquitin-antibody and therefore also cross-reaction with UCRP-conjugates. In the work of Hinchey , no cross-reaction with ubiquitin was described for the Sentrine SUMO-1-2 and the SUMO-3. The Sentrine family members have a sequence homology of 16% to ubiquitin. This makes a cross-reaction very improbable with ubiquitin-antibody and was also confirmed experimentally from Hinchey . These are convincing arguments against the cross-reaction of sentrines and their conjugates with ubiquitin-antibody. In literature cross-reactions of ubiquitin-like proteins with ubiquitin-antibody are described as improbable.
The muscles were presumably degraded by the 26S proteasome in the situation of denavation , fasting , azidosis , tumor illness  and burning . Furthermore, first results for disassembly of muscle proteins via the 26S proteasome may be arranged: actin (42 kDa), myosin (510 kDa), troponine (78 kDa) and tropomyosine (64 kDa) . The ubiquitylated proteins found in this work are in accordance with the literature. However, in this study additional ubiquitylated proteins were detected which have not been described in the literature mentioned above. A possible interpretation is the different extraction conditions of muscles in the experimental settings. In the study of G. Tiao  septic rats were investigated. Lecker et al.  employed streptozotocin induced rats which developed diabetes mellitus. All organs employed in this work were gained from sacrificed rabbits under Nembutal®-sedation without induction of any stress factors and without further preparatory treatment. Ubiquitin is a heat shock protein and therefore induced in stress situations. The extraction conditions are crucial for the ubiquitylation pattern in organ tissues. The different "ubiquitylated organ patterns" could consequently be explained through the different experimental setups and different animal models. Presumably there are differences in the ubiquitylated organ patterns between two species.
The trypsin-incubation of organ tissues is a procedure to prove the existence of "internal" ubiquitin-conjugates. Here, trypsin (see material and methods) cleaves the last two amino acids of the ubiquitin and/or of the ubiquitin-conjugates and ubiquitin-T (des Gly-Gly ubiquitin) results. The protein peaks not detectable after incubation with trypsin are ubiquitin-conjugates. After trypsin-incubation decomposition products can often be observed. These decomposition products are splitted ubiquitin-T from the ubiquitin chains or the targeted protein. In all employed organ tissues a signal reduction of ubiquitylated proteins could be observed. Various decomposition partners (11 and 13 kDa) were detectable. Both proteins were cleaved by the ubiquitylprotein-isopeptidase and were detected by a calmodulinantibody (data not shown). This indicates that these proteins could presumably be composition products of ubiquitin-calmodulin conjugates. Trypsin itself generated new protein peaks initially not detectable with the ubiquitin-antibody. The protein peak with a molecular weight of 6 kDa could be the Sentrin NEDD8 and/or RUB1 . Both proteins have a molecular weight of 6 kDa. As described before a cross reaction by sentrine with an ubiquitin-antibody is improbable. Presumably it is a degradation product of ubiquitin.
This work is the first attempt to detect ubiquitylcalmodulin conjugate in vivo via ubiquitylprotein isopeptidase incubation. Further experiments had to be done to go more in detail for this question.
This work was supported by grants (Je 84/8-1, Je 84/9-1) from the Deutsche Forschungsgemeinschaft. The authors appreciate the excellent technical assistance of G. Botzet †.
