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)

In attempts to produce fragments of an allergenic molecule which would retain allergenic and/or antigenic determinant(s), the cytochrome c of ryegrass (RG) pollen, which had been shown to be an allergenic constituent of this pollen, was digested with trypsin and chymotrypsin and the resulting fragments were separated by high performance liquid chromatography. Several of these fragments were shown, with the aid of the radioallergosorbent test and solid phase radioimmunoassays, to bind IgE antibodies present in a pool of six sera from grass-sensitive patients and three murine monoclonal antibodies, designated as Mab 41, Mab 42 and Mab 43, which had been originally produced against the crossreacting cytochrome c of Kentucky bluegrass (KBG). In summary, (i) fragments C-67 and C-74 reacted with all antibodies, (ii) fragments T-45, T-46 and C-69 bound to human IgE antibodies as well as to Mab 41 and Mab 42, but not to Mab 43, (iii) fragment T-44 reacted only with Mab 41 and Mab 42, and (iv) fragment C-83 bound only Mab 42 and Mab 43. On the basis of these results, it is concluded that (i) immunochemically active fragments of the RG cytochrome c can be readily produced by enzymatic degradation, (ii) there is significant crossreaction between the antigenic determinants of RG and KBG cytochromes c, (iii) whereas all fragments possessed at least two of the original antigenic determinants, fragments C-83 and T-44 were devoid of allergenic determinants, (iv) the antigenic determinants recognized by Mab 41 and Mab 42 were different from those reacting with human IgE antibodies and Mab 43, (v) each of the three monoclonal antibodies recognized a distinct antigenic determinant, (vi) fragments C-67 and C-74 possessed all determinants recognized by the human IgE and mouse antibodies used.
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PMID:Determinants of ryegrass pollen cytochrome c recognized by human IgE and murine monoclonal antibodies. 620 97

A specific antibody subpopulation(s) in antihorse cytochrome c serum was detected for peptide fragment 81-104 of cyanogen bromide (CNBr) cleaved horse cytochrome c (HCytc). This antiserum was made in the rabbit against polymeric horse cytochrome c. The presence of the peptide-specific antibody subpopulation(s) was demonstrated utilizing HCytc, CNBr-peptide 81-104 and isolated chymotrypsin-digested HCytc fragments 60-67, 83-97 and 98-104 to compete with radio-labeled peptide 81-104 and antiHCytc serum in a competitive radioimmunoassay (RIA). This antibody subpopulation(s) in antiHCytc serum was demonstrated to be specific for peptide 81-104. At the 50% inhibition level in competitive RIA, 100- and 1000-fold molar excesses of HCytc and its peptide 1-65, respectively, were required to affect an equivalent binding to that of the HCytc peptide 81-104. Competitive RIAs have been performed utilizing three different kinds of antigen to compete with HCytc peptide 81-104 and antihorse cytochrome c sera. These three kinds of antigens are: endopeptidase digests of HCytc, cytochrome c peptides 81-104 of several species and several isolated chymotryptic peptide fragments of HCytc. The results have indicated that this peptide-specific antibody subpopulations(s) in antiHCytc serum is similar to antibodies made against peptide 81-104-BSA. Regions of antigenicity have been identified at positions 92, 100, 103 and 104 with both antisera.
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PMID:Antibody subpopulation in antihorse cytochrome c serum. 620 69

Myoglobin was purified from a muscle extract of lace monitor lizard, Varanus varius, by Sephadex G-75, followed by DEAE-cellulose column chromatography. The apomyoglobin was cleaved with cyanogen bromide. The largest fragment was further digested with pepsin, trypsin, and alpha-chymotrypsin. From the amino acid sequence of the cyanogen bromide fragments, together with those of tryptic peptides of apomyoglobin, the complete amino acid sequence of lizard myoglobin was deduced. To investigate the tetrapod and amniote origins, many possible phylogenetic trees were constructed using the myoglobin sequences, including those of map turtle and lace monitor lizard. The tree that requires the minimum number of nucleotide substitutions in their genes for the myoglobin sequences to have evolved from a common ancestor was different from the similarly most parsimonious trees for cytochrome c or for alpha-hemoglobin. The trees were different from each other and from the tree that best reflects current biological opinions.
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PMID:Amino acid sequence of a myoglobin from lace monitor lizard, Varanus varius, and its evolutionary implications. 626 Jul 92

