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)

Rat ovarian granulosa cells synthesize two distinct species of plasma membrane-intercalated heparan sulfate (HS) proteoglycans; glycosylphosphatidylinositol (GPI)-anchored and core protein-intercalated HS proteoglycans. Both species of HS proteoglycans are primarily localized on the plasma membrane. Cell surface localization of GPI-anchored and protein-intercalated HS proteoglycans can be determined by their accessibility to exogenously added phosphatidylinositol-specific phospholipase C (PI-PLC) and trypsin, respectively. Kinetic parameters for the processes involving their transfer from the Golgi to the cell surface, endocytosis and secretion, and the modes of intracellular degradation were determined by metabolic labeling experiments using [35S]sulfate and various chase protocols in combination with the use of PI-PLC and trypsin in rat ovarian granulosa cells. The experiments demonstrated that (i) both HS proteoglycan species are transferred from the Golgi to the cell surface with an average transit time of approximately 12 min. (ii) GPI-anchored HS proteoglycans are endocytosed with a t1/2 approximately 3 h, without being shed into the medium, and they are rapidly degraded, t1/2 approximately 25 min, without generating recognizable degradation intermediates. (iii) Protein-intercalated HS proteoglycans are partly (approximately 30%) shed from the cell surface into the medium and the remaining approximately 70% are endocytosed with a t1/2 approximately 4 h. After endocytosis, they undergo a slow (t1/2 approximately 4 h) stepwise degradation generating distinct HS oligosaccharides as degradation intermediates. These results indicate that the GPI-anchored and the protein-intercalated HS proteoglycans have distinct secretory, endocytotic, and intracellular degradation pathways probably due to the differences in their anchor structures.
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PMID:Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways. 153 36

The 145-kDa phospholipase C isozyme, PLC-gamma, is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. We now demonstrate that EGF treatment of HSC-1 cells, a human squamous cell carcinoma-derived cell line that expresses high levels of the EGF receptor, rapidly induces tyrosine phosphorylation of two-thirds of the total cellular PLC-gamma pool. A two-step immunoaffinity protocol was used for large-scale isolation of phosphorylated PLC-gamma from the cytosol of EGF-treated HSC-1 cells. Phosphorylated PLC-gamma was digested with trypsin, then phosphotyrosine-containing peptides were purified by phosphotyrosine affinity chromatography and reverse-phase high performance liquid chromatography. The two major phosphotyrosine-containing tryptic peptides were sequenced. Comparison of the sequence data with the bovine brain PLC-gamma amino acid sequence indicated that the major, EGF-sensitive tyrosine phosphorylation sites of human PLC-gamma correspond to the bovine brain PLC-gamma tyrosine residues 771 and 1254. The former residue is adjacent to regions of PLC-gamma that contain high homology to the non-catalytic, amino-terminal region of the src tyrosine kinase. The latter residue lies near the carboxyl terminus of the PLC-gamma molecule. The accompanying manuscript (Kim J.W., Sim, S.S., Kim, U-H., Nishibe, S., Wahl, M. I., Carpenter, G., and Rhe, S. G. (1990) J. Biol. Chem. 265, 3940-3943) identifies these same 2 residues plus 2 additional tyrosine phosphorylation sites through large-scale in vitro phosphorylation of purified bovine brain PLC-gamma by the EGF receptor.
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PMID:Identification of two epidermal growth factor-sensitive tyrosine phosphorylation sites of phospholipase C-gamma in intact HSC-1 cells. 168 11

