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)

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.
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PMID:Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved. 404 Dec 37

A cell-surface modulator with the ability to mimic the reciprocal effects of cell density on cell growth and liver-specific functions of mature rat hepatocytes in primary culture (Nakamura, T., Yoshimoto, K., Nakayama, Y., Tomita, Y., and Ichihara, A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7229-7233) was solubilized with 4% octyl glucoside and 4 M guanidine HCl from plasma membranes purified from adult rat liver. The membrane-free modulator showed activities for inhibition of DNA synthesis stimulated by insulin with epidermal growth factor and stimulation of tyrosine aminotransferase induction by dexamethasone. The apparent Mr of the factor was estimated as 670,000 by gel filtration on a Sephacryl S-400 column equilibrated with 0.5% octyl glucoside and 4 M guanidine HCl. The modulator was heat-labile and sensitive to trypsin. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated via cell to cell contact by a cell-surface modulator(s) that is an integral membrane protein.
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PMID:Reciprocal modulation of growth and liver functions of mature rat hepatocytes in primary culture by an extract of hepatic plasma membranes. 614 8

The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.
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PMID:Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease. 635 Feb 93

In the presence of nerve growth factor (NGF), PC12 pheochromocytoma cells undergo neuronal differentiation with a concomitant 3- to 5-fold increase in the specific level of an Mr = 230,000 cell surface component named the NGF-inducible large external, or NILE, glycoprotein. Antisera raised against NILE glycoprotein (NILE GP) purified from PC12 cells have been found to recognize most, if not all, neurons derived from the peripheral and central nervous systems. In the current studies several of the biochemical properties of NILE GP were investigated. NILE GP was found to be phosphorylated in NGF-treated and -untreated PC12 cells and in cultured rat sympathetic neurons. The phosphate moiety of NILE GP is almost completely alkali labile, suggesting that phosphoserine groups predominate. Immunoprecipitation experiments revealed that incorporation of [32P]phosphate into NILE GP relative to total PC12 cell phosphoprotein was not significantly altered at 12 and 24 hr of NGF treatment but was enhanced 3-fold after 7 days and up to 5-fold after 2 to 3 weeks of NGF exposure. These changes in phosphorylated NILE GP paralleled, and therefore appeared to be mainly a consequence of, the NGF-induced increase in total cellular levels of NILE GP. By two-dimensional gel analysis, anti-NILE GP selectively immunoprecipitated two NGF-inducible spots (apparent Mr = 230,000; pI = 6.4 to 6.6) from PC12 cells labeled with either [3H] fucose, [35S]methionine, or [32P]phosphate. Anti-NILE GP immunoprecipitated a single band (apparent Mr = 205,000) from extracts of rat brain labeled with [3H] glucosamine. This confirms the previously established apparent molecular weight difference between central and peripheral NILE GP cross-reactive material. When PC12 cells, cerebellar cultures, and cultured cerebral cortex were treated with tunicamycin and labeled with [35S]methionine, nonglycosylated bands each with Mr = 160,000 were immunoprecipitated, implying that the differences in the mobilities on sodium dodecyl sulfate gels of cross-reactive NILE GP from different tissues is due to variation in glycosylation rather than to large differences in apoprotein structure. Prolonged treatment of PC12 cells with trypsin produced an immunoreactive fragment of NILE GP of apparent Mr = 28,000 that was phosphorylated but not glycosylated, and that remained in the membrane. NILE GP remained predominantly membrane associated under a variety of aqueous extraction conditions, suggesting that it is an integral membrane protein.
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PMID:Biochemical properties of the nerve growth factor-inducible large external (NILE) glycoprotein. 665 95

