The sequence of amino acids in insulin isolated from islet tissue of the cod (Gadus callarias)

1. S-Aminoethylcysteinyl derivatives of the A and B chains of cod insulin were prepared from the individual S-sulpho chains. 2. Studies on small peptides derived from the S-aminoethylated peptide chains by treatment with trypsin allowed the amino acid sequences in the region of the cysteinyl residues of the A and B peptide chains to be defined. 3. The six amide groups in cod insulin were located by complete digestion of small peptides from the A and B chains with aminopeptidase followed by amino acid analyses. 4. The results, together with previous studies on the oxidized A and B chains, define the sequences of the 51 amino acids that constitute cod insulin.     

Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding

1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s(20,w) and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD(+)-sulphite complex/subunit but with affinity decreased 10(3)-10(4)-fold. 6. The inhibited enzymes fail to bind C(2) and C(3) molecules to give the ternary complexes enzyme-NAD(+)-pyruvate, enzyme-NADH-oxamate and enzyme-NADH-oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.     

Effects of intravenous injection of butyrate, valerate and their isomers on endocrine pancreatic responses in conscious sheep (Ovis aries)

1. The effects of intravenous injection of n-butyrate, iso-butyrate, n-valerate and iso-valerate on insulin and glucagon secretion was examined in conscious sheep. 2. Each sodium salt of the short chain fatty acids increased plasma insulin and glucagon concentrations in a dose-dependent manner (312-1250 mumol/kg body wt). 3. Both butyrate and valerate isomers with branched carbon chains had larger insulin releasing activity than isomers with straight carbon chains. 4. The glucagon responses to butyrate or valerate did not differ between the isomers with straight carbon chains and those with branched carbon chains. 5. Our results suggest that the receptive mechanism to short chain fatty acids, which may involve the nervous system, differs between the A cell and the B cell in sheep in vivo.     

Intramolecular cross-linking of insulin. Preparation and properties of oxalyl- and malonyl-bis(methionyl) insulin

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.     

The insulin A and B chains contain structural information for the formation of the native molecule. Studies with protein disulphide-isomerase.

It has been shown previously [Tang, Wang & Tsou (1988) Biochem. J. 255, 451-455] that, under appropriate conditions, native insulin can be obtained from scrambled insulin or the S-sulphonates of the chains with a yield of 25-30%, together with reaction products containing the separated A and B chains. The native hormone is by far the predominant product among the isomers containing both chains. It is now shown that the presence of added C peptide has no appreciable effect on the yield of native insulin. At higher temperatures the content of the native hormone decreases whereas those of the separated chains increase, and in no case was scrambled insulin containing both chains the predominant product in the absence of denaturants. Both the scrambling and the unscrambling reactions give similar h.p.l.c. profiles for the products. Under similar conditions cross-linked insulin with native disulphide linkages can be obtained from the scrambled molecule or from the S-sulphonate derivative with yields of 50% and 75% respectively at 4 degrees C, and with a dilute solution of the hexa-S-sulphonate yields better than 90% can be obtained. The regenerated product is shown to have the native disulphide bridges by treatment with CNBr to give insulin and by the identity of the h.p.l.c. profile of its peptic hydrolysate with that for cross-linked insulin. It appears that the insulin A and B chains contain sufficient information for the formation of the native molecule and that the role of the connecting C peptide is to bring and to keep the two chains together.     

Application of a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate for detection of cystine-containing peptides

A simple, sensitive and reliable method for the detection of cystine-containing peptides has been developed. A peptide bridged with a disulfide bond was reduced and cleaved with tributylphosphine, and then coupled with a thiol specific reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, under alkaline conditions. After incubation at 60 degrees C for 1 h, the fluorescent derivative formed was measured with excitation at 385 nm and emission at 515 nm. The intensity of fluorescence labeled to the peptide was very stable and the peptide containing disulfide was quantitatively determined in the range of 100 pmol to 10 nmol, when oxidized glutathione was used as a standard. This method was very useful for specific detection of cystine-containing peptides in the column effluents on reverse phase high performance liquid chromatography.     

2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations.

S-2-Sulphobenzylcysteine and S-2-(sulphomethyl)benzylcysteine are prepared by alkylation of cysteine with omega-toluenesultone and 2,3-benzo-1,4-butanesultone respectively. Owing to the presence of the sulphonic acid group, these protected cysteine derivatives are extremely water-soluble and are stable to acid hydrolysis. The groups can be removed by treatment with sodium in liquid NH3. Reduction with tributylphosphine and simultaneous alkylation of insulin with toluenesultone under mild conditions (pH 8.3, aq. 50% propanol) gives the fully S-substituted derivatives in excellent yield; they can be separated by isoelectric precipitation of the S-sulphobenzylated B-chain. Treatment of the latter with sodium in liquid NH3 led simultaneously to the removal of the protecting groups and to the well-documented cleavage at the threonine-proline bond which can be prevented by addition of sodium amide. When deprotected A-chain was recombined with B-chain, insulin was isolated in the same yield and with the same degree of biological activity as that in the control experiment.     