- Ciechanover A: Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Exp Biol Med (Maywood) 2006,231(7):1197–211.Google Scholar
- Jennissen HP: Ubiquitin and the enigma of intracellular protein degradation. Eur J Biochem 1995,231(1):1–30. 10.1111/j.1432-1033.1995.tb20665.xPubMedGoogle Scholar
- Fitzgerald J, Lamande SR, Bateman JF: Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains. J Biol Chem 1999,274(39):27392–8. 10.1074/jbc.274.39.27392PubMedGoogle Scholar
- Ferber S, Ciechanover A: Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system. J Biol Chem 1986,261(7):3128–34.PubMedGoogle Scholar
- Strous GJ, et al.: The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. Embo J 1996,15(15):3806–12.PubMed CentralPubMedGoogle Scholar
- Scheffner M, et al.: The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 1990,63(6):1129–36. 10.1016/0092-8674(90)90409-8PubMedGoogle Scholar
- Glotzer M, Murray AW, Kirschner MW: Cyclin is degraded by the ubiquitin pathway. Nature 1991,349(6305):132–8. 10.1038/349132a0PubMedGoogle Scholar
- Ciechanover A, et al.: Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc Natl Acad Sci USA 1991,88(1):139–43. 10.1073/pnas.88.1.139PubMed CentralPubMedGoogle Scholar
- Wiertz EJ, et al.: The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 1996,84(5):769–79. 10.1016/S0092-8674(00)81054-5PubMedGoogle Scholar
- Huibregtse JM, Yang JC, Beaudenon SL: The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase. Proc Natl Acad Sci USA 1997,94(8):3656–61. 10.1073/pnas.94.8.3656PubMed CentralPubMedGoogle Scholar
- Sudakin V, et al.: The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol Biol Cell 1995,6(2):185–97.PubMed CentralPubMedGoogle Scholar
- Bond U, Schlesinger MJ: Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol Cell Biol 1985,5(5):949–56.PubMed CentralPubMedGoogle Scholar
- May MJ, Ghosh S: Signal transduction through NF-kappa B. Immunol Today 1998,19(2):80–8. 10.1016/S0167-5699(97)01197-3PubMedGoogle Scholar
- Browatzki M, et al.: Endothelin-1 induces functionally active CD40 protein via nuclear factor-kappaB in human vascular smooth muscle cells. Eur J Med Res 2007,12(3):129–33.PubMedGoogle Scholar
- Browatzki M, et al.: Endothelin-1 induces CD40 but not IL-6 in human monocytes via the proinflammatory transcription factor NF-kappaB. Eur J Med Res 2005,10(5):197–201.PubMedGoogle Scholar
- Mayer RJ, et al.: Ubiquitin in health and disease. Biochim Biophys Acta 1991,1089(2):141–57.PubMedGoogle Scholar
- Staub O, et al.: Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. Embo J 1997,16(21):6325–36. 10.1093/emboj/16.21.6325PubMed CentralPubMedGoogle Scholar
- Madura K, Prakash S, Prakash L: Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle. Nucleic Acids Res 1990,18(4):771–8. 10.1093/nar/18.4.771PubMed CentralPubMedGoogle Scholar
- Sung P, Prakash S, Prakash L: Mutation of cysteine88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions. Proc Natl Acad Sci USA 1990,87(7):2695–9. 10.1073/pnas.87.7.2695PubMed CentralPubMedGoogle Scholar
- Jennissen HP, Laub M: Ubiquitin-calmodulin conjugating activity from cardiac muscle. Biol Chem Hoppe Seyler 1988,369(12):1325–30.PubMedGoogle Scholar
- Mori S, Heldin CH, Claesson-Welsh L: Ligand-induced ubiquitination of the platelet-derived growth factor beta-receptor plays a negative regulatory role in its mitogenic signaling. J Biol Chem 1993,268(1):577–83.PubMedGoogle Scholar
- Laub M, Jennissen HP: Ubiquitination of endogenous calmodulin in rabbit tissue extracts. FEBS Lett 1991,294(3):229–33. 10.1016/0014-5793(91)81436-CPubMedGoogle Scholar
- Ziegenhagen R, Gehrke P, Jennissen HP: Covalent conjugation of mammalian calmodulin with ubiquitin. FEBS Lett 1988,237(1–2):103–7. 10.1016/0014-5793(88)80180-7PubMedGoogle Scholar
- Ziegenhagen R, Jennissen HP: Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase. Biol Chem Hoppe Seyler 1988,369(12):1317–24.PubMedGoogle Scholar
- Ziegenhagen R, et al.: Multiple ubiquitination of calmodulin results in one polyubiquitin chain linked to calmodulin. FEBS Lett 1990,271(1–2):71–5. 10.1016/0014-5793(90)80374-RPubMedGoogle Scholar
- Ziegenhagen R, Jennissen HP: Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2(+)-dependent manner. FEBS Lett 1990,273(1–2):253–6. 10.1016/0014-5793(90)81097-8PubMedGoogle Scholar
- Jennissen HP, et al.: Ca(2+)-dependent ubiquitination of calmodulin in yeast. FEBS Lett 1992,296(1):51–6. 10.1016/0014-5793(92)80401-2PubMedGoogle Scholar
- Majetschak M, Laub M, Jennissen HP: A ubiquitylcalmodulin synthetase that effectively recognizes the Ca(2+)-free form of calmodulin. FEBS Lett 1993,315(3):347–52. 10.1016/0014-5793(93)81192-3PubMedGoogle Scholar
- Majetschak M, et al.: The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties. Eur J Biochem 1998,255(2):492–500. 10.1046/j.1432-1327.1998.2550492.xPubMedGoogle Scholar
- Majetschak M, et al.: The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitinbinding first component, a ubiquitin-activating enzyme. Eur J Biochem 1998,255(2):482–91. 10.1046/j.1432-1327.1998.2550482.xPubMedGoogle Scholar
- Andersen MW, et al.: Protein A24 lyase activity in nucleoli of thioacetamide-treated rat liver releases histone 2A and ubiquitin from conjugated protein A24. Biochemistry 1981,20(5):1100–4. 10.1021/bi00508a009PubMedGoogle Scholar
- Andersen MW, Goldknopf IL, Busch H: Protein A24 lyase is an isopeptidase. FEBS Lett 1981,132(2):210–4. 10.1016/0014-5793(81)81162-3PubMedGoogle Scholar
- Matsui S, et al.: Isopeptidase: a novel eukaryotic enzyme that cleaves isopeptide bonds. Proc Natl Acad Sci USA 1982,79(5):1535–9. 10.1073/pnas.79.5.1535PubMed CentralPubMedGoogle Scholar
- Ciechanover A, et al.: ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation. Proc Natl Acad Sci USA 1980,77(3):1365–8. 10.1073/pnas.77.3.1365PubMed CentralPubMedGoogle Scholar
- Wilkinson KD, et al.: Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase. Biochemistry 1986,25(21):6644–9. 10.1021/bi00369a047PubMedGoogle Scholar
- Rose IA, Warms JV: An enzyme with ubiquitin carboxy-terminal esterase activity from reticulocytes. Biochemistry 1983,22(18):4234–7. 10.1021/bi00287a012PubMedGoogle Scholar
- Mayer AN, Wilkinson KD: Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus. Biochemistry 1989,28(1):166–72. 10.1021/bi00427a024PubMedGoogle Scholar
- Wilkinson DA, McIntosh TJ: A subtransition in a phospholipid with a net charge, dipalmitoylphosphatidylglycerol. Biochemistry 1986,25(2):295–8. 10.1021/bi00350a002PubMedGoogle Scholar
- Pickart CM, Rose IA: Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides. J Biol Chem 1985,260(13):7903–10.PubMedGoogle Scholar
- Wilkinson KD, et al.: The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 1989,246(4930):670–3. 10.1126/science.2530630PubMedGoogle Scholar
- Doran JF, et al.: Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis. J Neurochem 1983,40(6):1542–7. 10.1111/j.1471-4159.1983.tb08124.xPubMedGoogle Scholar