Cytochrome c1 is a subunit of ubiquinol--cytochrome c reductase (EC 1.10.2.2). In Neurospora crassa wild type 74A grown in the presence of chloramphenicol, the subunit is inserted only into the bilayer of the mitochondrial inner membranes without associating with other proteins. From these modified membranes a monodisperse (cytochrome c1)-Triton complex was isolated by subjecting the Triton-solubilized membranes to affinity chromatography on immobilized cytochrome c. A water-soluble pentamer of cytochrome c1 was prepared from the (cytochrome c1)-Triton complex by removing the detergent. By limited proteolytic digestion of the cytochrome c1-Triton complex with chymotrypsin, a water-soluble monomeric cytochrome c1 was prepared which has a molecular weight of only 24 000 as compared to 31 000 of the membrane-bound cytochrome c1. The 24 000-Mr cytochrome c1 and the 31 000-Mr cytochrome c1 have same light absorption spectra and cytochrome-c-binding properties. These results are used to propose the following model. Cytochrome c1 consists of a large hydrophilic part and a small hydrophobic part. The hydrophilic part extends from the mitochondrial inner membrane into the intermembrane space. This part carries the heme and interacts with cytochrome c. The hydrophobic part anchors the cytochrome c1 to the bilayer.
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PMID:Membrane-bound and water-soluble cytochrome c1 from Neurospora mitochondria. 626 10

A recently developed photometric version of polyelectrolyte titration was applied for the determination of the number of charged residues on globular proteins. Based on the observation that oppositely charged polyelectrolytes form, in general, stoichiometric polyelectrolyte complexes, the protein solutions were incubated in excess with an oppositely charged polyelectrolyte, and the residual amount was back-titrated using o-toluidine blue for end point detection. It was found that within the range pH 2 to pH 9 the interaction of the polyelectrolytes, potassium polyvinylsulfate, polydiallylammonium chloride, and N-methylglycolchitosan iodide, with various proteins of known amino acid composition (ribonuclease A, trypsin, chymotrypsin A, pepsin, cytochrome c) occurs stoichiometrically through 1:1 ion pair interaction, irrespective of the spatial distribution of the interacting ionic sites. The close correspondence between the experimental data for the net charge and the calculated balance of ionized residues for the proteins at a given pH indicates that in the native structure of these proteins oppositely charged ionic functions are largely neutralized by the formation of intramolecular salt linkages. It is concluded that polyelectrolyte titration offers an easy access to the determination of the surface charge of proteins and other biopolymers. The data further support the notion of the importance of electrostatic cooperative interactions in biological systems.
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PMID:Charge determination of proteins with polyelectrolyte titration. 629 8

The amino acid sequence of the soluble monohaem cytochrome c-556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl-Edman degradation and automatic liquid-phase sequence analysis. The main characteristic of this cytochrome is that the haem-binding sequence Cys-Xaa-Yaa-Cys-His occurs in the C-terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C-terminal lysine-122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c-556 is the first example known of a class II cytochrome of the low-spin type isolated from an obligate aerobic organism.
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PMID:The complete amino-acid sequence of the low-spin class II cytochrome c-556 from Agrobacterium tumefaciens strain B2a. 629 89

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.
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PMID:Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography. 652 98

The variable hydrophobic nature of proteins allows their separation through differential hydrophobic surface interactions. From these observations two modes of protein chromatography have been developed, hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC). Selectivity of the HIC column can be easily manipulated by changing mobile phase variables. Protein retention was increased by decreasing the pH from neutrality or by using a salt with a greater "salting-out" ability. In addition, selectivity can be altered through chemical modification of the matrix surface. Protein retention and resolution decreased concomitantly with matrix ligand density. There were several major differences in HIC and RPC selectivity. Hydrophilic proteins such as cytochrome c and myoglobin were weakly retained on the HIC column but strongly retained on the RPC column. In contrast, a hydrophobic protein such as beta-glucosidase was strongly retained on the HIC column and only weakly retained on the RPC column. Other proteins were retained equally by RPC and HIC columns. Load capacity on the HIC column was determined by plotting resolution as a function of protein load. Resolution decreased significantly after 7.5 mg of total protein had been loaded onto the column per cm3 of column material. Samples of lactic dehydrogenase and alpha-chymotrypsin ranging in size from 10-200 micrograms were recovered from an HIC column with greater than 86% enzymatic activity in all cases. The recovery of enzymatic activity of alpha-chymotrypsin ranged from 55-91%, while none of the activity of beta-glucosidase was recovered from the RPC column.
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PMID:Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins. 653 Apr 30

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.
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PMID:Human NADH-cytochrome b5 reductases: comparison among those of erythrocyte membrane, erythrocyte cytosol, and liver microsomes. 684 58

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.
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PMID:The site of alpha-chymotryptic activation of pigeon erythrocyte adenylate cyclase. 737 11


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