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 possible involvement of a 15-kDa phosphotyrosyl protein, pp15, in insulin action was investigated by using the insulin-mimetic agent, vanadate. Vanadate, a phosphotyrosine phosphatase inhibitor, was found to mimic insulin in 3T3-L1 adipocytes by three criteria. First, kinetic and concentration-dependence studies verified the insulin-like effect of vanadate in activating 2-deoxyglucose uptake. Insulin had an additive activating effect at a submaximal vanadate concentration, but showed no further activation at a saturating vanadate concentration. The trivalent arsenical, phenylarsine oxide (PAO) which forms complexes with vicinal dithiols, markedly inhibited vanadate-activated hexose transport in agreement with our previous studies in which PAO abolished the insulin-activated component of sugar uptake. Second, in situ phosphorylation experiments showed that vanadate activated tyrosine phosphorylation of the insulin receptor's beta-subunit. Exposure of vanadate-treated cells to PAO further increased the level of beta-subunit phosphorylation. The increased level of phosphorylation in the presence of PAO occurred only on tyrosyl residues. Third, vanadate caused the accumulation of a phosphorylated 15-kDa protein in the presence of PAO, but not in its absence. The characteristics of this protein were identical to those of pp15: 1) both proteins behaved identically by two-dimensional gel electrophoresis, 2) digestion of both proteins with trypsin gave rise to apparently identical phosphopeptides, and 3) both proteins contained phosphotyrosine as the only phosphoamino acid. The results indicate that both vanadate and insulin stimulate the accumulation of pp15 in the presence of PAO. The dithiol,2,3-dimercaptopropanol, but not a monothiol, reversed the effects of PAO on the inhibition of vanadate-induced hexose transport and the accumulation of pp15, thus implicating a vicinal dithiol in these actions of vanadate and insulin. Our results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin receptor tyrosine kinase action, is coupled to signal transmission to the glucose transport system.
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PMID:Effect of vanadate on the cellular accumulation of pp15, an apparent product of insulin receptor tyrosine kinase action. 245 50

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.
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PMID:Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin. 265 75

Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half. 280 17

The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors. 302 49

A glycopeptide from adenocarcinoma tissue of human lung was extracted by protein digestion with papain (EC 3.4.22.2) and trypsin (EC 3.4.21.4). This component was isolated by Pevikon block electrophoresis. It possessed hexose, glucosamine, fucose, galactosamine, and sialic acid. In its carbohydrate composition and also the abundance of threonin in the peptide moiety it was quite similar to the glycoprotein from gastrointestinal tract. The physical characteristic of the two, such as their optical rotation and viscosity, were also very similar each other.
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PMID:A glycopeptide from adenocarcinoma tissue of human lung. 321 27

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.
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PMID:The interaction of phosphorylated sugars with human hexokinase I. 325 34

After denaturation in 0.6 M guanidine hydrochloride, rat brain hexokinase becomes highly susceptible to proteolysis by trypsin. Glucose 6-phosphate (Glc-6-P) and its analog, 1,5-anhydroglucitol 6-phosphate, selectively protect the N-terminal half of the molecule from proteolysis. These compounds do not protect the C-terminal half of the molecule, nor do they protect enzyme activity; the Glc analog, N-acetylglucosamine, does protect the C-terminal domain and catalytic activity, but does not prevent proteolysis of the N-terminal half of the molecule. These results are consistent with previous work [M. Nemat-Gorgani and J. E. Wilson (1986) Arch. Biochem. Biophys. 251, 97-103; D. M. Schirch and J. E. Wilson (1987) Arch. Biochem. Biophys. 254, 385-396] demonstrating that binding sites for both hexose and nucleotide substrates, and thus catalytic function, are associated with a 40-kDa domain located at the C-terminus of the enzyme. They further demonstrate that the binding site for the allosteric effector, Glc-6-P, lies in the N-terminal half of the molecule and is distinct from the catalytic site. Using protection against proteolysis as a reflection of binding, it is shown that the Glc-6-P binding site in the N-terminal region has all the characteristics described for the allosteric effector site on this enzyme in terms of affinity for Glc-6-P, specificity, and synergistic interactions with the hexose binding site in the C-terminal region of the molecule. This disposition of catalytic and regulatory functions in discrete halves of the molecule is consistent with suggestions by several investigators that mammalian hexokinases evolved by a process of duplication and fusion of an ancestral gene coding for a hexokinase similar to the present-day yeast enzyme, with the regulatory site of mammalian hexokinases having evolved from what was originally a catalytic site.
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PMID:Rat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme. 342 36

The cleavage of the human erythrocyte hexose transporter by the proteinases trypsin and thermolysin has been studied. When red cell membranes are treated with trypsin, washed and then photolabelled with cytochalasin B, a labelled peak at 18 kDa is obtained. This labelling of the cleaved transporter is D-glucose inhibitable. This probably indicates that the residual 36 kDa portion of the transporter is not required for binding of ligands. Extensive cleavage of the transporter with low concentrations of thermolysin only occurs when transporter is prelabelled with cytochalasin B. This indicates that covalently bound cytochalasin B can cause a conformational change which exposes the thermolysin cleavage site.
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PMID:Binding of cytochalasin B to trypsin and thermolysin fragments of the human erythrocyte hexose transporter. 362 Apr 69

Phloridzin-insensitive, Na+-independent D-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed a Kt of 48 mM and a Vmax of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lower Kt of 23 mM and a Vmax of 0.20 nmol glucose/mg protein/sec. D-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. The Ki for cytochalasin B as an inhibitor of glucose transport was 0.11 microM and the KD for binding to the carrier was 0.08 microM. D-glucose-sensitive sensitive binding of cytochalasin B to the membrane preparation was maximized with L- and D-glucose concentrations of 1.25 M. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na+-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and the Kt for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.
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PMID:The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics. 362 59

Previous work from this laboratory has demonstrated the persistence of Bacteroides intermedius in the livers of mice receiving an intraperitoneal inoculum of B. intermedius and Fusobacterium necrophorum. This study was undertaken to determine whether F. necrophorum enhanced the in vitro growth of B. intermedius. Tryptose phosphate broth did not support the growth of B. intermedius alone, but the bacterium did survive in a tryptose phosphate broth culture of F. necrophorum. B. intermedius cultured in F. necrophorum-conditioned tryptose phosphate broth grew impressively, reaching maximal absorbance at 24 h after inoculation. The growth of B. intermedius in F. necrophorum-conditioned tryptose phosphate broth was proportional to the amount of conditioned medium present. The B. intermedius growth-stimulating factor was detectable in conditioned medium 8 h after inoculation with F. necrophorum and could be detected throughout the 96-h incubation period. Growth-factor-active fractions eluted from a Sephadex G-100 column did not absorb at 280 nm and were retained on the column until 4 column volumes were eluted. The growth factor was nondialyzable and stable to boiling, lyophilization, extraction with hot aqueous phenol, and trypsin digestion. The factor was inactivated by exposure to pH 2.0 in the pepsin digestion protocol. Significant amounts of hexose, methyl pentose, and 2-keto-3-deoxyoctonate were detected in pooled growth-factor-active fractions eluted from the Sephadex column. This pool was also active in the Limulus lysate endotoxin assay. These results suggest that the B. intermedius growth-stimulating factor produced by F. necrophorum is a lipopolysaccharide.
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PMID:Enhancement of Bacteroides intermedius growth by Fusobacterium necrophorum. 370 Jun 5


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