Figure 9a

Trypsin-incubation of heart muscle-extract and APFII. Figure 9a shows the cleavage of internal and external ubiquitin-conjugates of the heart muscle with trypsin. The incubation batches have a final concentration of 5 μg/ml leupeptine. The batches were incubated for 30 min in 37°C in a waterbath. The reaction was stopped by adding trichloracetic acid (final concentration 5% w/v TCA, 20 min 0°C). After centrifugation for 5 min in an Eppendorf centrifuge 5415 (Eppendorf, Hamburg) at 16000 × g the pellet were solved in the sample buffer for Laemmli system and then blotted on PVDF-membrane (see materials). Lane 1: 8.5 μg ubiquityl-calmodulin conjugates and 2 μg unconjugated ubiquitin. Lane 2: 200 μg red muscle-extract, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 3: 200 μg heart muscle extract and 65 μg trypsin. Lane 4: 200 μg heart muscle extract. Lane 5: 200 μg heart muscle APFII, 8.5 μg ubiquityl-calmodulin conjugates and 65 μg trypsin. Lane 6: 200 μg heart muscle APFII and 65 μg trypsin. Lane 7: 200 μg heart muscle APFII.