Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.
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PMID:Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. 242 14

The functional domains of the in situ red cell membrane calcium pump were mapped by a double labeling technique. In inside-out vesicles (IOVs) the calcium pump was phosphorylated by [gamma-32P]ATP, the proteins blotted onto nitrocellulose and tagged by monoclonal antibodies raised against the purified pump protein. After proteolytic treatment of the IOVs by trypsin, chymotrypsin, or calpain-I, the fragmentation pattern of the enzyme was followed on the double-labeled immunoblots. The changes in the kinetics of the pump were examined by parallel measurements of the active calcium uptake in IOVs. By analysis of the results of tryptic digestion, it was possible to show that the antibodies recognized three different domains of the pump: 1) a Mr = 10,000-15,000 fragment (not seen directly) which includes the calmodulin-binding domain, 2) a nonphosphorylated Mr = 35,000 tryptic fragment, and 3) a phosphorylated fragment of Mr = 76,000-81,000. Chymotrypsin or calpain-I digestion of the membranes produced one major, Mr = 125,000 fragment, which had lost antibody-binding region 1. Production of this fragment coincided with the loss of calmodulin dependence and with a calmodulin-like activation of IOV calcium uptake (high Vmax, cooperativity in calcium activation). The Mr = 125,000 fragment was further activated by acidic lipids producing high Vmax and low K 1/2 (Ca2+) with no cooperativity. Based on these data a kinetic model and a functional map of the plasma membrane calcium pump is suggested.
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PMID:Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. Possible sites for regulation by calpain and acidic lipids. 253 49

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.
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PMID:Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct. 316 Jun 95