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 folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

Apolipoprotein A-I-containing lipoproteins (high density lipoproteins, HDL) can be separated into two subfractions, which have pre-beta and alpha electrophoretic mobilities, respectively. These fractions differ in both composition and structure. Some preparations of pre-beta-migrating HDL, but not alpha-migrating HDL, were found to contain two polypeptides with Mr of approximately 26 and 14 kDa, which are scission products of apolipoprotein (apo) A-I. They are recognized by monospecific antibodies to apo A-I and have N-terminal sequences identical to those of mature apo A-I. This proteolytic scission of apo A-I occurs primarily after venipuncture. Immediate addition of protease inhibitors minimized the appearance of the fragments in plasma. To study the relative susceptibilities of pre-beta and alpha HDL to proteolysis, the lipoproteins were incubated in vitro with plasmin. The apo A-I in pre-beta HDL was extensively degraded, but that in alpha-migrating HDL was degraded to a much lesser extent, indicating that the appearance of apo A-I fragments in pre-beta HDL was due to enhanced sensitivity to proteolysis. To varying degrees, thrombin, kallikrein, elastase, arginine C endoprotease, and chymotrypsin also appear to cleave pre-beta HDL faster than alpha HDL. Most of the proteases generated a 12 to 14 kDa peptide fragment under conditions of limited cleavage. These results suggest that the conformational state of apo A-I in pre-beta-migrating HDL or its spatial relationship to lipids is significantly different from that of apo A-I in alpha-migrating HDL.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pre-beta high density lipoprotein. Unique disposition of apolipoprotein A-I increases susceptibility to proteolysis. 213 93

The digestive tracts of adult and juvenile Dover sole were examined for protease activities. A pepsin-like protease with an optimal pH value of 1.7 predominated in the stomach region, but the main endoprotease action in the foregut, midgut and hindgut regions was optimal in the range of pH 9.5-10.5 and showed good activity towards elastin orcein. Experiments using synthetic substrates suggested the presence of chymotrypsin- and trypsin-like activities optimal between pH 7 and 8. Collagenase activity was also shown to exist in this pH region. The presence of enzymes corresponding to carboxypeptidases a and b and leucine aminopeptidase was indicated. The possible significance of these results to the farming of Dover sole is discussed.
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PMID:Metabolism in marine flatfish. II. Protein digestion in Dover sole (Solea solea L.). 299 Aug 7

Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material.
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PMID:Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells. 352 12

Amino-acid sequence of kynureninase purified from rat liver cytosol was determined by an amino-acid sequencer. The enzyme was degraded to small peptides with cyanogen bromide, TPCK-trypsin, endoproteinase Glu-C, lysyl endoprotease and alpha-chymotrypsin. The enzyme subunit consisted of 464 amino acids, and the molecular weight of subunit was determined to be 52,510. The coenzyme pyridoxal phosphate-binding residue was lysine of which position was 276, and the N-terminal residue was N-acetylmethionine. The homology search between this enzyme and the other pyridoxal phosphate-dependent enzymes showed that kynureninase was similar to mitochondrial aspartate aminotransferase, and also to cystathionine gamma-synthase and gamma-lyase to a lesser extent.
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PMID:Amino-acid sequence of rat liver kynureninase. 757 21

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is an important pharmacological target of antiviral nucleoside drugs and it uniquely possesses both a thymidine kinase and a thymidylate kinase activity. The structural relationship between these two activities is addressed in this study using a combination of active-site directed photoaffinity analogs, proteases, and tricine-SDS-polyacrylamide gel electrophoresis. For analysis of the thymidylate binding site, the thymidylate analog [32P]5-azido-dUMP was specifically photocrosslinked to the active site of HSV-1 TK. Because the amino acid sequence of HSV-1 TK is known, endoprotease Lys-C, V8 protease, trypsin, or chymotrypsin was used to generate a proteolytic map of photoincorporated peptides by separation on high-resolution tricine-SDS-polyacrylamide gels. Analysis of the resulting peptides indicated that the photoprobe was localized to one region comprising amino acids Ile112-Tyr132. Photolabeling of this region indicates that the thymine base of thymidine and TMP bind at one shared site in HSV-1 TK. In addition, the results reported in this study demonstrate that photolabeling with azidonucleotides can be used to identify photolabeled peptides by proteolytic mapping. This technique bypasses the problems of peptide purification and sequencing and yields rapid results when the primary amino acid structure of the protein of interest is known.
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PMID:Proteolytic mapping of the thymidine/thymidylate binding site of herpes simplex virus type 1 thymidine kinase: a general photoaffinity labeling method for identifying active-site peptides. 866 May 48

