Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The rod prepared from chicken gizzard myosin has been found to have two sites sensitive to limited digestion with chymotrypsin; these sites were located at a subfragment 2/light meromyosin junction (site 1), and at a site 10 kDa remote from either C-terminal or N-terminal of light meromyosin (site 2). The site 1 was more sensitive to the digestion than the site 2. The cleavage at site 2 of the light meromyosin yielded a 74-kDa fragment that was soluble in a low ionic strength solution, contrary to the insolubility of the parent light meromyosin in the same solution. Studies on the effects of MgCl2, ATP and pH on the susceptibilities of these sites to chymotrypsin have given following results. (a) Millimolar concentrations of MgCl2 protected site 1 and site 2 from the chymotryptic cleavage. (b) The cleavage at site 1 of myosin rod in the low salt solution free of Mg2+ at pH 7.0 and pH 8.5, was not affected by the presence of 5 mM ATP. However, MgCl2-induced protection of site 1 was relieved by addition of ATP. On the other hand, the cleavage at site 2 was stimulated by addition of ATP, irrespective of the presence or absence of MgCl2. (c) The alkaline condition of pH 8.5 was more favorable for the chymotryptic cleavages at both site 1 and site 2 than the neutral condition of pH 7.0. These results suggest that myosin rod contains two flexible regions, the structures of which are influenced by such an ambient factor as MgCl2, ATP or pH.
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PMID:Two chymotrypsin-susceptible sites of myosin rod from chicken gizzard. 399 92

Lens membranes, purified from calf lenses, have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated two major 125I-containing products of Mr = 49 000 and 36 000. That the formation of these two components was specifically inhibited by unlabeled calmodulin, or calmodulin antagonists, would indicate that the formation of these components was calmodulin-specific. The size of these two 125I-labeled components was unchanged over a range of 125I-calmodulin or dithiobis(succinimidyl propionate) concentrations indicating that they represent 1:1 complexes between 125I-calmodulin (Mr = 17 000) and Mr-32 000 and Mr-19 000 lens membrane components respectively. Although formation of both cross-linked components exhibited an absolute dependence on Mg2+, the autoradiographic intensity of these components was enhanced when Ca2+ was included with Mg2+ during the cross-linking reaction. Labeling was maximal in 10 mM MgCl2 and approximately 1 microM Ca2+. Treatment of lens membranes with chymotrypsin resulted in the cleavage of MP26 (the major lens membrane protein), with the appearance of a major proteolytic fragment of Mr = 22 000. This proteolysis was not associated with any significant change in either the size or amount of the 125I-calmodulin-labeled membrane components. These results suggest that calmodulin interacts with two membrane proteins, but not significantly with MP26, in the intact lens cell membrane. Our results indicate the need to maintain caution in interpreting direct calcium plus calmodulin effects on MP26 and lens cell junctions.
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PMID:Identification of the calmodulin-binding components in bovine lens plasma membranes. 401 84

The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, alpha-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.
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PMID:Evidence that neutral sphingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane. 609 59

1. DNase I from porcine pancreas, if Mg2+ was present, hydrolyzed both sDNA and dDNA, whether free or bound to Sepharose. The hydrolysis rates were maximum at pH 7.5 with the bound DNAs and at pH 7.0 with the free DNAs negligible at pH 4.0 and pH 10.5 with the free and bound DNAs. The hydrolysis was completely inhibited by 50 mM sodium citrate. 2. With 50 mM citrate buffer (Ph 4.0), DNase I was effectively adsorbed on the DNA-Sepharoses in the absence of 5 mM Mg2+. The adsorbed enzyme was effectively eluated by the buffer containing 1 M KCl (eluate). The amounts of the eluated enzyme were approximately 1.5 X 10(5) units/mg DNA with sDNA-Sepharose and approximately 3.0 X 10(5) units/mg DNA with dDNA-Sepharose. This simple adsorption-elution of the pancreas extract resulted in approximately 300-fold purification of DNase I with a yield of 95%. In the elute, the ratios in activity of trypsin, chymotrypsin and RNase to DNase I were 1/(4.0 X 10(5)), 1/(5.3 X 10(3)), and 1/(4.1 X 10(2)) as low as in the extract, respectively. In addition, the eluate was not contaminated by kallikrein or carboxypeptidases A and B. 3. Upon repeating the adsorption-elution described above, the adsorbing capacities of DNA-Sepharoses gradually deteriorated with the whole pancreas extract, but not with the precipitate of the extract formed on 60% ammonium sulfate saturation, which contained 90% of the DNase I. With the precipitate, one dDNA-Sepharose solumn was repeatedly usable at least 20-times without deterioration. The DNase I preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis. 4. Conceivably, the above-mentioned adsorption of DNase I on DNA-Sepharoses was mainly due to the steric and electrostatic affinity of a relatively large moiety of the DNA molecule to the substrate-binding site, but not to the catalytic site, of the enzyme.
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PMID:Affinity chromatography of porcine pancreas deoxyribonuclease I on DNA-binding sepharose under non-digestive conditions, using its substrate-binding site. 625 6

