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 binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.
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PMID:Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum. 142 Feb 53

1. Patch-clamp recording techniques were used, to examine the effects of diazoxide on KATP currents in CRI-G1 insulin-secreting cells in the presence of non-hydrolysable nucleotides. 2. In the presence of non- or slowly-hydrolyzed ATP analogues, bathing the intracellular aspect of cell-free membrane patches diazoxide inhibited KATP channel activity. 3. Under whole-cell recording conditions, with various non-hydrolysable nucleotides present intracellularly (after dialysis), diazoxide induced KATP current activation. The largest activation occurred with Mg-adenylyl-(beta, gamma-methylene) diphosphate (Mg-AMP-PCP) present in the dialysing solution. This activation was diazoxide- and nucleotide-concentration-dependent. 4. In the absence of Mg2+, or in the presence of manganese (Mn2+) ions intracellularly, diazoxide did not induce KATP current activation, regardless of the species of nucleotide present in the pipette. 5. Intracellularly applied trypsin prevented the activation of KATP currents by diazoxide in the presence of Mg-AMP-PCP, an effect reversed by co-application of intracellular polymethylsulphonyl fluoride with the trypsin. 6. The application, by dialysis, of a CRI-G1 cell lysate, with negligible Mg-ATP, resulted in a substantial activation of the KATP current by diazoxide. 7. It is concluded that diazoxide can activate KATP channel currents by two separate pathways, one requiring a phosphorylation process, the other the presence of an intracellular protein coupled with a Mg-purine nucleotide.
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PMID:Nucleotide-dependent activation of KATP channels by diazoxide in CRI-G1 insulin-secreting cells. 142 77

We previously demonstrated that insulin accumulated in the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time, temperature, and insulin concentration dependent, but apparently nonsaturable. This study investigated further the initial endocytotic pathways that contribute to the nuclear accumulation of insulin using trypsin treatment of the cells to prevent insulin binding to its plasma membrane receptor. Total cell-associated, intracellular, and nuclear insulin were compared in control and trypsin-treated H35 hepatoma cells. Trypsin treatment markedly decreased total cell-associated and intracellular insulin as well as the nuclear accumulation of insulin when cells were incubated with 2.8 ng/ml insulin. When the cells were incubated with 100 ng/ml insulin, trypsin treatment totally inhibited insulin binding to the plasma membrane for at least 90 min. However, intracellular accumulation of insulin was reduced by only 50% at 60 min, and trypsin treatment failed to inhibit the nuclear accumulation of insulin. Chemical extraction and Sephadex G-50 chromatography revealed nuclear associated insulin in trypsin-treated cells was identical to that in control cells incubated with either 2.8 or 100 ng/ml insulin. These results suggest that a nonreceptor mediated uptake pathway, i.e., fluid-phase endocytosis, contributed significantly to the nuclear accumulation of insulin at high insulin concentrations, but at lower insulin concentrations the receptor-mediated pathway predominated. No matter which initial endocytotic route was used to internalize insulin, the insulin apparently associated with the same nuclear matrix proteins. This association of insulin with the nuclear matrix may be involved in regulation of nuclear events such as cell growth and differentiation or gene transcription.
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PMID:Nonreceptor mediated nuclear accumulation of insulin in H35 rat hepatoma cells. 144 21

1. Three different proteinases were isolated from intestinal extracts of Hirudo. 2. The action on synthetic substrates and the effect of various inhibitors indicate that one is a trypsin-like enzyme whereas the other two are chymotryptic. 3. This was confirmed by analysing the degradation of the insulin B-chain. 4. The relative molecular masses of all enzymes are approximately 35,000 and the isoelectric points 9.03, 9.12 and 9.41, respectively. 5. The specific activities assayed with SAPPNA are very high in comparison with proteinases of other animals.
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PMID:Proteinases of the medicinal leech, Hirudo medicinalis: purification and partial characterization of three enzymes from the digestive tract. 149

