The bifunctional reagents, oxalyl-(Met-ONp)2 and malonyl-(Met-ONp)2 have been prepared and investigated as reversible cross-linking reagents for insulin and model compounds. The removal of the cross-linking residues was demonstrated by the cyanogen bromide cleavage of oxalyl-(Met-Phe-OMe)2 and malonyl-(Met-Phe-OMe)2. Zinc-insulin reacted with a molar equivalent of oxalyl-(Met-ONp)2 or malonyl-(Met-ONp)2 in presence of excess triethylamine to yield oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin, respectively. In these derivatives the N-terminal phenylalanine (B1 residue) was free. Thus the cross-link was between A1 and B29 residues in insulin. All three disulfide bonds of these insulin derivatives undergo reduction with tributylphosphine to give six sulfhydryls. Air-oxidation of reduced oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin in 0.05 M disodium phosphate, pH 9.5, yielded products which were indistinguishable from oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin respectively, as measured by physicochemical and biological methods. Cyanogen bromide cleavage of reduced and reoxidized malonyl-(Met)2-insulin in 70% formic acid regenerated insulin quantitatively, but only 40% of insulin was determined from similar treatment of oxalyl-(Met)2-insulin. The regenerated insulins exhibited the biological activity of native insulin. These studies strongly suggest that disulfide bonds formed during oxidation of reduced oxalyl-(Met)2-insulin and malonyl-(Met)2-insulin are identical to those found in insulin.

Leucine aminopeptidase (LAP) is a protease requiring two divalent metal cations per subunit for activity. Zn2+, Mg2+, and Co2+ metal-substituted forms of LAP have been prepared and investigated kinetically. Substitution of metal into the two binding sites independently resulted in the preparation of Zn2+Zn2+, Mg2+Zn2+, Co2+Co2+, Zn2+Co2+, Mg2+Co2+, and Co2+Zn2+ LAP derivatives that were characterized by atomic absorption spectrophotometry. Kinetic analysis of the metal-substituted enzymes indicated that site 1 (fast exchanging) metal substitution results in a Km decrease in the relative order Zn2+ greater than Mg2+ greater than Co2+. Similar comparisons for the site 2 metal (slow exchanging) involved only Zn2+ and Co2+, since only these metals have been shown to compete effectively for this site. Substitution of these two metals into site 2 revealed a Km decrease in the order Zn2+ greater than Co2+. It was suggested previously [e.g., Thompson, G. A., & Carpenter, F. H. (1976) J. Biol. Chem. 251, 1618-1624] that the fast-exchanging site 1 metal predominantly effects kcat while the slow-exchanging metal in site 2 exerts effects exclusively on Km. The present study, the first direct comparison of Km change resulting from metal substitution into both sites, clearly indicates that both metal sites exert significant effects on Km. In addition, the data suggest a more complex interaction between the two bound metals than previously suspected.

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn2+-Mg2+ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The Km and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than Km, Δϵ at 295 nm of 2000 m−1 cm−1, and V of 300 μmol min−1 mg−1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with Ki values approximately their Km values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the Km values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of Km values. Although the Km values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the Km for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.

ADVERTISEMENT RETURN TO ISSUEPREVArticleSemisynthesis of insulin: specific activation of the arginine carboxyl group of the B chain of desoctapeptide-(B23-30)-insulin (bovine)Eleanor Canova-Davis and Frederick H. CarpenterCite this: Biochemistry 1981, 20, 24, 7053–7058Publication Date (Print):November 1, 1981Publication History Published online1 May 2002Published inissue 1 November 1981https://doi.org/10.1021/bi00527a044RIGHTS & PERMISSIONSArticle Views80Altmetric-Citations9LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts

A modified procedure for the preparation of the S-sulfonates of the A- and B-chains of insulin and their conversion to the sulfhydryl forms by tri-n-butylphosphine is described. Air oxidation of the sulfhydryl forms of the A-chain in dilute solution (0.2 mg/ml) either in the presence or absence of urea at pH 9.0 yields primarily monomeric, intrachain disulfides. Similar treatment of the reduced B-chain yield monomeric, intrachain disulfide in 7 M urea but a large number of oligomeric, interchain disulfides in the absence of urea. Electrolytic reduction of insulin in 7 M urea of pH 8.5, followed by oxidation of the sulfhydryls in dilute solution in 7 M urea at pH 9.0 yields primarily a mixture of the monomeric, intrachain disulfides of the A-chain and of the B-chain which can be separated by chromatography on Sp-Sephadex in acidic urea. The rate of the oxidation of the sulfhydryls of the two separate chains was much slower and less complete than that reported for the two chains crosslinked by the carbonylbismethionyl residue.