- Choi J, et al.: Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases. J Biol Chem 2004,279(13):13256–64.PubMedGoogle Scholar
- Lincoln S, et al.: Low frequency of pathogenic mutations in the ubiquitin carboxy-terminal hydrolase gene in familial Parkinson's disease. Neuroreport 1999,10(2):427–9. 10.1097/00001756-199902050-00040PubMedGoogle Scholar
- Naze P, et al.: Mutation analysis and association studies of the ubiquitin carboxy-terminal hydrolase L1 gene in Huntington's disease. Neurosci Lett 2002,328(1):1–4. 10.1016/S0304-3940(02)00231-8PubMedGoogle Scholar
- Chen Z, Pickart CM: A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. J Biol Chem 1990,265(35):21835–42.PubMedGoogle Scholar
- Eytan E, et al.: Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex. J Biol Chem 1993,268(7):4668–74.PubMedGoogle Scholar
- Mahaffey D, Yoo Y, Rechsteiner M: Ubiquitin metabolism in cycling Xenopus egg extracts. J Biol Chem 1993,268(28):21205–11.PubMedGoogle Scholar
- Ugai S, et al.: Purification and characterization of the 26S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins from rat liver. J Biochem 1993,113(6):754–68.PubMedGoogle Scholar
- Moskovitz J: Characterization of the 30-kDa enzyme from red blood cells that cleaves ubiquitin-protein conjugates. Biochem Biophys Res Commun 1994,205(1):354–60. 10.1006/bbrc.1994.2672PubMedGoogle Scholar
- Falquet L, et al.: A human de-ubiquitinating enzyme with both isopeptidase and peptidase activities in vitro. FEBS Lett 1995,359(1):73–7. 10.1016/0014-5793(94)01451-6PubMedGoogle Scholar
- Gehrke PP, Jennissen HP: ATP-dependent proteolysis and the role of ubiquitin in rabbit cardiac muscle. Biol Chem Hoppe Seyler 1987,368(6):691–708.PubMedGoogle Scholar
- Rabinovitz M, Fisher JM: Characteristics of the inhibition of hemoglobin synthesis in rabbit reticulocytes by threo-alpha-amino-beta-chlorobutyric acid. Biochim Biophys Acta 1964, 91: 313–22.PubMedGoogle Scholar
- Lew VL, Garcia-Sancho J: Measurement and control of intracellular calcium in intact red cells. Methods Enzymol 1989, 173: 100–12.PubMedGoogle Scholar
- Sairam MR, Porath J: Isolation of antibodies to protein hormones by bioaffinity chromatography on divinylsulfonyl sepharose. Biochem Biophys Res Commun 1976,69(1):190–6. 10.1016/S0006-291X(76)80290-2PubMedGoogle Scholar
- Jennissen HP: Evidence for negative cooperativity in the adsorption of phosphorylase b on hydrophobic agaroses. Biochemistry 1976,15(26):5683–92. 10.1021/bi00671a001PubMedGoogle Scholar
- Ciechanover A, et al.: "Covalent affinity" purification of ubiquitin-activating enzyme. J Biol Chem 1982,257(5):2537–42.PubMedGoogle Scholar
- Hemdan ES, et al.: Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc Natl Acad Sci USA 1989,86(6):1811–5. 10.1073/pnas.86.6.1811PubMed CentralPubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970,227(259):680–5. 10.1038/227680a0PubMedGoogle Scholar
- Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979,76(9):4350–4. 10.1073/pnas.76.9.4350PubMed CentralPubMedGoogle Scholar
- Armbruster FP, et al.: A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies. Eur J Med Res 2001,6(1):1–9.PubMedGoogle Scholar
- Autric F, et al.: Large-scale purification and characterization of calmodulin from ram testis: its metal-ion-dependent conformers. Biochim Biophys Acta 1980,631(1):139–47. 10.1016/0304-4165(80)90062-8PubMedGoogle Scholar