The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [32P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. 32P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.
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PMID:Identification of Ser-Pro and Thr-Pro phosphorylation sites in chicken neurofilament-M tail domain. 900 38

Mammalian reovirus virions undergo partial disassembly of the outer capsid upon exposure to proteases in vitro, producing infectious subvirion particles (ISVPs) that lack protein sigma3 and contain protein mu1/mu1C as endoprotease-generated fragments mu1delta/delta and phi. ISVPs are thought to be required for two early steps in reovirus infection: membrane penetration and activation of the particle-bound viral transcriptase complexes. Genetic and biochemical evidence implicates outer-capsid protein mu1 in both these steps. To determine whether the cleavage of mu1/mu1C is relevant to the unique properties of ISVPs, we analyzed the properties of novel subvirion particles that lacked sigma3 yet retained mu1/mu1C in an uncleaved but cleavable form. These detergent-plus-protease subvirion particles (dpSVPs) were produced by treating virions with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents. Infections with dpSVPs in murine L or canine MDCK cells provided evidence that the cleavage of mu1/mu1C during viral entry into these cells is dispensable for reovirus infection. Additionally, dpSVPs behaved like ISVPs in their capacity to permeabilize lipid bilayers and to undergo transcriptase activation in vitro, supporting the conclusion that cleavage of mu1/mu1C to mu1delta/delta and phi during viral entry is not required for either membrane penetration or transcriptase activation in cells. The capacity of alkyl sulfate detergents to inhibit the cleavage of mu1/mu1C in a reversible fashion suggests a specific association between virus particle and detergent micelles that may mimic virus particle-phospholipid membrane interactions during reovirus entry into cells.
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PMID:Protease cleavage of reovirus capsid protein mu1/mu1C is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle. 942 Feb 47

An extracellular 1,3-specific lipase with molecular weight of 35.5 kDa and an isoelectric point of 4.4 from Aspergillus niger has been purified 50-fold by pH precipitation followed by a series of chromatographic steps with an overall yield of 10%. The enzyme was homogeneous as judged by denaturing polyacrylamide gel electrophoresis and size-exclusion fast-performance liquid chromatography. It contained 2.8% sugar which was completely removed by endoglycosidase F treatment, and the deglycosylated enzyme retained full activity. The native lipase showed optimal activity between temperatures 35 and 55 degrees C and pH 5.0 and 6.0. The amino acid composition and the N-terminal sequence were found to be different from lipases previously purified from A. niger. The enzyme was resistant to trypsin, chymotrypsin, endoprotease Glu-C, thrombin, and papain under native conditions but was susceptible to cleavage by the same proteases when heat-denatured.
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PMID:Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger. 1090 84

Proteolytic labeling in H2(18)O has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic 16O-to-18O exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger KM for the Lys-peptide (4400 +/- 700 microM), when compared to that of the Arg-peptide (KM 1300 +/- 300 microM), was mainly responsible for the slower reaction with YGGFMK (kcat/KM 0.64 +/- 0.14 microM(-1)min(-1)) compared to YGGFMR (kcat/KM 2.6 +/- 0.9 microM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous 18O2-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin 18O2-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic 18O labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to 18O2-code peptides for comparative studies after proteolysis has taken place.
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PMID:Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates. 1271 28


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