Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteolytic cleavage of Chlamydia trachomatis LGV-434 surface proteins and resultant effects on infectivity and association with cultured human epithelial (HeLa) cells have been examined. Of several proteases examined, trypsin, chymotrypsin, and thermolysin extensively cleaved the chlamydial major outer membrane protein (MOMP). Two proteases, trypsin and thermolysin, cleaved the MOMP to the extent that monomeric MOMP was not detectable by immunoblotting with monospecific polyclonal antibodies. In the case of thermolysin, not even antigenic fragments were detected. Surprisingly, infectivity toward HeLa cells was not diminished. In addition, the association of intrinsically 14C-radiolabeled elementary bodies (EBs) with HeLa cells or their dissociation by proteinase K was not measurably affected by prior trypsinization of the EBs. Trypsinization of lactoperoxidase surface-iodinated elementary bodies demonstrated that most of the 125I-labeled surface proteins were cleaved. In all cases, however, a number of proteolytic cleavage fragments remained associated with the EB surface after surface proteolysis. When trypsinized EBs were electrophoresed under nonreducing conditions and immunoblotted with either polyclonal or type-specific monoclonal MOMP antibodies, MOMP was found in a large oligomeric form that failed to enter the polyacrylamide stacking gel. Additionally, trypsinized viable EBs bound radioiodinated type-specific MOMP monoclonal antibody as efficiently as did the control nontrypsinized organisms. Taken together, the findings indicate that although the MOMP is highly susceptible to surface proteolysis, the supramolecular structure of the protein on the EB surface is apparently maintained by disulfide interactions. Thus, if surface-exposed chlamydial proteins are involved in the initial interaction of chlamydiae with eucaryotic cells, the functional domains of these proteins which mediate this interaction must be resistant to proteolysis and remain associated with the EB surface.
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PMID:Effect of proteolytic cleavage of surface-exposed proteins on infectivity of Chlamydia trachomatis. 258 Jul 94

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.
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PMID:Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage. 271 76

Radiolabeled insulin was affinity cross-linked to purified insulin receptor with six separate bifunctional N-hydroxysuccinimide esters of different lengths. Results were qualitatively identical for each cross-linker in that insulin was predominantly cross-linked through its B chain to the receptor's alpha subunit. The maximum efficiencies of cross-linking were 10-15% for the most effective reagents, and this value was dependent upon the concentration and length of the cross-linker. In an effort to locate the cross-linking site, monoiodoinsulin was cross-linked to affinity-purified insulin receptor with disuccinimidyl suberate. Limited proteolysis of the hormone/receptor adduct with Staphylococcus aureus V8 protease, chymotrypsin, or thermolysin in an SDS-containing buffer rapidly generated a 55-kDa, insulin-labeled fragment as shown by SDS-polyacrylamide gel electrophoresis. We reported earlier that the 55-kDa chymotryptic fragment contained multiple internal disulfide bonds as evidenced by its shifting mobility on an SDS gel after dithiothreitol treatment [Boni-Schnetzler et al. (1987) J. Biol. Chem. 262, 8395-8401]. Here we show that the 55-kDa fragment is also formed by proteolysis of the receptor in the absence of prior insulin cross-linking. This fragment was prepared in amounts sufficient for sequence analysis and was purified by passage successively over gel permeation and reverse-phase HPLC columns. The sequence of the fragment's amino terminus corresponds to that of the amino terminus of the receptor's alpha subunit. This fragment also reacts with an antibody raised against a synthetic peptide corresponding to residues 242-253 of the receptor's alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation of a proteolytically derived domain of the insulin receptor containing the major site of cross-linking/binding. 274 47

The primary structure of human epidermal growth factor (hEGF), which was produced by Escherichia coli using recombinant DNA technique, has been studied by tandem mass spectrometry. The molecular weight of hEGF (about 6200 amu) was determined by fast atom bombardment mass spectrometry. Then reduced and carboxymethylated hEGF was digested by chymotrypsin into seven peptides which could cover the whole sequence of hEGF. The amino acid sequences of five of these seven peptides could be confirmed by tandem mass spectrometry with or without isolation by high-performance liquid chromatography (HPLC). After isolation by HPLC, the other two peptides were digested with trypsin or thermolysin into small peptides, and sequenced by tandem mass spectrometry.
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PMID:The primary structure of human EGF produced by genetic engineering, studied by high-performance tandem mass spectrometry. 278 14

The occasional cleavage of the Pseudomonas cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) molecule into two well-defined fragments during the preparation of the enzyme is shown to be identical to that caused by elastase isolated from the culture solution of Pseudomonas aeruginosa. A cyanogen bromide fragmentation of proteolytically cleaved and of intact enzyme shows the cleaved peptide bond to be situated in cyanogen bromide fragment II. The amino-acid sequence of this fragment was established by sequencing peptides obtained with trypsin, thermolysin, chymotrypsin and o-iodosobenzoate. It is concluded from the sequence homology that the polypeptide chain of Pseudomonas peroxidase is wrapped around the high-potential heme in a similar manner as in high-potential cytochromes c in general. The specific proteolytic cleavage occurs at a Ser-Val (Leu-Pro) region which is assumed to be the site of attachment between enzyme and membrane. The cleavage of the Ser-Val bond renders the peroxidase molecule enzymatically inactive by impeding the conformational changes essential for the function of the native enzyme.
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PMID:Specific cleavage of Pseudomonas cytochrome-c peroxidase by elastase from Pseudomonas aeruginosa. 282 23

Plasma fibronectin (pFN) has been shown to mediate phagocytosis of several types of artificial particles and tissue debris by macrophages. In the present investigation some of the dynamic aspects of this receptor-mediated cellular process have been studied. Plasma fibronectin did not bind specifically to fibronectin (FN)-receptors of rat peritoneal macrophages at either 4 degrees C or 37 degrees C. On the other hand, pFN aggregated on the surface of gelatin-coated latex beads (gLtx) and 125I-labeled pFN covalently coupled to latex beads (pFN-Ltx) bound strongly to macrophages at both temperatures. Both of these particles were also internalized at 37 degrees C. Treatment of macrophages by chymotrypsin, thermolysin, or trypsin in a protein-free tissue culture medium did not affect either of the above reactions; however, pronase treatment strongly reduced both the binding and internalization of the pFN-coated particles. The pronase-treated macrophage monolayers in time regained their ability to bind and internalize pFN-gLtx when incubated in fresh tissue culture medium. Such recovery, however, did not take place when the medium contained cycloheximide. On the other hand, phagocytosis of pFN-gLtx was not affected directly by cycloheximide with untreated macrophages; this suggests that the FN-receptor recycles during sustained phagocytosis. This assumption was substantiated by the observations that some of the established lysosomotropic amines--i.e., chloroquine, dansylcadaverine, and dimethyldansylcadaverine--caused total inhibition of internalization without affecting the binding of particles to macrophages. Furthermore, chloroquine protected the FN-receptors against destruction by pronase. Together these results suggest that macrophage receptors for FN are protein, present both on the cell surface and intracellularly, and recycle between the plasma membrane and intracellular sites during phagocytosis.
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PMID:Evidence for the recycling nature of the fibronectin receptor of macrophages. 295 89

Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [14C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis (EP + H2O in equilibrium E + Pi) without significant incorporation of the 14C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [14C]DCCD under more drastic conditions produced inhibition of calcium binding, 14C radioactive labeling of tryptic fragments A1 and A2 (but not of B), and extensive cross-linking. Intermolecular and, to some extent, intramolecular cross-linking were prevented by exogenous nucleophiles. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A2, and internal cross-linking of fragment A1. It is proposed that both A1 and A2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A2 are directly involved in calcium complexation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase. 296 40

The blood cell of the horseshoe crab, Limulus, is packed with granules that can be stimulated to release their contents by exocytosis. We have identified a family of proteinase inhibitors in the released materials. Included is a factor similar to the alpha 2-macroglobulin homologue present in the plasma and acid-stable and acid-instable active-site inhibitors. The acid-stable factor is active against both serine (trypsin, chymotrypsin) and metal (thermolysin) proteinases. The trypsin- and chymotrypsin-inhibitory activity has a molecular weight of 6100, as determined by gel-filtration chromatography.
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PMID:Proteinase inhibitory activity released from the horseshoe crab blood cell during exocytosis. 298 12

A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.
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PMID:Human von Willebrand factor: a multivalent protein composed of identical subunits. 301 99

The renaturation of the tetrameric enzyme phosphoglycerate mutase from baker's yeast after denaturation in guanidinium chloride was studied. Three proteinases (trypsin, chymotrypsin and thermolysin) cause extensive loss of activity of samples taken during the early stages of refolding. As judged by SDS/polyacrylamide-gel electrophoresis, the proteinases cause substantial degradation of the polypeptide chain with no evidence for large quantities of fragments of Mr greater than 6500. These data suggest that the early intermediates in the refolding, especially the folded monomer, possess a number of sites that are susceptible to proteolysis.
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PMID:The susceptibility towards proteolysis of intermediates during the renaturation of yeast phosphoglycerate mutase. 301 21


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