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)

Oleic acid and dioleoyl phosphatidic acid at low concentrations (20 and 0.5 mu g/ml, respectively) agglutinate rabbit and rat erythrocytes, while dioleoyl phosphatidylcholine is not hemagglutinating up to 0.5 mg/ml. Palmitic acid is not a hemagglutinin and dipalmitoyl phosphatidic acid is a very poor one. A polar lipid fraction obtained from calf thymocytes and a commercial preparation of gangliosides also exhibit pronounced hemagglutinating activity. Modification of the erythrocytes by either trypsin or neuraminidase causes a marked increase in agglutination only with oleic acid, whereas glutaraldehyde fixation of the cells significantly decreases agglutination with oleic acid, dioleoyl phosphatidic acid and calf thymocyte lipids. None of the lipids tested agglutinate freshly drawn human and sheep erythrocytes, but agglutination occurs following fixation of the sheep cells with glutaraldehyde. Lipid-mediated hemagglutination is strongly inhibited by fetuin and bovine submaxillary mucin (0.5 mg/ml). Defatted bovine serum albumin, also at 0.5 mg/ml, inhibits agglutination by oleic acid, whereas agglutination by other lipids is only poorly inhibited if at all. Monosaccharides at concentration up to 0.25 M do not inhibit the hemagglutinating activity of the lipids. Comparison of the hemagglutinating properties of lipids and lectins raises the possibility that the agglutinating activity of crude biological extracts which is not inhibited by mono- or oligosaccharides may be due to lipid constituents. Since agglutination by lipids is species specific, they may serve as mediators in intercellular recognition. The mechanism of lipid-mediated hemagglutination is discussed in terms of current concepts of the fusogenic activity of these compounds.
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PMID:Lipid-mediated hemagglutination and its relevance to lectin-mediated agglutination. 728 60

P0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P0 was labeled in rat sciatic nerve slices with [3H]palmitic acid and subsequently treated with various reagents. The protein-bound palmitate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydroxylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 mM NaBH4 with the concomitant production of [3H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [14C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [14C]carboxyamidomethylated P0 was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys153 in rat P0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmembrane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.
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PMID:Identification of the palmitoylation site in rat myelin P0 glycoprotein. 750 74

The intrinsic polypeptide D1, isolated from photosystem (PS) II-particles of the cyanobacterium Oscillatoria chalybea, was obtained by electroelution and fractionated extraction with organic solvents. Purification was demonstrated by Western blotting and amino acid sequencing. By carrying out D1-immunization in rabbits a polyclonal monospecific D1-antiserum was obtained. For the qualitative characterization of D1 as a lipid-binding peptide, the effect of the lipids phosphatidylglycerol (PG), monogalactosyldiacylglyceride (MGDG) and phosphatidylcholine (PC) on PSII-oxygen evolution was analysed in reconstitution experiments. In these experiments purified photosystem II (PSII)-particle preparations were treated with the enzyme phospholipase A2 and supplemented with lipid emulsions. We were able to show that the inhibition of electron transport, as the consequence of this lipase treatment, was only relieved, if phosphatidylglycerol was added to the preparation. A model was proposed, in which phosphatidylglycerol is a functional effector for the optimal conformation of D1 in the PSII core complex. Phosphatidylglycerol molecules are unusually tightly bound to the D1 peptide by hydrophobic interactions. A covalent binding seems not probable. The localisation of phosphatidylglycerol binding sites was found by trypsin treatment of D1 and analysis of the obtained oligopeptides with HPLC and immunoblotting. The binding sites could be confined to the hydrophobic amino acid section between arginine 27 and arginine 225, which is known to be the membrane anchor of D1. This has led us to the conclusion that the phospholipid phosphatidylglycerol plays an important role for anchoring the D1-peptide and for its orientation in the thylakoid membrane. Phosphatidylglycerol with its high amount of palmitic acid has in prokaryotic cyanobacteria apparently a role in stabilization and orientation. The high turn-over of D1 and the spatial separation of the synthesis- and incorporation-site in the membrane, developed during evolution in eukaryotic organisms, might have changed the requirement on the mobility and the orientation of D1 in photosynthetic membranes.
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PMID:The role of phosphatidylglycerol as a functional effector and membrane anchor of the D1-core peptide from photosystem II-particles of the cyanobacterium Oscillatoria chalybea. 754 31

Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10-3H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C-terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.
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PMID:Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus. 755 84

Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies. Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system. The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies. Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes. PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol. In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles. Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb. Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein. The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.
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PMID:Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing. 768 Jan 92

cAMP-binding ectoprotein (Gce1) and lipoprotein lipase (LPL) are anchored to plasma membranes of rat adipocytes by glycosylphosphatidylinositol (GPI) moieties as demonstrated by cleavage by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), reactivity with anti-crossreacting determinant antibodies (anti-CRD), and metabolic labeling with radiolabeled palmitic acid and myo-inositol. Quantitative release from the membrane of LPL and Gce1 requires both lipolytic removal of their GPI anchors and the presence of either 2 M NaCl or 1 mM inositol 1,2-cyclic monophosphate or inositol 1-monophosphate. PI-PLC-cleaved and released LPL or Gce1 reassociates with isolated plasma membranes of rat adipocytes and, less efficiently, with membranes of 3T3 fibroblasts. The specificity of the reassociation is demonstrated (i) by its inhibition after pretreatment of the membranes with trypsin, (ii) by its competition with inositol 1,2-cyclic monophosphate and inositol 1-monophosphate in a concentration-dependent manner, and (iii) by the limited number of binding sites. Enzymic or chemical removal as well as masking with anti-CRD antibodies of the terminal inositol (cyclic) monophosphate moiety of hydrophilic Gce1 and LPL significantly impairs the reassociation. These data suggest that in rat adipocytes GPI-proteins are not readily released from the cell surface upon lipolytic cleavage, but remain associated through a receptor which specifically recognizes the terminal inositol (cyclic) monophosphate epitope of the (G)PI-PLC-cleaved GPI moiety. This interaction may have implications for the regulated membrane release of GPI-proteins and for their possible internalization.
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PMID:Membrane association of lipoprotein lipase and a cAMP-binding ectoprotein in rat adipocytes. 791 36

