title: Tryptic cleavage inhibits but does not uncouple Ca2+ATPase of sarcoplasmic reticulum.
abstract: Sarcoplasmic reticulum Ca2+ATPase is cleaved by trypsin at two sites, T1 and T2. Cleavage at T1 is complete, whereas only about 50% of the Ca2+ATPase is digested at the T2 site. In the absence of Ca2+ ionophor, Ca2+-ATPase activity of the digested enzyme remains virtually unchanged. In the presence of Ca2+ ionophor, however, the calculated specific activity of the doubly cleaved Ca2+ATPase is decreased by about 40%. The decrease in Ca2+ transport activity is much more rapid than cleavage of the T2 site, and could be correlated with an increased leak of Ca2+ from the digested vesicles. We obtained evidence that this leakiness is independent of the digestion of the Ca2+ATPase itself and is presumably due to the digestion of some other components of the sarcoplasmic reticulum vesicles. Examination of steady-state phosphoenzyme levels resulting from phosphorylation by ATP and Pi, or dephosphorylation by ADP or ADP/EGTA revealed no difference between the digested and the undigested Ca2+ATPase indicating no change in the equilibria caused by the T2 cleavage. Analysis of the substrate concentration dependence of the Ca2+ATPase activity also led to the conclusion that the digestion at T2 reduced the Vmax of ATP hydrolysis but leaves the Km unchanged. The above results are consistent with the model that cleavage at the T2 site reduces the turnover rate of the Ca2+ATPase reaction cycle by about 40% by slowing down or altering the rate-limiting step without affecting the equilibrium constants of the examined steps. We found no evidence of true uncoupling of Ca2+ transport from ATP hydrolysis correlated with cleavage at the T2 site.
authors.affiliation: Laboratory of Protein Structure, National Institute for Medical Research, London, England.
authors.forename: K
authors.initials: K
authors.lastname: Török
authors.forename: B J
authors.initials: BJ
authors.lastname: Trinnaman
authors.forename: N M
authors.initials: NM
authors.lastname: Green
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D006868:Hydrolysis; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D010710:Phosphates; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases