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 specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.
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PMID:Proteins of the hepatoma tissue culture cell plasma membrane. 0 57

A polar, nonpenetrating compound of high specific activity, diazotized (125I)-diiodosulfanilic acid (DD125ISA), has been developed as a label for exposed proteins of the human platelet plasma membrane, and platelet proteins and the pattern of labeling have been studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). That DD125ISA binds specifically to membrane proteins was demonstrated by: (1) the specific activity of isolated membrane protein was five to seven times that of whole platelet protein and (2) no proteins of intact platelets were labeled which were not represented in the isolated plasma membrane. That the DD125ISA-labeled membrane proteins were exposed on the cell surface was demonstrated by: (1) DD125ISA-labeled proteins were altered by trypsin treatment of intact, labeled platelets and (2) the pattern of labeling produced by reaction of isolated membranes with DD125ISA was quite different from that produced by the labeling of intact platelets. Analysis of platelet membrane proteins by SDS-PAGE demonstrated the glycoproteins previously described at 150,000 daltons (termed glycoprotein I) and 92,000 daltons (glycoprotein III) but we could discriminate two apparently distinct glycoproteins in the intermediate region (IIa: 125,000 daltons, and II: 118,000 daltons). Glycoproteins I and III were constant whereas IIa was clearly visible only in unreduced samples and II was predominant in reduced samples. Reaction of DD125ISA with intact platelets resulted in equal labeling of three of these four membrane glycoproteins (IIa, II, and III). The pattern of exposed proteins on the platelet surface labeled by DD125ISA was different from lactoperoxidase-131I, which labeled predominantly the 92,000 dalton glycoprotein, as demonstrated by simultaneous SDS-PAGE analysis. Therefore three glycoproteins of the human platelet plasma membrane are exposed to a radioisotope probe on the platelet surface and are accessible for contact interactions.
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PMID:Studies on platelet plasma membranes. I. Characterization of surface proteins of human platelets labeled with diazotized (125i)-diiodosulfanilic acid. 6 Apr 57

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.
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PMID:Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes. 6 76

Biochemical characterization of serologically detected human melanoma antigens was undertaken for the development of immunodiagnostic assays in melanoma. An antiserum from a human melanoma patient, which detected melanoma antigens expressed on a large proportion of different melanoma cells, was used in leucocyte-dependent cytotoxic antibody (LDA) 51Cr-release assays to monitor the purification of melanoma antigens in urea/acetate extracts of lactoperoxidase 125I-labelled melanoma cell membranes. The separation procedures included affinity chromatography on Concanavalin A, gel filtration on porous polyacrylamide beads and preparative isoelectric focusing. The fractions were also monitored by polyacrylamide electrophoresis in sodium dodecyl sulphate and by measurement of beta 2 microglobulin and carcinoembryonic antigen content. The antigens detected by this antiserum appeared to be acidic (pI 3.5) low-mol.-wt glycoproteins of approximately 15,000 daltons which were resistant to heating at 56 degrees C and digestion with neuraminidase, but susceptible to repeated freeze-thawing and trypsin digestion. They did not appear to be related to HLA antigens, beta 2 microglobulin or known foetal antigens. The nature of the antigens detected in these studies is as yet unknown, but they appear similar to those described in the sera and urine of melanoma patients in previous reports. Thes combined results and the frequent expression of these antigens on melanoma cells from different patients suggest that assays to detect this antigen may provide a valuable immunodiagnostic aid in the management of melanoma.
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PMID:Detection of a low-molecular-weight antigen on melanoma cells by a human antiserum in leukocyte-dependent antibody assays. 9 79