The topology of beef heart Complex III has been studied by tryptic and chymotryptic digestion of isolated Complex III, Mg2+-ATP submitochondrial particles, and mitoplasts. Degradation products were detected by the immunoreplication technique using specific antibodies against core protein 1 (50 K) and core protein 2 (47 K). It can be shown that both peptides are digested from the matrix side of the inner membrane. However, no evidence was found that these peptides were digested by trypsin or chymotrypsin from the cytoplasmic side. It is concluded that the beef heart core proteins are membrane-bound peptides containing tryptic and chymotryptic digestion sites only on the matrix surface of the inner membrane. The data also suggest that beef heart core protein 2 contains multiple domains which are inserted into the membrane from the matrix surface. Proteolytic treatment of submitochondrial particles under conditions which digested at least 50% of the core proteins from the matrix surface did not, however, influence NADH oxidation rates or the respiratory control ratios.
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PMID:Studies on beef heart ubiquinol-cytochrome c reductase. Topological studies on the core proteins using proteolytic digestion and immunoreplication. 630 20

Friend murine erythroleukemia cells (MEL cells) contain a cAMP-independent protein kinase which phosphorylates the 100,000-Da catalytic subunit of the (Na,K)-ATPase both in living cells and in the purified plasma membrane (Yeh, L.-A., Ling, L., English, L., and Cantley, L. (1983) J. Biol. Chem. 258, 6567-6574). We have taken advantage of the selective phosphorylation of the 100,000-Da subunit in purified plasma membranes and the similarity between the proteolysis patterns of the MEL cell and dog kidney (Na,K)-ATPase to map the site of kinase phosphorylation on the MEL cell enzyme. The chymotryptic and tryptic cleavage sites of the dog kidney (Na,K)-ATPase have previously been located (Castro, J., and Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228). The 100,000-Da catalytic subunits of the dog kidney and MEL cell enzymes were specifically labeled at the active site aspartate residue by incubation with (32P)orthophosphate in the presence of Mg2+ and ouabain. Digestion of these two enzymes with chymotrypsin or trypsin revealed similar active site aspartate containing proteolytic fragments indicating a similar structure for the two enzymes. Chymotryptic digestions of MEL cell (Na,K)-ATPase labeled in vitro with [gamma-32P]ATP localize the region of kinase phosphorylation to within a 35,000-Da peptide derived from the middle of the 100,000-Da subunit. Tryptic digestion of the MEL cell plasma membranes degraded the 100,000-Da subunit to an NH2-terminal 43,000-Da peptide which contained the active site aspartate but which did not contain the kinase-labeled region. These results further locate the region of kinase phosphorylation to the COOH-terminal half of the 35,000-Da chymotryptic peptide. This location places the site of phosphorylation between the active site aspartate residue which accepts the phosphate of ATP during turnover and an ATP-binding site which has previously been located by labeling with fluorescein 5'-isothiocyanate (Carilli, C. T., Farley, R. A., Perlman, D. M., and Cantley, L. C. (1982) J. Biol. Chem. 257, 5601-5606). Phosphorylation of the (Na,K)-ATPase in this region may serve to regulate the activity of this enzyme.
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PMID:The (Na,K)-ATPase of Friend erythroleukemia cells is phosphorylated near the ATP hydrolysis by an endogenous membrane-bound kinase. 632 56

