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)

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.
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PMID:Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides. 59 Sep 40

Using a reaction suite which was suggested by Ruttenberg [5] for the semisynthesis of insulin variants, insulin hexamethyl ester was digested by trypsin, then the N-terminal amino groups of the resulting desoctapeptide insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the arginyl residue (B22) of this product was coupled to two different series of synthetic peptide methyl esters: I) Gly-OMe, Gly-Phe-OMe, Gly-Phe-Phe-OMe, Gly-Phe-Phe-Tyr-OMe and II) Gly-Ala-OMe, Gly-Phe-Ala-OMe, Gly-Phe-Phe-Ala-OMe, Gly-Phe-Phe-Tyr-Ala-OMe. Removal of all protecting groups yielded the corresponding insulin variants. The syntheses of these peptide methyl esters are described. Following the original prescription of Ruttenberg[5], we were not able to prepare the desired variants. That is why we were forced to change some important details of the Ruttenberg[5] recipe. The activity determinations by the mouse fall test showed the weak activity (ca. 4%) of the desoctapeptide insulin (C-terminus Arg B22). This activity increases drastically in three steps, when the amino acids Phe, Phe, Tyr (B24-26) are added successively to the insulin trunk. Coupling of Gly-Phe yields 14%, -Gly-Phe-Phe 36%, and -Gly-Phe-Phe-Tyr 61% of the biological activity (cryst. insulin=100%). The same peptides, elongated at their C-terminis with an alanyl residues (see above, series II) yield higher activities. Coupling these peptides to the arginyl residue B22 increases the activity as follows: -Gly-Phe-Ala, 36%, -Gly-Phe-Phe-Ala, 59%, and -Gly-Phe-Phe-Tyr-Ala, 91%. Comparing the activities of the variants with the C-termini-Gly-Phe-Phe (36%) and -Gly-Phe-Ala (36%) or -Gly-Phe-Phe-Tyr (61%) and -Gly-Phe-Phe-Ala (59%), it becomes clear that the aromatic amino acids Phe (B25) and Tyr (B26) can be substituted by Ala without loss of activity. In our preceding work (published 1969-1973 [3, 6-8]), we synthesized successively shortened insulin B-chains which yielded, after combination with natural A-chain, practically the same activity values as we have now obtained with the Ruttenberg semisynthesis. As we have already mentioned l.c.[1-4], it is obvious that the activity of insulin proceeds from the arginyl residue (B22) and is only intensified by the aromatic amino acids (B24-26). We[2,3] observed the same three-step increase in activity in the case of our synthetic oligopeptides Arg-Gly-Phe, Arg-Gly-Phe-Phe and Arg-Gly-Phe-Phe-Tyr (B22-26), which we assume to be the active region of insulin (1971[2]).
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PMID:Further studies on the three-step-increase in activity due to the aromatic amino acids B24-26 (-Phe-Phe-Tyr-). 125 46

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

Unprotected porcine desoctapeptide(B23-30) insulin (DOPI) and the synthetic Gly-Phe-Phe were used as substrates for the trypsin-catalyzed synthesis of despentapeptide(B26-30) insulin (DPPI). The DPPI synthesis was accompanied by a moderate oligomerization and by the formation of a side produce which was identified as a DOPI derivative having an extra peptide bond between the Gly(A1) and Arg(B22) and which was named des(23-63) proinsulin (1). Despite side reactions, the conditions were found where the overall DPPI yields were comparable to those obtained via di-Boc DOPI, and these procedures were faster and simpler since the Boc protection and deprotection steps were omitted. The reaction progress was directly monitored by HPLC.
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PMID:Enzymatic semisynthesis of porcine despentapeptide (B26-30) insulin using unprotected desoctapeptide (B23-30) insulin as a substrate. Model studies. 352 39

Semisynthetic analogues of insulin were prepared from derivatives of desoctapeptide-(B23-30)-insulin (DOI). A1, B1-(Boc)2-DOI (di-Boc-DOI) was converted to A1, B1-(Boc)2-DOI-B22-phenylhydrazide (di-Boc-DOI-NHNH-C6H5) by the trypsin-catalyzed addition of phenylhydrazine in aqueous organic solvents at pH 6.5 [Canova-Davis, E., & Carpenter, F. H. (1981) Biochemistry 20, 7053-7058]. Treatment of di-Boc-DOI-NHNH-C6H5 with BNPS-skatole produced the phenyldiimide. The latter was coupled with a variety of protected peptides that, after removal of protecting groups, yielded the following compounds whose biological activities were compared to that of insulin in binding, in stimulation of hexose transport (), and in the stimulation of lipogenesis [)), in terms of percent of insulin activity, all in the isolated epididymal fat cell: di-Boc-DOI 0.2, (0.1), [0.2]; di-Boc-DOI-NHNH-C6H5 0.5, (0.2), [0.5]; DOI 0.2, (0.2), [0.1]; DOI-(Gly)B23 0.2, (0.2), [0.1]; DOI-(Gly-Phe)B23-24 6.3, (6.3), [8.0]; DOI-(Gly-Phe-Phe)B23-25 17.0, (25.6), [24.7]; DOI-(Gly-Phe-Phe-Tyr)B23-26 59.0, (50.0), [69.0]. The semisynthetic derivatives represent a stepwise readdition of the aromatic residues near the C terminus of the B chain. A given analogue demonstrated comparable activity in all three biological assays. The results indicate that the stepwise addition of aromatic residues to the B-chain C terminus of DOI produces an increase in insulin-like activity. The biological activity of DOI-(Gly-Phe-Phe-Tyr)B23-26, the derivative in which the aromatic region has been completely reassembled, is the same order of magnitude as that of insulin.
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PMID:Preparation of semisynthetic insulin analogues from bis(tert-butyloxycarbonyl)-desoctapeptide-insulin phenylhydrazide: importance of the aromatic region B24-B26. 634 Jul 39

The semisynthesis of a series of B23-D-Ala deshexapeptide and despentapeptide analogues with B22-arginine replaced are described. The following semisynthetic scheme is used: desnonapeptide insulin pentamethyl ester is prepared from insulin hexamethyl ester through the action of trypsin and carboxypeptidase B, the amino groups are protected with Boc-groups, the protected product is condensed with synthetic peptides, and the protecting groups are removed with trifluoroacetic acid treatment and by saponification. The biological activities of these analogues indicate that the B22-Arg is non-essential and can be replaced by Lys or even Asp without any influence on the biological activity. The activity is reduced to one half when it is replaced by valine or to a very low level when replaced by Gly or D-Arg. The reason for the low activity of guinea pig insulin is discussed.
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PMID:Effect of B22-arginine replacement on the biological activity of insulin. 702 77

The resealing of a split peptide bond (B22/23) in a crosslinked insulin derivative by a trypsin-mediated coupling reaction in Tris buffer/glycerol/dimethylsulphoxide 1 : 1 : 1 at pH 6.5 is described. The coupling yield obtained with N alpha A1, N epsilon B29-(carbonyl-bis-methionyl) insulin [OC(Met)2] was 55-60%, and reproducible yields of OC(Met)2-insulin were 40%. After removing the crosslink and purification, crystalline resynthesized insulin of high biological activity was obtained in a yield of 43% and an overall yield, based on split OC(Met)2-insulin, of 18%.
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PMID:Intramolecular enzymatic peptide synthesis: trypsin-mediated coupling of the peptide bond between B22-arginine and B23-glycine in a split crosslinked insulin. 702 86