Gq alpha and G11 alpha differ from other G protein alpha subunits in that they have unique, conserved 6 residue amino-terminal extensions. Wild-type and amino-terminal mutants of Gq alpha expressed in COS cells were analyzed for their ability to functionally couple with co-expressed neurokinin NK2 receptor. Wild-type, T2A and delta 2-7 Gq alpha were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino-terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with G beta gamma subunits and GTP gamma S-induced trypsin resistance are also similar to the wild-type alpha subunit. This demonstrates that the NK2 receptor is able to functionally interact with the alpha subunit of Gq and that the first seven amino-acids of Gq alpha are not required for any of the alpha subunit functions tested. In contrast to the T2A and delta 2-7 mutants, a C9,10A Gq alpha mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high-affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTP gamma S-induced trypsin-resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild-type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino-terminus of Gq alpha results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.
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PMID:Palmitoylation but not the extreme amino-terminus of Gq alpha is required for coupling to the NK2 receptor. 795 23

Severe deterioration of surfactant function is noted under conditions of plasma protein leakage into the alveolar space; moreover, fibrinogen has previously been reported to possess strong surfactant inhibitory capacity. Dissolution of alveolar deposits of fibrinogen and fibrin (e.g., hyaline membranes) requires enzymatic degradation by the plasminogen/plasmin system or by leukocyte-derived proteases. We investigated the surfactant inhibitory properties of differently prepared sets of fibrinogen cleavage products. Proteolysis was performed with plasmin, with predominant split products D (mol wt 85,000) and E (mol wt 50,000). In addition, fibrinogen was cleaved by leukocyte elastase and trypsin, with fragments ranging mainly between mol wt of 30,000 and 50,000. To provide split products of even lower molecular weight, fibrinogen was incubated sequentially with trypsin and endoproteinase (split products < mol wt 25,000). Natural surfactant extracts used in clinical replacement studies (CLSE, Alveofact, Curosurf, Survanta) as well as an apoprotein-based phospholipid mixture (PLM-C/B; DPPC:PG:PA = 68.5:22.5:9 with 2% [wt/wt] nonpalmitoylated recombinant human SP-C and 1% [wt/wt] natural bovine SP-B) were employed. Experiments were performed in a pulsating bubble surfactometer (standard phospholipid concentration 2 mg/ml) with assessment of surfactant activity measuring adsorption and dynamic surface tension. Fibrinogen caused dose-dependent, severe deterioration of the surface activities of Curosurf and Survanta, whereas CLSE, Alveofact, and PLM-C/B were only moderately affected up to protein-surfactant ratios of 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteolytic cleavage of fibrinogen: amplification of its surfactant inhibitory capacity. 839 60

A tightly membrane-associated form of spectrin (TMA-spectrin) was labeled when human red blood cells were incubated with [3H]palmitic acid. About 90% of spectrin was not fatty acid-acylated and was extracted from membranes by low salt buffers. The 3H-palmitoylated TMA-spectrin, however, resisted low and even high salt extraction and remained associated with inside-out vesicles that were generated in the process of spectrin-actin extraction from membranes. TMA-spectrin was preferentially extracted from KCl-stripped vesicles by 5 M urea at low ionic strength. TMA-spectrin was purified by gel filtration and by ion exchange chromatography in the presence of urea and a non-ionic detergent. Purified TMA-spectrin was 3H-palmitoylated exclusively in the beta subunit to 0.28 mol/mol after a 12-h incubation of red cells. The labeled palmitate may be bound as an ester or thioester, since hydroxylamine (1 M, pH 7.5) released the label completely. Peptide maps of 3H-palmitoylated TMA-spectrin showed three or two labeled peptides from the beta subunit, when generated by V8 protease and trypsin, respectively. Two types of antibodies to spectrin reacted with purified TMA-spectrin, and TMA-spectrin contained the same antigenic peptides as low salt-extractable spectrin. Rabbit anti-ankyrin antibodies did not bind to TMA-spectrin. The substoichiometric incorporation of [3H]palmitic acid into TMA-spectrin could result from the slow turnover of endogenously bound fatty acids. Generation of the tightly membrane-associated and 3H-palmitoylated subpopulation of spectrin cannot be due to entrapment of an unmodified residual fraction of spectrin in right-side-out vesicles. Instead, the data suggest the existence of a subpopulation of spectrin molecules that undergo a covalent fatty acid modification and thereby alter their binding properties. This may offer a new, metabolically dependent mechanism for dynamic interactions between spectrin and the membrane lipid bilayer.
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PMID:A tightly membrane-associated subpopulation of spectrin is 3H-palmitoylated. 850 31

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.
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PMID:Purification of Borrelia burgdorferi outer surface protein A (OspA) and analysis of antibody binding domains. 855 77


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