Monoclonal antibodies against human alpha-fetoprotein (AFP) were obtained by the hybridoma technique and studied with regard to their reactivities with the human hepatoma cell lines PLC/PRF/5 and KN, and a spontaneously immortalized cell line derived from fetal liver, NuE, all of which synthesize AFP. One of the monoclonal antibodies, 19F12 (IgG2b) became bound to free AFP which was used as the immunogen with an affinity constant of 3.4 X 10(8) M-1. This value was not much higher than those of two other antibodies, 19B1 (IgG1) and 9D12 (IgG2b). However, only antibody 19F12 showed definite reactivity with AFP-producing cells in analysis using flow cytometry. Immunofluorescence microscopy showed that antibody 19F12 detected AFP over the surface of NuE and PLC/PRF/5 cells with a uniform distribution, whereas definite reactivities of antibodies 19B1 and 9D12 to these cells were not detected. These antibodies did not show the specific binding to a non-AFP-producing human lung cancer cell line, PC-9, or to human peripheral blood lymphocytes. The binding ability of 19F12 to hepatoma cells was shown in both viable and fixed cells. Addition of free AFP inhibited the binding of antibody 19F12 to PLC/PRF/5 cells in a concentration-dependent manner. The specific reactivity of 19F12 to human AFP was also confirmed by immunostaining of a tissue section of human cancer proved to be AFP positive with AFP-specific antisera. In two-dimensional polyacrylamide gel electrophoresis of the antigen (from membrane fraction of PLC/PRF/5 cells)-antibody (19F12) complex, spots derived from the antibody and a spot (pI 4.7, Mr 65,000) corresponding in pI and molecular weight to AFP were detected. Western blot analysis showed that material in the membrane fraction of PLC/PRF/5 cells recognized by antibody 19F12 has the same molecular weight as human AFP derived from placenta. In a study of reactivities to PLC/PRF/5 cells treated with various enzymes, the reactivity of this antibody decreased when cells were treated with protease and trypsin and increased when lipase was used. The binding of 19F12 to AFP was not inhibited by concanavalin A. The antibody 19F12 appeared to recognize an epitope that is considered to be part of the peptide area of AFP. These results indicate that the reactivity, the amount of bound antibodies, and the distribution of monoclonal antibodies on antigen-producing cells vary, respectively, even though these antibodies were produced using the same antigen as an immunogen.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Detection of membrane-bound alpha-fetoprotein in human hepatoma cell lines by monoclonal antibody 19F12. 246 75

Serine protease inhibitors with a specificity for trypsin inhibit interferon-gamma (INF-gamma)-induced HLA-DR expression on a hybrid human epidermal cell line (H12), dermal fibroblasts, and primary keratinocytes. Protease inhibitors with a specificity for chymotrypsin or papain fail to inhibit IFN-gamma. The inhibitory effect of the trypsin inhibitors is similar to that of glucocorticoids in that it is a transient event, fading with length of exposure to IFN-gamma, and is reversed by the addition of dibutyryl cyclic AMP (dbcAMP) and phospholipase C(PLC) from Clostridium perfringens. In H12 cells, dbcAMP and PLC enhance the IFN-gamma induction of HLA-DR, but do not induce in the absence of INF-gamma. Evidence suggests that the protease inhibitors, as well as dbcAMP and PLC, may modulate HLA-DR expression at a post-translational site as well as during IFN-gamma signal transduction. These results suggest that trypsin-like protease activity may be required for cellular HLA-DR antigen expression following exposure to IFN-gamma.
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PMID:Trypsin inhibitors inhibit induction by interferon-gamma of HLA-DR antigen expression on human skin cells. 247 85

In order to study hepatocellular carcinoma-associated antigens, screening of sera and ascites was done from hepatocellular carcinoma patients having antibodies reactive with three hepatocellular carcinoma cell lines (PLC/PRF/5, Hep 3B and HA22T/VGH). The indirect immunofluorescent antibody test was used. Ten of 86 (11.6%) sera and 3 of 14 (21.4%) ascites from hepatocellular carcinoma patients showed positive bindings, whereas only 1 of 35 (2.8%) sera, none of 4 (0%) ascites from chronic hepatitis patients and 3 of 60 (5%) normal human sera had positive immunofluorescent antibody activity. The binding specificities of these positive specimens were further defined by other human cancer cell lines and mouse NIH/3T3 fibroblasts. The antinuclear antibody test against mouse liver sections was also performed. The results suggested that antigens identified by the two tests may not be identical. The nature of nuclear antigens reactive with one of the serum samples, S83, and ascites A83 were characterized. These antigens were sensitive to trypsin but not to RNase A and DNase I. Further studies by radioimmunoprecipitation and two-dimensional gel electrophoresis with serum S83 and ascites A83 showed two acidic phosphorylated antigens with molecular weights of 77 and 79 kd, which had a pI around pH 5.2. The presence of a large amount of these two phosphorylated proteins in 5 of 7 human hepatocellular carcinoma cell lines suggests that these two antigens might play some roles in the carcinogenesis or progression of human hepatocellular carcinoma.
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PMID:Nuclear antigens reacted with sera and ascites of hepatocellular carcinoma patients. 283 90