The (Na+,K+)-ATPase from dog kidney has been reconstituted into egg lecithin vesicles (Goldin, S. M. (1977) J. Biol. Chem. 252, 5630-5642). Using sucrose density gradient centrifugation, we have isolated sealed vesicle populations in which the protein molecules have defined orientations. Sealed vesicles sedimented at higher density than unsealed vesicles after equilibration with CsCl. Vesicles containing inside-out oriented enzyme sedimented at lower density than vesicles containing right-side-out oriented enzyme after the internally trapped Cs+ had been pumped out during an incubation with Mg2+ and ATP. Pools of gradient fractions representing unsealed vesicles and sealed vesicles containing inside-out and right-side-out oriented protein were characterized with respect to orientation and degree of sealing by determination of the ATPase activity, the rate of ATP-dependent Na+ uptake, and the inhibition of ATPase activity by ouabain. The accessibilities of sialic acid and of a tryptic site in the vesicle populations were in agreement with the proposed orientations of the protein. The structure of the reconstituted (Na+,K+)-ATPase was examined by proteolysis with trypsin and chymotrypsin over a range of reconstitution protocols. The fragmentation patterns demonstrate that the cholate-reconstituted enzyme, although functionally competent, differs in structure from the native purified enzyme.
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PMID:Purification and proteolysis of vesicles containing inside-out and right-side-out oriented reconstituted (Na+, K+)-ATPase. 632 27

Reproducing forms of Trypanosoma lewisi isolated from X-irradiated rats and adult forms from intact rats were not lysed by fresh mammalian sera. Treating parasites with trypsin or chymotrypsin, but not with neuraminidase, under conditions which did not impair viability rendered the parasites sensitive to lysis by rat, mouse, rabbit, and human sera. Serum from animal strains or humans genetically deficient in complement component C3, C5, or C6 did not lyse protease-treated parasites. The lytic factors in serum displayed the heat sensitivity and the Mg2+ requirement characteristic of the alternate complement pathway. Lysis was resolved into two phases, Mg2+-dependent binding of serum factors to parasites and subsequent C5-dependent, Mg2+-independent lysis. Allowing protease-treated parasites to readsorb host proteins did not block lysis by serum. Protease-treated parasites regenerated components which prevented complement-mediated lysis during 2 h in culture at 37 degrees C. This regeneration was inhibited by cycloheximide but not by tunicamycin. Ten major components were resolved in radioautographs of sodium dodecyl sulfate-polyacrylamide gels of extracts of radioiodinated intact cells. Protease treatment before radioiodination reduced the amount of radioactivity associated with these components disproportionately. Components with apparent molecular weights of 102,000, 88,000, and 47,000 were strongly labeled in intact cells, poorly labeled after enzyme treatment, and again labeled in cells that were cultured at 37 degrees C after enzyme treatment. Cycloheximide blocked the reappearance of these components on cultured cells. The presence of these three components was therefore correlated with resistance to complement-mediated lysis.
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PMID:Externally disposed membrane polypeptides of intact and protease-treated Trypanosoma lewisi correlated with sensitivity to alternate complement pathway-mediated lysis. 635 42

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.
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PMID:The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro. 645 76

A cyclic nucleotide-independent protein kinase, protease-activated kinase II, which incorporates up to four phosphates into 40 S ribosomal protein S6, has been purified from the postribosomal supernatant of rabbit reticulocytes. Protease-activated kinase II was purified as an inactive proenzyme by chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-150, and hydroxylapatite. The enzyme was activated in vitro by limited digestion with trypsin or chymotrypsin. No other mode of activation for protease-activated kinase II in vitro was identified. The proenzyme had a molecular weight of 80,000 as measured by gel filtration; following tryptic digestion, the molecular weight of the activated protein kinase was 45,000-55,000. Protease-activated kinase II required Mg2+ for activity but was inhibited by other divalent cations, monovalent cations, and fluoride ion. ATP was the phosphoryl donor in the phosphorylation reaction; GTP had no effect. In vitro, multiple phosphorylation of S6 was observed with some phosphate incorporated into S10. Phosphorylation of S6 by protease-activated kinase II has been shown to be stimulated in serum-starved 3T3-L1 cells by insulin (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 9589-9592) and in reticulocytes by altering the pH of the incubation medium (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 13998-14002.
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PMID:Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Purification and characterization of protease-activated kinase II. 664 62


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