The quaternary structure of leucine aminopeptidase was studied by electron microscopy of negatively stained single molecules and of both stained and unstained thin sections of crystals. The predominant images observed in micrographs of single molecules were triangles, many of which had a more dense triangle inset into the outer triangle and rotated by 60° from it. Measurements performed on electron micrographs of thin sections of the crystals cut parallel to the (0001) and (1120) planes gave unit cell parameters of a = 120 Å (±10 Å) and c = 110 Å (±15 Å). A quaternary structure composed of six asymmetric bilobal subunits arranged so that the principal lobes are eclipsed and the minor lobes are staggered in a manner similar to that of aspartate carbamoyltransferase is the model that best explains the images observed.

The preparation of a series of X-Met-Gly-OEt and X-Met-Phe-OMe and their treatment with CNBr in either 70% or 97-100% formic acid at 25 degrees C are described where X is methanesulfonyl (mesyl), p-nitrobenzyloxycarbonyl, phthaloyl, trifluoroacetyl, acetyl, formyl, or tert-butyloxycarbonyl. Total cleavage of the peptide esters was found with mesyl-, p-nitrobenzyloxycarbonyl-, phthaloyl-, and trifluoroacetylmethionyl derivatives which indicated the suitability of these derivatives as amino protecting groups in peptide synthesis. Treatment of the acetylmethionyl peptide esters with CNBr in 70 and 97-100% formic acid resulted in 92 and 98% cleavage, respectively. With formylmethionyl peptide esters, about 85-95% cleavage was estimated when either 70 or 97-100% formic acid was used as the solvent. With the tert-butyloxycarbonylmethionyl derivatives, CNBr treatment in 70% formic acid resulted in about 93% cleavage of peptides, while treatment in 97-100% formic acid led to only 30-33% release of C-terminal amino acid esters. Quantitative cleavage of the carbonylbis(methionyl peptide esters) was observed. The reaction of CNBr with N-terminal methionyl derivatives containing free alpha-amino groups revealed that free methionine was quantitatively converted to homoserine lactone, whereas methionine ethyl ester and methionyl peptides (Met-Gly and Met-Phe) disappeared from the reaction mixture in 70% formic acid with only partial splitting of the ester (16%) or peptide bond (45%).

Bovine lens leucine aminopeptidase (EC no. 3.4.1.1) crystallizes in the hexagonal space group P6322 with unit cell dimensions a = 132 Å and c = 122 Å. The asymmetric unit consists of one protomer of molecular weight 54,000. The 32 point group symmetry of the hexameric molecule establishes the presence of two types of isologous interactions between dyad-related subunits.

The preparation and use of carbonylbis (L-methionine p-nitrophenyl ester) as a reversible cross-linking reagent for insulin are described. The reaction of 1 equiv of reagent with zinc insulin in dimethylformamide in the presence of triethylamine yields as one of the products NalphaA1, NepsilonB29-carbonylbis(methionyl)insulin, (CBM-insulin). The CBM-insulin was characterized by end group analysis and by the products formed on tryptic and chymotryptic cleavage. It possessed 91% of the immunological and 6.5% of the hormonal activity of insulin. Treatment of CBM-insulin with cyanogen bromide (CNBr) in 70% formic acid for 1 h resulted in nearly complete removal of the methionine bridge to yield insulin. A small amount of a side product was removed on DEAE-cellulose at pH 7.2 to give an overall recovery of insulin of 70-80%. Oxidative sulfitolyses of CBM-insulin gave the hexa(S-sulfonate) which was reduced with dithiothreitol to yield reduced CBM-insulin. The latter compound, containing 6 sulfhydryls, exhibited a pH-dependent circular dichroic spectrum. The form at pH 10 exhibited a spectrum typical of random coil which was converted to a form at pH 7.8 which was characterized by a negative extremum at 213 nm. The change in the spectrum at 213 nm with pH was characterized by an apparent pKa of 8.5. Studies on the reoxidation of reduced CBM-insulin were performed at pH values between 7.8 and 10 and at protein concentrations of 0.01-1 mg/ml. The best yields (ca. 85%) of the correctly paired disulfide bonds were obtained in reoxidations at pH 9.5-10 at protein concentration of 0.01-0.1 mg/ml. CBM-insulin, which had been isolated from reoxidation at high pH of the reduced CBM-insulin, was cleaved by CNBr to yield a fully active insulin in an overall yield of 60% from the reduced CBM-insulin.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.

