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)

E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.
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PMID:[Studies on genetic engineering of human insulin-purification and characterization of human proinsulin and insulin]. 141 26

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

We previously reported on patients with severe insulin resistance due to unprocessed insulin proreceptors. A structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G----T point mutation appeared to be the cause for failure to process the proreceptors. To determine whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin receptors and to elucidate the structural and binding characteristics of the proreceptors, we transfected cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in cells transfected with either normal or mutated cDNA, and insulin binding was 40 and 14 times higher than that of nontransfected cells, respectively. The declining rate of insulin binding after reaching the maximum was delayed in cells transfected with mutated cDNA. Affinity cross-linking and surface-labeling studies showed a 135-kilodalton (kD), normal alpha-subunit in the cells transfected with normal cDNA and a 210-kD proreceptor in the mutant cells. The proreceptors were cleaved by trypsin to yield normal-sized alpha- and beta-subunits. The sensitivity to trypsin was similar to that demonstrated in patients' cells, and the most effective concentration for the cleavage was 0.025%. Autophosphorylation resulted in decreased 32P incorporation into proreceptors of cells transfected with mutated cDNA at both basal and insulin-stimulated states, without a change in insulin sensitivity. Competitive binding studies with insulin, proinsulin, and miniproinsulin showed that the proreceptors had a lower relative affinity for proinsulin, but this characteristic disappeared after trypsin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of unprocessed insulin proreceptors in COS 7 cells transfected with cDNA with Arg735----Ser735 point mutation at the cleavage site. 164 Aug 58

Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1-29)-A(1-21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr-X (X = blocking residue) (two-step reaction) or (2) subjecting Ala-B30 of porcine insulin or Gly-A1 of the single chain insulin precursor to transpeptidation with Thr-X (one-step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two-step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two-step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.
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PMID:Enzymatic semisynthesis of human insulin: an update. 209 84

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.
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PMID:Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes. 228 41

Human proenkephalin generated by means of a recombinant vaccinia virus expression vector was used as the substrate for a putative processing enzyme obtained from bovine adrenal chromaffin granules. The adrenal enzyme successfully cleaved proenkephalin to generate low mol wt enkephalins as well as other enkephalin-containing intermediates. Radioactively labeled proenkephalin prepared with this system was also cleaved; however, under identical conditions bovine proinsulin was not cleaved. These results provide support for the notion that the adrenal trypsin-like enzyme is involved in the processing of proenkephalin in vivo and demonstrate the usefulness of protein substrates prepared by expression vector systems in testing the reactivity and specificity of proposed prohormone processing enzymes in vitro.
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PMID:Cleavage of proenkephalin by a chromaffin granule processing enzyme. 229

Sera from patients of biliary, alcoholic, and idiopathic acute pancreatitis with severity scored from 1 to 5 based on the Ranson criteria were tested for proinsulin/insulin degrading activity. Proinsulin degrading activity by normal controls was 8 +/- 4% as compared with 22-78 +/- 17% with a mean of 45% by the patient sera. An order of magnitude increase of proinsulin degrading activity was accompanied by an order of magnitude increase of immunoreactive pancreatic cationic trypsin(ogen) and (pro)elastase-2 as determined by radioimmunoassay with day 1 sera. Proinsulin degrading activity also showed a negative correlation with the clinical time course and dropped to normal by 6 days after admission. The decrease of proinsulin degrading activity was concomitant with a decrease of serum immunoreactive pancreatic serine proteases. High-performance liquid chromatography analysis of the proteolysis products showed the appearance of insulin and smaller peptides with no proinsulin conversion intermediates. Ninety to ninety-eight percent of proinsulin degrading activity was inhibited by anti-alpha 2-macroglobulin (alpha 2-M) antiserum, or (Ac)Eglin-C(J141), and 52% by an elastase and chymotrypsin-specific inhibitor, MeOSuc-Ala-Ala-Pro-boroVal-pinacol. E64c, TLCK, alpha 1-protease inhibitor (alpha 1-PI), or Trasylol inhibited proinsulin degrading activity by 10-17%, and anti-cathepsin B antiserum by 9%. The observed proinsulin degrading activity did not correlate with the Ranson's scores, age, sex, etiology, total serum immunoreactive insulin, calcium, albumin or alpha 2-M but had a negative correlation with serum alpha 1-PI (r = -0.55) and a positive correlation with serum esterase activity (r = .62).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteolytic degradation of human recombinant proinsulin/insulin by sera from acute pancreatitis patients and complete inhibition by Eglin-C. 240 52