The addition of Ca2+ to aqueous dispersions of cardiolipin triggers complete hexagonal (HII) phase formation at Ca2+/cardiolipin molar ratios greater than or equal to 1.0 as detected by 31PNMR and freeze-fracture electron microscopy. Incorporation of the integral membrane protein glycophorin prevents the bilayer leads to hexagonal (HII) phase transition at Ca2+/cardiolipin ratios as high as 15:1. Removal of the outwardly oriented, negatively charged sialic-acid-containing sugar groups of glycophorin with trypsin had little effect on the bilayer-stabilizing capacity of the protein. As the Ca2+ binding was found to be similar in both the cardiolipin and the cardiolipin-glycophorin systems, it can be concluded that the protein exerts a bilayer-stabilizing effect on the cardiolipin. In addition, the possibility that glycophorin may prevent vesicle fusion is also discussed.
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PMID:The effect of an integral membrane protein on lipid polymorphism in the cardiolipin-Ca2+ system. 682 77

Previous studies in this laboratory have shown that membranes derived from dorsal root ganglia (DRG) neurites are mitogenic for cultured Schwann cells derived from the same source [Salzer et al (1980): J Cell Biol 84:767-778]. Improved procedures are described for preparing Schwann cells derived from dorsal root ganglia that are highly responsive to various mitogens. Under these conditions, the cells respond not only to the neurite mitogen but also to pituitary extracts, dibutyryl cyclic AMP, and cholera toxin that have been shown previously to be good mitogens for Schwann cells derived from sciatic nerve [Raff et al (1978): Cell 15:813-822], thus reconciling discrepancies in the response of these different Schwann cell preparations to mitogens. Searching for a source of membranes more suitable for biochemical characterization of the neurite mitogen, we found that bovine brain axolemma, prepared by the method of DeVries et al [(1977):Brain Res 147:339-352] is highly mitogenic for Schwann cells. The mitotic index of Schwann cells was increased by the addition of axolemma from 0.5%-2% to 30%-50% during 24-h incubation with [3H]thymidine. Half maximal effect was obtained at about 0.4 microgram axolemma protein per microwell containing 2-4 X 10(3) cells. The axolemma mitogen appears to be an integral membrane protein that remains bound to the membrane under various ionic conditions but can be extracted in a partially active form with deoxycholate. Like the DRG neurite mitogen, the mitogenic activity of axolemma was abolished by trypsin treatment. Unlike the neurite preparation, however, the mitogenic activity of axolemma was only partially inactivated by heat treatment (60%-70% inactivation). A significant difference between the mitogenic activity of axolemma membranes and neurite membranes is the fact that axolemma membranes fail to stimulate Schwann cell proliferation in a defined, serum-free medium (N-2), whereas neurites show significant mitogenic activity in this medium. These findings indicate a possible difference between DRG neurites and brain axolemma either in the mitogen itself or surface components responsible for recognition between the membranes and the cells.
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PMID:Mitogenicity of brain axolemma membranes and soluble factors for dorsal root ganglion Schwann cells. 708 77

As part of a study of the mechanism whereby organic anionic dyes such as sulfobromophthalein and bilirubin enter hepatocytes, the binding of [35S]sulfobromophthalein and of its glutathione conjugate to two rat liver plasma membrane fractions were studied in vitro. Both fractions reversibly and saturably bound conjugated and unconjugated sulfobromophthalein. Three classes of binding site were necessary to account for the observed sulfobromophthalein binding, their maximal binding capacities being 3.5 . 10(-11), 1.6 . 10(-7) and 5.4 . 10(-7) mol/mg membrane protein. The corresponding association constants were 5.5 . 10(7), 1.5 . 10(5) and 1.3 . 10(3) M-1. Binding of the glutathione conjugate could be accounted for by two classes of binding site only, their association constants being 2.0 . 10(8) and 1.9 . 10(3) M-1 and their maximal binding capacities 5.0 . 10(-11) and 2.2 . 10(-7) mol/mg protein, respectively. Conjugated and unconjugated sulfobromophthalein mutually competed for binding, KI being 7.8 . 10(-7) and 5.5 . 10(-5) M for conjugated and unconjugated sulfobromophthalein, respectively. Similarly, bilirubin and indocyanine green, but not taurocholate, competitively inhibited sulfobromophthalein binding. Treatment with trypsin and phospholipases reduced, while treatment with neuraminidase did not affect binding. Neither changes in pH nor substitution of other cations for Na+ in the incubation mixture significantly affected sulfobromophthalein binding. Heating the membranes increased binding. This was due to an increase in maximal binding capacity of the low-affinity, high-capacity sites. The description of saturable binding sites on hepatocellular surface membranes, the affinity of one of the sites exceeding the reported affinities for albumin and ligandin, supports the hypothesis that a membrane-located membrane carrier is responsible for hepatic uptake and biliary excretion of organic anionic dyes. Based on the studies with enzyme treatments, it is speculated that this carrier is a phospholipid-dependent, integral membrane protein.
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PMID:Binding of unconjugated and conjugated sulfobromophthalein to rat liver plasma membrane fractions in vitro. 721 88

