Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues.
Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40,000 x g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4-80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3-4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15-20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin-125I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.
Laub
M
M
Institut für Physiologische Chemie, Universität-GHS-Essen, Germany.
Jennissen
H P
HP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002474:Cell-Free System; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D010453:Peptide Synthases / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D013154:Spleen / Q000201:enzymology; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D002147:Calmodulin; D014452:Ubiquitins; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D044767:Ubiquitin-Protein Ligases; C059404:ubiquitin-calmodulin ligase; D010450:Endopeptidases; D044764:Ubiquitin-Activating Enzymes; D010453:Peptide Synthases; D002118:Calcium