Latent ATPase, located on the inner surface of protoplast ghosts of Mycobacterium phlei, was unmasked either by trypsin or an impermeable form of trypsin, ethylene maleic anhydride-trypsin. Density gradient experiments showed that the ghost preparations remained intact following trypsin treatment. Evidence was obtained that 125I-trypsin failed to penetrate the ghost membranes. Thus, attempts were made to determine whether the ATPase molecule in the ghost membranes is accessible from the outer surface. Treatment of protoplast ghosts and trypsin-treated ghosts with 125I by the lactoperoxidase method resulted in the labeling of ATPase only in the trypsin-treated ghost preparations. The antibody to latent ATPase inhibited ATPase activity in trypsin-treated ghosts. The changes in the fluorescence polarization of diphenyl hexatriene indicated that trypsin treatment of the ghost membranes resulted in an increase in membrane fluidity. These studies suggest that the latent ATPase moiety has undergone translocation to the outer surface or it became accessible to trypsin digestion from the outer surface of the membranes as a result of removal of some proteins covering ATPase molecule in the membranes.
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PMID:Trypsin-induced changes in the orientation of latent ATPase in protoplast ghosts from Mycobacterium phlei. 14 35

Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances.
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PMID:Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm. 15 36

The relationship between activation of resting chick embryo fibroblasts by proteases and proteolytic alteration of the cell surface has been investigated. Five different proteases were examined: trypsin, collagenase, plasmin, alpha-chymotrypsin, and thrombin. All of these proteases, when added to the culture medium at concentrations of 0.08-2.2 mug/ml, stimulated deoxyglucose uptake and induced cell division. The absolute levels of stimulation depended on the specific protease. Activation ranged from a doubling in cell number in 24 hr for trypsin and thrombin down to a 47% increase in cell number for alpha-chymotrypsin. Except in the case of thrombin, the stimulatory effects of these proteases correlated with breakdown of Z, a protein which is the major chick surface protein as revealed by lactoperoxidase-catalyzed iodination and which disappears upon transformation. In the case of thrombin, stimulatory concentrations brought about no detectable loss of surface components. Thus loss of Z is not a necessary condition for activation of chick fibroblasts; it may be a sufficient condition for activation of part of the cell population.
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PMID:Effect of proteases on activation of resting chick embryo fibroblasts and on cell surface proteins. 17 Oct 80

It was previously shown that the fibroblast surface antigen (SF antigen, SFA) is composed of polypeptides of high molecular weight 210,000 (SF210) and 145,000 (SF145) and that both of these decrease in quantity after transformation of the fibroblasts by Rous sarcoma virus (RSV). The present experiments show that SF210 is a glycoprotein. It is accessible to surface labelling by lactoperoxidase catalyzed iodination. The SF210 molecule is highly susceptible to trypsin on cell surface. Anti-SFA antibodies specifically precipitated the surface labelled polypeptide. The lactoperoxidase iodinated SF210 polypeptide was greatly reduced in cells transformed by RSV. It is concluded from these studies that the large external transformation sensitive (LETS) protein detected by other workers is the same molecule as SF210. Part of the label of surface iodinated fibroblasts did not enter the polyacrylamide gels. This high molecular weight material is also susceptible to trypsin treatment and decreases in quantity after transformation by RSV. The data suggest that it may be antigenically related to SF protein. Treatment of surface of 35S-methionine-labelled cultures with trypsin in concentrations able to initiate proliferation of density-inhibited cells rapidly released SF210 from fibroblast surface. A single high molecular weight polypeptide (mol. wt about 200,000, SF200) was detected in the culture medium. SF210 may thus be a major target molecule of trypsin action. Treatment of cultures with insulin that also stimulated the fibroblasts to initiate proliferation did not result in any detectable alteration in the external glycoprotein SF210. It is concluded that although release of SF210 may be a sufficient trigger to stimulate proliferation in stationary cells, this molecule appears not to be directly involved in initiation of fibroblast proliferation from the G1 (or G0) phase of the cell cycle.
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PMID:Fibroblast surface antigen (SF): the external glycoprotein lost in proteolytic stimulation and maligant transfromation. 17 31

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cells cultured at the permissive (32 degrees C) and non-permissive (39 degrees C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3-4 generations in medium containing radioactive leucine (32 degrees C and 39 degrees C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (congruent to 100 000-160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growth (39 degrees C). A decrease in amount of a 120 000 molecular weight glycopeptide at 39 degrees C was the most prominent of these alterations. We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 degrees C, using lactoperoxidase-catalyzed iodination, NaBH4 reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.
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PMID:Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties. 17 78

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.
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PMID:Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells. 17 96


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