- Haas AL, Murphy KE, Bright PM: The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts. J Biol Chem 1985,260(8):4694–703.PubMedGoogle Scholar
- Urso ML, et al.: Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components. J Appl Physiol 2006,101(4):1136–48. 10.1152/japplphysiol.00180.2006PubMedGoogle Scholar
- Lecker SH, Goldberg AL, Mitch WE: Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. J Am Soc Nephrol 2006,17(7):1807–19. 10.1681/ASN.2006010083PubMedGoogle Scholar
- Seiffert M, et al.: Regulation of the ubiquitin proteasome system in mechanically injured human skeletal muscle. Physiol Res 2006.Google Scholar
- Attaix D, et al.: The ubiquitin-proteasome system and skeletal muscle wasting. Essays Biochem 2005, 41: 173–86. 10.1042/EB0410173PubMedGoogle Scholar
- Bamezai S, Tate S, Breslow E: Inhibition of ubiquitin-dependent proteolysis by des-Gly-Gly-ubiquitin: implications for the mechanism of polyubiquitin synthesis. Biochem Biophys Res Commun 1989,162(1):89–94. 10.1016/0006-291X(89)91966-9PubMedGoogle Scholar
- Goldknopf IL, et al.: Loss of endogenous nuclear protein A24 lyase activity during chicken erythropoiesis. Biochem Biophys Res Commun 1981,100(4):1464–70. 10.1016/0006-291X(81)90683-5PubMedGoogle Scholar
- Busch H, Goldknopf IL: Ubiquitin--protein conjugates. Mol Cell Biochem 1981,40(3):173–87.PubMedGoogle Scholar
- Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem 1998, 67: 425–79. 10.1146/annurev.biochem.67.1.425PubMedGoogle Scholar
- Koegl M, et al.: A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 1999,96(5):635–44. 10.1016/S0092-8674(00)80574-7PubMedGoogle Scholar
- Finley D, et al.: Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol Cell Biol 1994,14(8):5501–9.PubMed CentralPubMedGoogle Scholar
- Spence J, et al.: A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol Cell Biol 1995,15(3):1265–73.PubMed CentralPubMedGoogle Scholar
- Dubiel W, Gordon C: Ubiquitin pathway: another link in the polyubiquitin chain? Curr Biol 1999,9(15):R554–7. 10.1016/S0960-9822(99)80353-4PubMedGoogle Scholar
- Li SJ, Hochstrasser M: A new protease required for cell-cycle progression in yeast. Nature 1999,398(6724):246–51. 10.1038/18457PubMedGoogle Scholar
- Yeh ET, Gong L, Kamitani T: Ubiquitin-like proteins: new wines in new bottles. Gene 2000,248(1–2):1–14. 10.1016/S0378-1119(00)00139-6PubMedGoogle Scholar
- Melchior F, Schergaut M, Pichler A: SUMO: ligases, isopeptidases and nuclear pores. Trends Biochem Sci 2003,28(11):612–8. 10.1016/j.tibs.2003.09.002PubMedGoogle Scholar
- Pichler A, Melchior F: Ubiquitin-related modifier SUMO1 and nucleocytoplasmic transport. Traffic 2002,3(6):381–7. 10.1034/j.1600-0854.2002.30601.xPubMedGoogle Scholar
- Hemelaar J, et al.: Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol Cell Biol 2004,24(1):84–95. 10.1128/MCB.24.1.84-95.2004PubMed CentralPubMedGoogle Scholar
- Bailey D, O'Hare P: Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1. J Biol Chem 2004,279(1):692–703.PubMedGoogle Scholar
- Hemelaar J, et al.: A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L. J Biol Chem 2003,278(51):51841–50. 10.1074/jbc.M308762200PubMedGoogle Scholar
- Haas AL, et al.: Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin. J Biol Chem 1987,262(23):11315–23.PubMedGoogle Scholar
- Haas AL, Reback PB, Chau V: Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. Comparison to their putative rabbit homologs, E2(20K) AND E2(32K). J Biol Chem 1991,266(8):5104–12.PubMedGoogle Scholar