Cells of Acinetobacter calcoaceticus contain a constitutive periplasmic metalloproteinase showing similar properties as the periplasmic metalloproteinase of Escherichia coli. The periplasmic proteinase of A. calcoaceticus was purified, starting from periplasm, by ammonium sulfate precipitation, hydrophobic interaction chromatography and chromatofocusing up to the homogeneity of the enzyme in SDS-electrophoresis with a yield of 6.7% and a purification factor of 417. The enzyme has a molecular mass of 108,000 (gel filtration) or 112,000 (native electrophoresis), and consists of four identical subunits with a molecular mass of 27,000 (SDS-electrophoresis). The purified enzyme degrades preferentially polypeptides such as glucagon and insulin. Larger proteins are accepted as substrates to a considerably lower extent. All tested synthetic substrates with trypsin, chymotrypsin, elastase and thermolysin specificity were not cleaved. Therefore, the described enzyme was designated "insulin-cleaving proteinase" (ICP).
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PMID:Purification of a periplasmic insulin-cleaving proteinase from Acinetobacter calcoaceticus. 151 May 71

The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins.
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PMID:A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex. 151 Sep 27

Qualitative disorders of an echopancreatogram are noted in half of patients with diabetes mellitus (both insulin dependent and noninsulin dependent). The most significant echopancreatographic quantitative and qualitative disorders were observed in diabetic patients with a maximal decrease in pancreatic enzyme excreting activity (on the basis of lipase and trypsin debit in a pancreozymin test, daily steatorrhea and chymotrypsin amount in daily feces). It has been assumed that a degree of ultrasound changes in the pancreas in diabetes depends on a degree of fibrosis of pancreatic exocrine tissue. Ultrasound investigations with quantitative and qualitative assessment of echopancreatograms is a valuable adjuvant diagnostic method in diabetes mellitus.
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PMID:[Echostructure of the pancreas. Comparison with exocrine secretory activity of the pancreas in patients with diabetes mellitus]. 151 83

In evaluating the purification of genetically engineered human insulin there is no plausible correlation between the yield of expression as determined by SDS-PAGE (taking into account all normally occurring losses during the several purification steps) and the actual yield, i.e., the final pure product. The aim of our work was to develop a fast, accurate, and reproducible method for the quantification of the initial yield of the intact insulin fusion protein directly after fermentation and without prior purification of the fermentation product. Therefore, a protocol for efficient tryptic digestion of protease-resistant inclusion bodies was established. The resulting crude peptide mixture was oxidized by performic acid and the insulin A-chain, which contains no cleavage side for trypsin, was quantified by HPLC in the form of a tetrasulfonate derivative to reduce artefacts due to free -SH groups. Compared with SDS-PAGE, this procedure allows sensitive monitoring of possible degradation at the C-terminus. Furthermore, quantification of expression products on the basis of the present method will provide better correlation between initial and actual yield.
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PMID:A method for directly determining intact recombinant insulin fusion proteins in crude fermentation extracts. 151 68

The effects of pancreatic duct anastomosis to stomach (stomach group) or duodenum (duodenal group) on pancreatic function were examined in dogs following two thirds pancreatectomy. Normal fasting blood glucose concentrations were maintained in both groups despite significant reductions in glucose tolerance in the stomach group, and reductions in fasting insulin and insulin peak response in both groups. Pancreatic exocrine function was significantly decreased in both groups, though plasma p-aminobenzoic acid (PABA) concentrations were generally higher in the duodenal group. A correlation was found between plasma trypsin-like immunoreactivity (TLI) and pancreatic weight. These results indicate that anastomosis of the pancreas to bowel can be undertaken with minimal postoperative complications and that the site of the anastomosis influences pancreatic function. They suggest that preservation of more than one third of the pancreas is required for optimal function. The complementary information provided by the PABA and TLI tests suggests their dual application will be clinically useful for the detection and characterisation of naturally occurring pancreatic diseases.
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PMID:Pancreatic function following partial pancreatectomy and anastomosis of the pancreatic duct to the stomach or duodenum in dogs. 155 43

We have used a preparation of soluble human insulin receptor ectodomain and a novel photoreactive, biotinylated derivative of insulin (4-azidosalicyloyl(B1-biocytinyl-B2-lysine)-insulin) to identify a new hormone contact site within the extracellular domain of the insulin receptor. The ectodomain was photoaffinity-labeled and digested to completion with trypsin, and the resulting tryptic fragment was purified by either HPLC or by streptavidin-affinity chromatography. The amino terminus of the fragment was identified as Gly390 within the alpha-subunit. These results suggest that residues that are carboxyl-terminal to the cysteine-rich domain, in addition to previously identified regions within the amino terminus of the alpha-subunit, contribute to the insulin binding site. The implications of these results for the de novo folding of the insulin receptor to constitute the hormone binding site are discussed.
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PMID:Detection of a new hormone contact site within the insulin receptor ectodomain by the use of a novel photoreactive insulin. 157 32


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