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

---

Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
...
PMID:Studies on beef heart ubiquinol-cytochrome c reductase. Topological studies on the core proteins using proteolytic digestion and immunoreplication. 630 20

Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM, and k3/Ki is 0.006 M-1 s-1. 3-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X isobutyramide, was retarded by NAD+, NADH, or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+, or NADH. Enzyme was inactivated with radioactive 3-bromopropionic acid, aminoethylated, and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.
...
PMID:Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid. 636 61

Pig heart citrate synthase was subjected to limited proteolytic attack by subtilisin, chymotrypsin, and trypsin in the presence of palmitoyl-CoA. Initial proteolysis by all three proteolytic enzymes resulted in cleavage of the monomeric subunit (Mr 45 000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000-12 000) fragment. Further proteolysis of the large subunit produced a secondary fragment (Mr 31 000-36 000). The small (Mr 9000-12 000) fragment was stable in the presence of subtilisin but was substantially degraded by both chymotrypsin and trypsin. The actual molecular weight of fragments varied with the choice of the proteolytic enzyme. Limited proteolysis was absolutely dependent on the presence of palmitoyl-CoA and resulted in complete inhibition of the catalytic activity of the enzyme. Citrate, ammonium sulfate, and especially oxaloacetate provided complete protection against proteolysis whereas acetyl-CoA, CoASH, NADH, and ATP were ineffective. Reaction of rabbit anti-citrate synthase with citrate synthase and its proteolytic fragments indicated that the main antigenic region lay primarily in the small fragment. The products of subtilisin cleavage were isolated by gel filtration under denaturing conditions. The large (Mr 35 000-38 500) fragment contained the amino-terminal (approximately)336 amino acids and the small fragment contained the remaining carboxyl-terminal amino acids. The results are discussed in relation to the structure of citrate synthase.
...
PMID:Limited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin. 677 58

NADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36,000 and 32,000 daltons, respectively, which are comparable to those of the detergent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver microsomes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.
...
PMID:Human NADH-cytochrome b5 reductases: comparison among those of erythrocyte membrane, erythrocyte cytosol, and liver microsomes. 684 58

Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.
...
PMID:An NADH-coupled assay for femtogram or nanogram quantities of chymotrypsin. 684 91

Treatment of intact pigeon erythrocytes with trypsin or alpha-chymotrypsin does not alter the isoproterenol-dependent adenylate cyclase activity in plasma membranes prepared after proteolysis. However, both proteases affect adenylate cyclase activity when isolated membranes are digested. Thus, the proteases probably act at the cytoplasmic side of the membranes. This conclusion is supported by the finding that proteases are able to inhibit NADH cytochrome c oxidoreductase, an enzyme located on the inner face of the plasma membrane. In isolated membranes, trypsin inhibits adenylate cyclase. Chymotrypsin (2.5 microgram/ml, 10 min, 37 degrees C) activates adenylate cyclase about 3-fold when the enzyme activity is measured with NaF, guanosine 5'-(beta, gamma-imino)-triphosphate, or guanosine 5'-(beta, gamma-imino)-triphosphate and isoproterenol. Chymotrypsin also activates adenylate cyclase in membranes pretreated with cholera toxin. Activation by chymotrypsin is not expressed when adenylate cyclase is assayed with 5 mM Mn2+ without guanine nucleotides or fluoride. However, the chymotryptic activation is expressed when guanosine 5'-(beta, gamma-imino)-triphosphate is present together with Mn2+. We conclude that interaction of the guanine nucleotide regulatory subunit with the catalytic subunit of adenylate cyclase is required for expression of chymotryptic activation.
...
PMID:The site of alpha-chymotryptic activation of pigeon erythrocyte adenylate cyclase. 737 11

Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [alpha-32P]nicotinamide-2-azidoadenine dinucleotide has been used to identify an NADH/NADPH binding site. Specificity of nucleotide interaction was demonstrated by saturation and protection of labeling at physiologically relevant concentrations. Saturation of photoinsertion was observed at approximately 100 microM probe with an apparent Kd of approximately 25 microM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 microM of the above-mentioned compounds. The protection obtained with NADP+ and NADPH was of the same order, respectively. The adenine ring binding domain of NADH/NADPH binding site was identified by trypsin and chymotrypsin digestion of the photolabeled prolactin and purification of the photolabeled peptide by boronate affinity chromatography and immobilized Fe3+ affinity chromatography. The peptide was identified to be Ala22-Tyr28. These studies demonstrate that prolactin contains an NADH/NADPH binding site which may be significant in the mechanism of action of this hormone.
...
PMID:Identification and characterization of a nucleotide binding site of ovine prolactin with 2-azido-NAD. 832 98

Sera from patients with a variety of cancers, including solid carcinomas, leukemias, and lymphomas, contain a ca. 33.5-kDa protein absent from sera of healthy volunteers or patients not diagnosed as having cancer. The protein exhibits an NADH oxidase activity inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin). The activity and the protein are resistant to digestion by proteases (trypsin, chymotrypsin, proteinase K, subtilisin) and to heat. Following protease digestion to reduce the content of major serum proteins, the 33.5-kDa protein could be detected on Western blots of SDS-PAGE transferred to nitrocellulose membranes using polyclonal antisera to a corresponding partially purified 33.5-kDa protein shed into culture media conditioned by growth of HeLa cells. No corresponding protein was seen with control sera. The findings confirm the capsaicin-inhibited NADH oxidase activity of cancer sera as a circulating marker potentially specific to sera of cancer patients and identify a ca. 33.5-kDa protein resistant to proteases and heat as the source of the circulating capsaicin-inhibited NADH oxidase activity.
...
PMID:A 33.5-kDa heat- and protease-resistant NADH oxidase inhibited by capsaicin from sera of cancer patients. 918 12

The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C. Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site. SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa. Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change. Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH. Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme. Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments. However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure. Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states. N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.
...
PMID:A thermally sensitive loop in clostridial glutamate dehydrogenase detected by limited proteolysis. 1241 8

Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate-aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. Modiro v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease.
...
PMID:Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampus. 2050 18


<< Previous 1 2 3 Next >>

---