Incubation of leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with various concentrations of Mg2+ at various pH values in 1 M KCl and 0.155 M trimethylamine-HCl at 37 degrees confirms that Mg2+ competes with Zn2+ for binding only 1 site per 54,000-dalton subunit. The ratio of the apparent association constants (1KZn:1KMg = 1KZn/Mg) at this site (site 1) was estimated to be 20,720 at pH 8.16, 10,570 at pH 8.44, 3,590 at pH 8.78, and 660 AT PH 9.14. The decrease in values of 1KZn/Mg with increasing pH in the activation of leucine aminopeptidase by Mg2+ is attributed to the lowering of the free Zn2+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH)2 complexes with increasing OH- concentration. When corrections are made for the binding of Zn2+ by OH- ions, the pH-independent ratio of association constants (1KZn:1KMg = 1KZn/Mg) for the relative binding of Zn2+ and Mg2+ at site 1 of leucine aminopeptidase in 29,800. From the effect of pH on the relative binding constant, a value (beta2) for the product of the two stepwise association constants for the formation of Zn(OH)2 from Zn2+ and OH- (Zn2+ + OH- in equilibrium ZnOH+; ZnOH+ + OH- in equilibrium Zn(OH)2) was estimated to be 4.42 X 10(10) M-2 at 37 degrees. Values of Km at pH 7.5 AND 30 degrees with L-leucine p-nitroanilide as substrate in the presence of 0.01 M NaHCO3 are 4.13 and 2.01 mM for the zinc-zinc and magnesium-zinc enzymes, respectively. Values for Vmax are 0.2 and 2.49 mumol/min/mg, respectively.

Chemischer InformationsdienstVolume 5, Issue 46 Natural Products ChemInform Abstract: CARBONYLBIS(L-METHIONINE P-NITROPHENYLESTER), A NEW REAGENT FOR THE REVERSIBLE INTRAMOLECULAR CROSS-LINKING OF INSULIN WOLF-DIETER BUSSE, WOLF-DIETER BUSSESearch for more papers by this authorFREDERICK H. CARPENTER, FREDERICK H. CARPENTERSearch for more papers by this author WOLF-DIETER BUSSE, WOLF-DIETER BUSSESearch for more papers by this authorFREDERICK H. CARPENTER, FREDERICK H. CARPENTERSearch for more papers by this author First published: November 19, 1974 https://doi.org/10.1002/chin.197446453AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume5, Issue46November 19, 1974 RelatedInformation

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCarbonylbis(L-methionyl)insulin. Proinsulin analog which is convertible to insulinWolf D. Busse, Susan R. Hansen, and Frederick H. CarpenterCite this: J. Am. Chem. Soc. 1974, 96, 18, 5949–5950Publication Date (Print):September 1, 1974Publication History Published online1 May 2002Published inissue 1 September 1974https://doi.org/10.1021/ja00825a050Request reuse permissionsArticle Views38Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (254 KB) Get e-Alertsclose Get e-Alerts

Leucine aminopeptidase (L-leucyl-peptide hydrolase EC 3.4.1.1) was shown to catalyze the exchange of oxygen-18 into and out of L-leucine. Incubation of L-leucine at 30° with Mg2+-activated leucine aminopeptidase in a solution that was enriched in oxygen-18 containing water for 24 h at pH 7.0 resulted in a substantial increase in the oxygen-18 content of the L-leucine as compared with blanks. When L-leucine, enriched with oxygen-18, was treated similarly with leucine aminopeptidase, a significant decrease in the oxygen-18 content occurred as compared with blank solutions and a heat-denatured leucine aminopeptidase control.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCyanogen bromide treatment of N-acetylmethionyl residues without cleavageFrederick H. Carpenter and Stanley M. ShiigiCite this: Biochemistry 1974, 13, 25, 5159–5164Publication Date (Print):December 1, 1974Publication History Published online1 May 2002Published inissue 1 December 1974https://doi.org/10.1021/bi00722a017RIGHTS & PERMISSIONSArticle Views105Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (685 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCarbonylbis(L-methionine p-nitrophenyl ester). New reagent for the reversible intramolecular crosslinking of insulinWolf D. Busse and Frederick H. CarpenterCite this: J. Am. Chem. Soc. 1974, 96, 18, 5947–5949Publication Date (Print):September 1, 1974Publication History Published online1 May 2002Published inissue 1 September 1974https://pubs.acs.org/doi/10.1021/ja00825a049https://doi.org/10.1021/ja00825a049research-articleACS PublicationsRequest reuse permissionsArticle Views74Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTZinc binding, circular dichroism, and equilibrium sedimentation studies on insulin (bovine) and several of its derivativesJose Goldman and Frederick H. CarpenterCite this: Biochemistry 1974, 13, 22, 4566–4574Publication Date (Print):October 1, 1974Publication History Published online1 May 2002Published inissue 1 October 1974https://pubs.acs.org/doi/10.1021/bi00719a015https://doi.org/10.1021/bi00719a015research-articleACS PublicationsRequest reuse permissionsArticle Views808Altmetric-Citations147LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts