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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic degradation of purple membrane sheets, reconstituted vesicles, and whole cells, with the following results: (i) Bacteriorhodopsin in purple membrane sheets is cleaved at a single site by Pronase or trypsin; a polypeptide segment of about 15 amino acids is lost from the carboxyl end. Carboxypeptidase A sequentially releases amino acids from the carboxyl end; the tetrapeptide sequence -Ala-Ala-Thr-Ser(COOH) was tentatively deduced for this terminus. (ii) The apomembrane, which lacks retinal, undergoes a second cleavage with trypsin releasing a fragment of approximately 6300 molecular weight from the amino terminus. (iii) Vesicles reconstituted from the purple membrane sheets and synthetic lecithins, in which the direction of proton pumping is opposite to that in the whole cells, have the carboxyl terminus of bacteriorhodopsin accessible to proteolysis. (iv) In envelope vesicles, which largely pump protons in the same direction as the whole cells, the carboxyl terminus is largely protected against proteolysis. (v) Treatment of whole cells with proteinase K hydrolyzes the cell wall proteins but has no effect on acteriorhodopsin. However, the same treatment after lysis of the cells results in degradation of the hydrophilic region at the carboxyl terminus. The results show that the carboxyl terminus as well as the additional cleavage site near the amino terminus observed in apomembrane are on the cytoplasmic side of the purple membrane.
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PMID:Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis. 27 65

Properties of carboxypeptidase A of cultured skin fibroblasts from control and cystic fibrosis patients were studied using alpha-N-carbobenzoxy-L-glutamyl-L-tyrosine as substrate. Carboxypeptidase A was inhibited by thiomersal, cyanide, iodoacetate and N-ethylmaleimide in a similar manner for control and cystic fibrosis fibroblasts. Both trypsin and dithiothreitol treatment activated the enzyme, but 1,10-phenanthroline inhibited only in the presence of dithiothreitol. Both Zn2+ and Co2+ reversed this inhibition. Trypsin treatment of carboxypeptidase A produced a form of the enzyme having a higher KM value for both control and cystic fibrosis fibroblasts. Dithiothreitol treatment of control fibroblasts resulted in a form with similar properties to the trypsin activated form, but cystic fibrosis fibroblasts yielded a variant form with even higher KM and Vmax values. Since other properties were similar, it seems likely that this difference reflected binding of a molecule to the enzyme rather than of a defect in the enzyme.
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PMID:Carboxypeptidase A activity of cultured skin fibroblasts and relationship to cystic fibrosis. 66 47

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.
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PMID:Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin. 171 14

Protein from hog which is recognized by human monoclonal antibody (HB4C5), generated from a patient with large cell lung carcinoma, was identified as carboxypeptidase A by comparison of the protein with carboxypeptidase A in enzymatic activity, immunologic reactivity, and amino acid sequence. Carboxypeptidase A activity was also found in human cancer tissue, and purified antigen from cancer tissue recognized by the antibody HB4C5 was reacted with rabbit anti-carboxypeptidase A serum, indicating that carboxypeptidase A is an antigen of HB4C5. Since large amounts of carboxypeptidase A can be obtained from porcine sources, a simple method for its purification was established. The fraction which was most reactive with HB4C5 was obtained from acetone powder of porcine pancreas by successive applications of water extraction, ammonium sulfate precipitation, trypsin treatment, and Mono Q column chromatography. Its apparent molecular weight was 40,000, according to SDS polyacrylamide gel electrophoresis. When the reactivity of IgG in sera with the purified carboxypeptidase A was measured, the detection rates for lung, ovary, larynx, uterus, and liver cancer were more than 50%, while the rates for stomach and breast cancer were around 30%, and pancreatic cancer, benign diseases, and normal controls were minimally detected.
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PMID:Serodiagnosis of cancer using porcine carboxypeptidase A as an animal antigen recognized by human monoclonal antibody HB4C5. 187 99

The "high-affinity Mn-binding site" in Mn-depleted photosystem II (PS II) membrane fragments isolated from Scenedesmus obliquus was examined by using the diphenylcarbazide (DPC)/Mn2+ non-competitive inhibition assay [Preston, C., & Seibert, M. (1991) Biochemistry (preceding paper in this issue)]. Different proteases were used to degrade lumenal surface protein segments from these PS II membranes, and a total of four independent high-affinity Mn-binding sites (ligands) were identified. Carboxypeptidase A, subtilisin, and Staphylococcus aureus V8 protease each degrade one of two high-affinity Mn-binding sites sensitive to the histidine chemical modifier diethyl pyrocarbonate (DEPC). However, sequential treatment experiments indicate that subtilisin degrades a DEPC-sensitive Mn-binding site that is different from the one degraded by the other two proteases. Trypsin also was found to degrade one of the DEPC-sensitive Mn-binding sites (that degraded by carboxypeptidase A and V8 protease). In addition, trypsin degrades one of two 1-ethyl-3-[(3-dimethylamino)propyl]carbodiimide (EDC) sensitive Mn-binding sites, but only in the absence of the 30-kDa extrinsic protein. Thus, the 30-kDa extrinsic protein associated with O2 evolution appears to protect the EDC-sensitive binding site from trypsin degradation. No protease has yet been identified that will degrade the trypsin-insensitive EDC-sensitive Mn-binding site. Under the conditions of the assay (high DPC concentration), more than three Mn per reaction center were found bound to the membrane with a KM of about 0.4 microM, as determined by direct metal analysis. This is consistent with the idea that each of the four high-affinity sites binds (or provides a ligand for) one of four Mn.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protease treatments of photosystem II membrane fragments reveal that there are four separate high-affinity Mn-binding sites. 191 48

Porcine pancreatic hydrolases in juice and homogenate surveyed by electrophoretic separation in agarose gel, at pH 8.6 and subsequently characterized using substrates of various specificity, either directly in the gel or after transfer to nitrocellulose (enzymoblotting) showed: Anodal and cathodal trypsin with Bz-Arg-pNA. Chymotrypsin A, B, and C with similar, but not identical, activities to Suc-Ala-Ala-Pro-Phe-pNA, Bz-Tyr-pNA, Suc-Phe-pNA and Ac-Phe-beta NE and with differences in their molecular weights and electrophoretical charges. Elastase I and protease E with Suc-(Ala)3-pNA and MeO-Suc-Ala-Ala-Pro-Val-pNA and elastase I also with elastin. Elastase II with the chymotrypsin substrates and with elastin. Carboxypeptidase A with CN-Phe. Amylase with blue starch polymer.
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PMID:Identification and characterization of eight porcine pancreatic proteinases, carboxypeptidase A and amylase after electrophoretic separation using specific substrates. 244 43

The effect of the proteolysis of aldolase on both the substrate specificity of the enzyme and binding capacity for actin have been studied. Carboxypeptidase A, trypsin, chymotrypsin and pepsin, all acted to cleave peptides from the C-terminal portion of the enzyme, resulting initially in a marked loss of activity towards fructose-1:6-bisphosphate (FBP), without impairment of activity towards fructose-1-phosphate (F1P). In some cases, however, further proteolysis caused reductions in activity with F1P as well. By correlating the size of the peptide fragments released by these enzymes with the known sequence of aldolase, evidence has been provided that cleavage of His-359 and/or Tyr-361 lead to the loss of FBP activity, while further cleavage of up to six amino acids begin to affect activity against F1P, as well. In regard to the ability of the proteolysed aldolase to bind to F-actin, it was evident from these studies that binding ability was not impaired in the initial stages of proteolysis referred to above, but was retained until the enzyme was extensively degraded. This differential behaviour of the active and binding sites on aldolase clearly establish their separate topographical localization. These results have been discussed in relation to the positioning of these separate sites on the enzyme, the nature of the interaction between aldolase and actin and the phenomenon of enzyme ambiquity in cells and tissues.
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PMID:Evidence for the spatial separation of the binding sites for substrate and for cytoskeletal proteins on the enzyme aldolase. 308 Mar 48

Unlike the pancreatic endopeptidase zymogens, procarboxypeptidase A is activated very slowly in vitro. The activation proceeds through the removal of about 100 amino acids away from the N-terminus of the chain. The cleavage of the susceptible bond(s) in monomeric and aggregated forms of bovine procarboxypeptidase A by catalytic amounts of trypsin was found to be very fast. However, as in the case of the porcine zymogen, the expression of the carboxypeptidase activity was considerably delayed by the inhibitory effect of the activation peptide which remains bound to the enzyme molecule after the trypsin treatment of the zymogen. alpha-Carboxypeptidase A was mainly formed under the relatively mild conditions used, indicating that the Arg-1-Ala+1 bond is probably the first to be cleaved during in vitro activation. The bovine carboxypeptidase activity was immediately and reversibly expressed upon dimethylmaleylation of the activation mixtures. This expression does not require full dissociation of the enzyme-peptide complex but merely a suitable change in its quaternary structure resulting from a modification of some electrostatic interactions upon dimethylmaleylation. Separation of bovine carboxypeptidase A from its activation peptide was only achieved upon filtration of the dimethylmaleylated mixtures in the presence of 6 M urea. The bovine activation peptide contains at least 93 amino acids compared to the 94 amino acids found by other authors for the rat and porcine peptides and sequencing of the first 53 amino acids showed a 75-85% homology with the latter two peptides.
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PMID:Further studies on the activation of bovine pancreatic procarboxypeptidase A by trypsin. 360 14

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.
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PMID:Amino acid sequence of human D of the alternative complement pathway. 638 66

The inside-out configuration of the patch-clamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic beta-cells. Trypsin (20 micrograms/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg2+ was observed: however, the protease did reduce the inhibitory effect of Mg2+ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 microM. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.
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PMID:Modification of K-ATP channels in pancreatic beta-cells by trypsin. 835 Dec 6


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