A library of murine monoclonal antibodies reactive with human hepatoma cells was generated following immunization of Balb/c mice with an intact cloned human hepatoma cell line, designated PLC/PRF/5-NR. We report the characterization of one such IgG2a antibody, designated anti-PLC1. This antibody specifically stains parental PLC/PRF/5 cell membranes and membranes of SK-Hep 1 and Mahlavu human hepatoma cells grown in culture, using indirect immunofluorescence and horseradish immunoperoxidase techniques. A similar pattern of membranous staining was observed in solid tumors derived from the three hepatoma cell lines which were injected subcutaneously into athymic nude rats and mice. Spontaneous capping on the cell surface was observed in 7 to 30% of the three human hepatocellular carcinoma cell types when incubated in suspension with monoclonal anti-PLC1 at 37 degrees C. Treatment of cells with trypsin or sustained growth in culture did not affect the intensity of membranous staining. Monoclonal anti-PLC1 appeared specific, and antibodies did not stain a variety of human carcinoma cell lines and primary tumors of nonhepatic origin, or several normal human and murine tissues. Purified 125I-labeled monoclonal anti-PLC1 bound specifically to the three hepatoma cell lines in culture. Specificity of the antigen-antibody reaction was demonstrated by competitive binding inhibition in experiments using unlabeled homologous antibody. Binding of 125I-anti-PLC1 was not inhibited by unlabeled monoclonal antibodies to HBsAg or to alpha-fetoprotein. Two hepatoma cell lines secrete a protein that specifically blocks binding of 125I-anti-PLC1 antibodies to cell surface antigenic determinants. This "hepatoma-associated" protein was subsequently purified by affinity chromatography from supernates derived from the three hepatoma cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human hepatoma-associated cell surface antigen: identification and characterization by means of monoclonal antibodies. 298 98

Interleukin 6 (IL6) is the new definition of a group of cytokines previously named according to their biological activity, e.g. B cell stimulatory factor 2 (BSF-2), hybridoma plasmocytoma-growth factor (HGF), interferon-beta 2 (IFN-beta 2), hepatocyte stimulating factor (HSF). It has recently been suggested that IL6 may represent the major mediator of acute-phase protein response whereas IL1 beta and TNF-alpha could play a minor role. We compared the effect of the three cytokines on hepatic protein synthesis by performing in vitro as well as in vivo experiments. Human hepatoma cells (PLC/PRF5) were exposed to each cytokine separately for 20 h, and the effect was then studied at the protein and RNA level. All three cytokines reduced albumin and increased C3 and ceruloplasmin biosynthesis. The cytokines induced the same effect at the RNA level indicating that the modulation was pretranslational. The effect of the cytokines was specific since actin gene expression was not changed; furthermore the effect was blocked by specific antibodies against the cytokines. The effect of the single cytokines was dose and time dependent, and quantitatively comparable. None of the cytokines was able to alter alpha 1-anti-trypsin synthesis. In vivo experiments with mice showed that IL1 beta and TNF-alpha both induce serum amyloid A (SAA) mRNA in the mouse liver and increase factor B (Bf) gene expression. Human recombinant IL6 induced SAA gene expression and it also had a weak positive effect on Bf gene expression after i.p. injection. These data demonstrate that the three cytokines studied are quantitatively and qualitatively comparable, and that all three are probably involved in acute-phase protein response.
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PMID:Interleukin 6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and mouse. Comparison with interleukin 1 beta and tumor necrosis factor-alpha. 313 37