The biosynthesis of the membrane-bound metalloendopeptidase meprin was studied after the introduction of cDNAs encoding the rat meprin alpha and beta subunits into human 293 cells. When expressed individually the meprin alpha subunit was found to be primarily secreted into the culture medium, while the beta subunit was determined to be an integral membrane protein. Coexpression of the alpha and beta subunits results in the localization of both subunits to the plasma membrane. In this case the alpha subunit is specifically released from the cell surface by dithiothreitol, indicating the alpha subunit is associated with the membrane via disulfide bond(s) to the beta subunit. Meprin expressed in 293 cells is similar to the rat kidney enzyme in that it forms disulfide-linked dimers and has a similar pattern of glycosylation. However, the expressed protein displayed no detectable peptidase activity against four meprin substrates. Processing of the alpha subunit was followed after the introduction of sequences coding for the human c-myc peptide epitope EQKLISEEDL into its cDNA in the region of its prosequence and at the COOH terminus. Detection of the resulting proteins using a monoclonal antibody specific for the c-myc epitope indicates the alpha subunit is processed at its COOH terminus but retains the prosequence which is absent from the enzyme purified from rat kidney. Limited trypsin digestion of meprin precursors expressed in 293 cells results in both the activation of the enzyme and the removal of the prosequence. This result supports the hypothesis of Bode et al. (Bode W., Gomis-Ruth, F. X., Huber, R., Zwilling, R., and Stocker, W. (1992) Nature 358, 164-167) that meprin and other astacin family proteases require removal of NH2-terminal prosequences at the junction of the astacin protease domain for zymogen activation. Trypsin-activated meprin was assayed with the protein substrate azocasein and three synthetic peptide substrates. The membrane-bound beta subunit was found to be more active than the secreted alpha subunit against azocasein but much less active toward the synthetic peptide substrates.
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PMID:Expression of meprin subunit precursors. Membrane anchoring through the beta subunit and mechanism of zymogen activation. 751 Feb 89

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.
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PMID:A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis. 752 2

Activated platelets and stimulated endothelial cells express P-selectin, an integral membrane protein receptor that binds monocytes and neutrophils. P-selectin mediates adhesion to glycoproteins with carbohydrate structures containing sialyl-Lewis X. Since many carcinoma cells also express these carbohydrate structures and are known to interact with platelets, we asked whether P-selectin may mediate this interaction. Both small cell lung cancer and neuroblastoma cell lines bound to activated platelets, and this interaction was blocked with inhibitory anti-P-selectin antibodies and by pretreatment of these cancer cells with neuraminidase or trypsin. Platelet binding to the small cell lung cancer cells was not inhibited with anti-GP IIb-IIIa antibody or Arg-Gly-Asp-Ser peptide. Pretreatment of the neuroblastoma cells with inhibitors of N-linked carbohydrate biosynthesis had little effect on binding to P-selectin, indicating that relevant carbohydrate ligand(s) may be O-linked. In addition, lipospheres containing P-selectin specifically bound to cryostat sections derived from a small cell lung tumor and two neuroblastoma tumors, but not to sections of normal lung. These observations demonstrate that P-selectin mediates binding of platelets to small cell lung cancer and to neuroblastoma and suggest a possible role for this lectin in metastasis.
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PMID:P-selectin mediates adhesion of platelets to neuroblastoma and small cell lung cancer. 768 63


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