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)

UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.
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PMID:Functional symmetry of UhpT, the sugar phosphate transporter of Escherichia coli. 983 61

C-terminal tails of plant hexose/H(+)-symporters of the major facilitator superfamily contain a highly conserved motif of four amino acids: HWFW. A deletion of these four amino acids in the Chlorella HUP1 protein leads to a decrease in transport activity by a factor of 3-4. The mutated tail is highly sensitive to trypsin; it does not show alpha-helical conformation in contrast to the wild type C-terminal peptide with an alpha-helical content of at least 15%. The production of monoclonal antibody 416B8 recognizing an epitope within the central loop of HUP1 protein has been a prerequisite for the experiments described.
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PMID:The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H(+)-symporter is essential for full activity and an alpha-helical structure of the C-terminus. 1069 91

Epithelial morphogenesis is supported by diffusible growth factors and by nondiffusible cell substrata, such as laminin and fibronectin. When embedded in a laminin-rich basement-membrane substratum, embryonic mouse submandibular epithelium undergoes cell proliferation and branching morphogenesis in response to epidermal growth factor (EGF) in mesenchyme-free culture but not in serum-free medium. In this study, we sought to identify the biologically active factor in serum. As this factor was heat-stable and trypsin-resistant, the lipid fraction was analyzed. Horse serum was fractionated by ethanol extraction, Folch partition with chloroform-methanol-water, and high-performance liquid chromatography, and we tested the branch-inducing activity of each fraction. We also analyzed the partially purified fraction with a mass spectrometer, indicating that the active fraction largely consisted of lysophosphatidyl-hexose. Finally we identified the molecule as lysophosphatidic acid (LPA), because, whereas lysophosphatidyl-inositol had only a slight branch-inducing activity, its relevant LPA fully substituted for serum and induced branching morphogenesis in cooperation with EGF. LPA receptor genes were expressed in submandibular epithelial cells. DNA-synthesizing cells were abundant only when cultured in the presence of both EGF and LPA, but not either singly.
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PMID:Lysophosphatidic acid cooperates with EGF in inducing branching morphogenesis of embryonic mouse salivary epithelium. 1631 26

Mice immunized with purified whole-cell ribonucleic acid (RNA), RNA from the bacterial "particulate" fraction, and ribosome-associated RNA obtained from Salmonella typhimurium were found to be resistant to subsequent challenge infection with virulent salmonellae. Chemically, the immunogenic nucleic acid fractions contained from 1 to 3% "contaminant" material defined (based on the mean of 19 different preparations) as protein (0.24%), deoxyribonucleic acid (0.43%), methyl pentose (0.64%), hexose (1.58%), and undefined carbohydrate (0.76%). Heptoses and lipoidal material were not detectable in any of the immunogenic preparations examined. Physically, the nucleic acid preparations, after analytical ultracentrifugation, exhibited three boundaries similar to those reported elsewhere in comparable systems: 4 to 5S, 16S, and 23S. An evaluation of the immunity induced by the ribosome-associated RNA established that the immune response was (i) comparable to that induced 15 days postimmunization with live salmonellae and by ribosomal vaccines, but greater at 30 days postimmunization than that in mice immunized with attenuated salmonellae; (ii) dependent on the quantity of immunogen administered; (iii) dependent on the size of the infective inocula; (iv) inhibited at 15 but not at 30 days postimmunization when the immunogenic nucleic acid preparations were incorporated into Freund's incomplete adjuvant, (v) reduced or lost by dialysis in relatively high or low immunizing doses, respectively; and (vi) unaffected by enzymatic treatment of the preparations with trypsin, deoxyribonuclease, Pronase plus pancreatic ribonuclease, or pancreatic ribonuclease alone. The possible mode of action of ribosome-associated RNA in inducing an immune response to subsequent challenge infection with the homologous organism is discussed.
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PMID:Immunogenicity of Ribonucleic Acid Preparations Obtained from Salmonella typhimurium. 1655 78

Plasma membranes were isolated under hypotonic conditions from rat and mouse livers and five hepatomas, i.e. one rather anaplastic rat hepatoma (and its subline) and three well-differentiated mouse hepatomas. All these membranes contained some 25% protein soluble in 0.15M NaCl. Evidence is presented that this protein is mainly, if not exclusively of nonmembranous origin. Protein/phospholipid P (P=phosphorus) ratios did not differ significantly for the various plasma membrane species except the rat-hepatoma subline, which showed a markedly lower ratio and was thus identified. Hepatoma membranes contained more P of a nonphospholipid nature than did liver membranes and to this increase contributed in all instances an increased RNA content and in some cases also an increased DNA content. The presence of DNA in these plasma membranes is artefactual, but that of RNA is more complicated. Artefactually, Ca(2+)-associated RNA of low mol wt and soluble in 0.15M NaCl, and residual RNA (genuine?, in liver membranes less than 1% in respect of protein) have been demonstrated. The increase in hepatoma-membrane RNA is attributed to the ribosomal RNA of the few microsomal vesicles which are structurally connected with these plasma membranes. The sialic acid content and the percentage of neuraminidase-resistant sialic acid of hepatoma as compared with liver membranes was either similar or changed, depending on the hepatoma strain. Gelfiltration of trypsin-released peptides of liver plasma membranes showed hexosamine and hexose to be confined to the sialic acidcontaining fractions. In spite of quantitative differences among fractions, the relative contents of the three carbohydrates in the combined fractions were (about) similar to those in intact liver membranes. Similar experiments with the rat-hepatoma membranes showed a changed carbohydrate expression.
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PMID:Studies on plasma membranes XVII. On the chemical composition of plasma membranes prepared from rat and mouse liver and hepatomas. 2417 41

Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.
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PMID:O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry. 2479 65


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