We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct phosphoinositide-specific phospholipases C (PLCs), PLC-I and PLC-II. In this report the subcellular distribution of PLC-I and PLC-II in brain homogenates was measured using RIA. Significant differences were found in the distribution of the two forms of PLC in 100,000 X g supernatants (cytosolic fraction) of brain homogenized in hypotonic buffer and 2 M KCl extracts of washed pellets (particulate fraction). More than 90% of PLC-II was found in the cytosolic fractions, whereas the PLC-I-like molecules were equally distributed between cytosolic and particulate fractions. Purification of PLC enzyme to near homogeneity from the particulate fractions yielded two PLC enzymes, both of which could be recognized by anti-PLC-I antibodies but not by anti-PLC-II antibodies. Their Mr values, determined under denaturing conditions, were 150,000 and 140,000. The polypeptide of the enzyme of Mr 150,000 seems to be the same as that of the cytosolic enzyme PLC-I: their Mr values were identical, and their trypsin-digested peptides yielded a similar elution profile on a C18 reverse-phase column. We propose, therefore, that PLC-I and its truncated form are weakly associated with membranes.
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PMID:Phospholipase C associated with particulate fractions of bovine brain. 347 91

The mechanism by which the rat T cell alloantigen, RT-6.2, is attached to the membrane was investigated. Treatment of rat lymph node and T-hybridoma cells with phosphatidylinositol-specific phospholipase C (PI-PLC) caused a substantial reduction in the amount of RT-6.2 on the cell surface. No significant release of a rat T helper marker (visualized by the mAb W3/25) was observed in response to PI-PLC treatment. This is in sharp contrast to the effects of trypsin, which removes most of the T helper marker but had little effect on RT-6.2. SDS-PAGE analysis of the RT-6.2 released by PI-PLC indicated that the Mr was not significantly changed by this treatment. Phase separation of the released RT-6.2 in Triton X-114 showed that the PI-PLC had converted it from an amphiphilic membrane form to a water-soluble form, apparently by removing its hydrophobic membrane anchoring domain. These results strongly suggest that RT-6.2, in common with Thy-1 and several other cell surface proteins, is anchored in the membrane by the 1,2-diacylglycerol moiety of a covalently attached phosphatidylinositol molecule.
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PMID:Release of the rat T cell alloantigen RT-6.2 from cell membranes by phosphatidylinositol-specific phospholipase C. 348 8

Gel filtration of human serum extracts on Bio-Gel P-30 columns produced two peaks of material reactive with insulin antisera. The earlier eluting fraction appeared at the elution position of proinsulin (serum proinsulin-like component, PLC) while the second fraction corresponded in elution volume to insulin. In assays using porcine insulin-(131)I and an antiserum against porcine insulin, human pancreatic proinsulin was less reactive than human insulin. Serial dilutions of the serum PLC in the immunoassay showed immunological identity with the human proinsulin standard. Partial tryptic digestion of the serum PLC yielded products with increased immunological reactivity as estimated with insulin as the standard. With larger amounts of trypsin, all the serum PLC was converted to insulin-like components (desthreonine and desoctapeptide insulin). On the basis of these results we conclude that the earlier eluting fraction of human serum extracts is proinsulin. The fasting values of proinsulin in normal subjects ranged between 0.05 and 0.4 ng/ml, representing from 5 to 48% of the insulin concentration. In one subject the values of proinsulin were higher than those of insulin. After oral administration of 100 g of glucose, the proinsulin levels tended to rise similarly to insulin. Three obese patients with hyperinsulinemia had higher fasting levels of proinsulin and a greater increase after glucose than the normal subjects. As the high levels of proinsulin coexisted with raised insulin concentration in these obese subjects, the relative proportions of the two hormones were in the same range observed in the normal group. Thus hyperinsulinemia in these obese subjects was not accompanied by an increase in the fraction of serum proinsulin. When the values for serum proinsulin were expressed as percentage of the insulin levels, there was a decrease in the per cent proinsulin in the first hour of the glucose tolerance test. After the second hour, the per cent tended to rise towards the fasting levels.
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PMID:Human serum proinsulin. 546 Dec 3


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