Capillary isotachophoresis (ITP) was applied to the qualitative and quantitative analysis of both natural and synthetic oligo- and polypeptides. Based on the mathematical model of acid-base equilibria for a general ampholyte, a procedure and a computer program for the calculation of the pH dependence of the effective and specific charge and effective mobility of peptides with known amino acid sequence were developed which allow the selection of electrolyte systems for peptide isotachophoretic analysis to be rationalized. Basic peptides (bovine pancreatic trypsin inhibitor, bull seminal isoinhibitors of trypsin, arginine vasopressin and adamantylamide-alanylisoglutamine) were analysed with a cationic ITP system at acidic pH. Neutral and acidic peptides (insulin, proinsulin, bull seminal isoinhibitors of trypsin, cow colostrum isoinhibitors of trypsin) were analysed with an anionic ITP system, mostly at alkaline pH. Peptide purity (electrophoretic homogeneity) was determined from the ITP degree of purity defined by a peptide itself and the zone length ratio of its admixtures. Enrichment of peptide in the sample during the purification procedure was measured by its zone length relative to unit mass of the amount of sample analysed.
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PMID:Isotachophoretic analysis of peptides. Selection of electrolyte systems and determination of purity. 266 86

An alteration of an amino acid sequence in the processing site of the insulin proreceptor by a point mutation of the insulin receptor gene produced extreme insulin resistance. We characterized functional properties of the unprocessed insulin receptor in transformed lymphocytes from a patient. Insulin binding to intact cells and to a partially purified insulin receptor preparation was radically decreased to 20% and 18% of the control values, respectively. In competitive insulin binding to intact cells, [LeuA3]-, [LeuB24]-, [SerB24]-insulin, and mini-proinsulin ([ B(1-29)-Ala-Ala-Lys-A(1-21)]-insulin) had the same relative binding activity in both the patient's and the control cells, but proinsulin and IGF-I were markedly less able to displace 125I-insulin in the patient's cells. In contrast to the study in intact cells, proinsulin and IGF-I as well as other insulin analogues had the same relative binding activity to bind to the partially lectin-purified insulin receptor preparations from both the patient's and the control cells. As regards the signal transduction after receptor binding, insulin-stimulated autophosphorylation of the unprocessed insulin proreceptor occurred proportionally to the amount of decreased insulin binding. With 0.025% trypsin treatment, the abnormal binding characteristics and autophosphorylation were normalized through conversion to functionally normal receptors. In spite of the abnormal processing, self-association of receptors into oligomeric structures was observed in the proreceptor. These results suggest that the unprocessed insulin proreceptor in the plasma membranes has an altered conformation which affects its binding characteristics but not its intramolecular signal transmission.
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PMID:Binding specificity and intramolecular signal transmission of uncleaved insulin proreceptor in transformed lymphocytes from a patient with extreme insulin resistance. 266 84

Incubation of pig desoctapeptide-(B23-30)-insulin with trypsin in solvent systems consisting of dimethyl sulphoxide, butane-1,4-diol and Tris buffer resulted in the formation of an extra peptide bond between Arg-B22 and Gly-A1 in the DOPI molecule. This DOPI derivative can also be regarded as pig des-(23-63)-proinsulin. The structure of the new, previously unreported, proinsulin analogue was determined on the basis of amino acid analysis, dansylation and digestion with Staphylococcus aureus V8 proteinase. Receptor-binding ability of des-(23-63)-proinsulin was 20% of that of pig desoctapeptide-(B23-30)-insulin and 0.02% of that of pig insulin.
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PMID:Trypsin-catalysed formation of pig des-(23-63)-proinsulin from desoctapeptide-(B23-30)-insulin. 352 94


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