- Lowe J, et al.: Immunohistochemical localization of ubiquitin cross-reactive protein in human tissues. J Pathol 1995,177(2):163–9. 10.1002/path.1711770210PubMedGoogle Scholar
- Matunis MJ, Coutavas E, Blobel G: A novel ubiquitin-like modification modulates the partitioning of the RanGTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 1996,135(6 Pt 1):1457–70.PubMedGoogle Scholar
- Boddy MN, et al.: PIC 1, a novel ubiquitin-like protein which interacts with the PML component of a multiprotein complex that is disrupted in acute promyelocytic leukaemia. Oncogene 1996,13(5):971–82.PubMedGoogle Scholar
- Mannen H, et al.: Cloning and expression of human homolog HSMT3 to yeast SMT3 suppressor of MIF2 mutations in a centromere protein gene. Biochem Biophys Res Commun 1996,222(1):178–80. 10.1006/bbrc.1996.0717PubMedGoogle Scholar
- Okura T, et al.: Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J Immunol 1996,157(10):4277–81.PubMedGoogle Scholar
- Shen Z, et al.: UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins. Genomics 1996,36(2):271–9. 10.1006/geno.1996.0462PubMedGoogle Scholar
- Kamitani T, Nguyen HP, Yeh ET: Preferential modification of nuclear proteins by a novel ubiquitin-like molecule. J Biol Chem 1997,272(22):14001–4. 10.1074/jbc.272.22.14001PubMedGoogle Scholar
- Lapenta V, et al.: SMT3A, a human homologue of the S. cerevisiae SMT3 gene, maps to chromosome 21qter and defines a novel gene family. Genomics 1997,40(2):362–6. 10.1006/geno.1996.4556PubMedGoogle Scholar
- Tsytsykova AV, et al.: The mouse genome contains two expressed intronless retroposed pseudogenes for the sentrin/sumo-1/PIC1 conjugating enzyme Ubc9. Mol Immunol 1998,35(16):1057–67. 10.1016/S0161-5890(98)00094-7PubMedGoogle Scholar
- Kamitani T, et al.: Characterization of a second member of the sentrin family of ubiquitin-like proteins. J Biol Chem 1998,273(18):11349–53. 10.1074/jbc.273.18.11349PubMedGoogle Scholar
- Kamitani T, et al.: Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein. J Biol Chem 1997,272(45):28557–62. 10.1074/jbc.272.45.28557PubMedGoogle Scholar
- Mizushima N, et al.: A protein conjugation system essential for autophagy. Nature 1998,395(6700):395–8. 10.1038/26506PubMedGoogle Scholar
- Saitoh H, Hinchey J: Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem 2000,275(9):6252–8. 10.1074/jbc.275.9.6252PubMedGoogle Scholar
- Furuno K, Goodman MN, Goldberg AL: Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy. J Biol Chem 1990,265(15):8550–7.PubMedGoogle Scholar
- Wing SS, Goldberg AL: Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting. Am J Physiol 1993,264(4 Pt 1):E668–76.PubMedGoogle Scholar
- Mitch WE, et al.: Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes. J Clin Invest 1994,93(5):2127–33. 10.1172/JCI117208PubMed CentralPubMedGoogle Scholar
- Llovera M, et al.: Ubiquitin gene expression is increased in skeletal muscle of tumour-bearing rats. FEBS Lett 1994,338(3):311–8. 10.1016/0014-5793(94)80290-4PubMedGoogle Scholar
- Fang CH, et al.: Burn injury stimulates multiple proteolytic pathways in skeletal muscle, including the ubiquitin-energy-dependent pathway. J Am Coll Surg 1995,180(2):161–70.PubMedGoogle Scholar
- Lecker SH, et al.: Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states. J Nutr 1999,129(1S suppl):227S-37S.PubMedGoogle Scholar
- Tiao G, et al.: Sepsis stimulates polyamine biosynthesis in the liver and increases tissue levels of ornithine decarboxylase mRNA. Shock 1995,4(6):403–10.PubMedGoogle Scholar