Vapor-phase modification of sulfhydryl groups in proteins

Proteins and peptides are readily and specifically modified at their sulfhydryl groups by the vapors of a mixture of 4-vinylpyridine and tributylphosphine. The phenylthiohydantoin derivative of S-beta-(4-pyridylethyl)cysteine formed during sequence analysis is easily detectable in current identification systems.     

Photoaffinity labeling of insulin receptor of rat adiopocyte plasma membrane

A photosensitive insulin derivative was synthesized by reacting radioactive iodinated bovine insulin with N-hydroxysuccinimide ester of 4-azidobenzolic acid. The photo-sensitivity and specificity of this insulin derivative were established by its covalent nonspecific cross-link to albumin and its covalent specific cross-link to the heavy and light chains of anti-insulin immunoglobulin. Plasma membrane preparations of rate adipocytes were incubated with the photosensitive insulin derivative and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization with sodium dodecyl sulfate and reduction with beta-mercaptoethanol showed that a protein having a molecular weight of 130,000 was specifically labeled by the radioactive photosensitive insulin, suggesting that this protein may be the insulin receptor.     

Kinetic analysis of the mechanism of insulin degradation by glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase).

Kinetic studies have been made with glutathione-insulin transhydrogenase, an enzyme which degrades insulin by promoting cleavage of its disulfide bonds via sulfhydryl-disulfide interchange. The degradation of 125I-labeled insulin by enzyme purified from beef pancreas was studied with various thiol-containing compounds as cosubstrates. The apparent Km for insulin was found to be a function of the type and concentration of thiol; values obtained were in the range from 1 to 40 muM. Lineweaver-Burk plots for insulin as varied substrate were linear, whereas those for the thiol substrates were nonlinears: the plots for low molecular weight monothiols (GSH and mercaptoethanol) were parabolic; those for low molecular weight dithiols (dithiothreitol, dihydrolipoic acid, and 2,3-dimercaptopropanol) were apparently linear modified by substrate inhibition; and the plots for protein polythiols (reduced insulin A and B chains and reduced ribonuclease) were parabolic with superposed substrate inhibition. The nonparallel nature of the reciprocal plots for all substrates shows that the enzyme does not follow a ping-pong mechanism. Product inhibition studies were performed with GSH as thiol substrate. Oxidized glutathione was found to be a linear competitive inhibitor vs. both GSH and insulin. The S-sulfonated derivative of insulin A chain was also linearly competitive vs. both substrates. Inhibition by S-sulfonated B chain was competitive vs. insulin; the data eliminated the possibility that this derivative was uncompetitive vs. GSH. Experiments with the cysteic acid derivatives of insulin A and B chains similarly excluded the possibility that these were uncompetitive vs. either substrate. These inhibition studies indicate that the enzyme probably follows a randdom mechanism.     

Tin-119 NMR studies on diorganoyltin(IV)dihalides and triorganoyltin(IV)halides; Formation and stereochemistry of adducts

The reactions of R₂SnX₂ (R = Ph, Me; X = Cl, Br) with excess halide, tributylphosphine, tricyclohexylphosphine and tributylphosphine oxide have been investigated in dichloromethane solution by tin-119 and phosphorus-31 NMR techniques. R₂SnX₂ form five coordinate 1:1 adducts with halide and phosphine (phos) ligands whilst both 1:1 and 1:2 adducts are formed with tributylphosphine oxide (L). Tin-119 spectra imply that Ph₂SnX₂(phos) has the phosphine in the equatorial position of a trigonal bipyramid. At low temperature there is evidence for a slow intramolecular twist mechanism between octahedral isomers of Ph₂SnCl₂L₂. The stereochemistry of the complexes Ph₂SnX₂L₂ differ between chloro and bromo compounds and no mixed halide complex is observed. In the case of the bromo system only, the 1:3 adduct [Ph₂SnBrL₃]⁺Br⁻ is formed. Ph₃SnCl does not react with phosphines but it does give 1:1 adducts with Cl⁻, L and pyridine. All the adducts have similar tin-119 chemical shifts which is consistent with the phenyl groups being equatorial in the five coordinate trigonal bipyramidal adducts. Ph₄Sn does not form adducts with X⁻, L or phosphine.     

Interaction and reconstitution of carboxyl-terminal-shortened B chains with the intact A chain of insulin

With the S-(thiomethyl)-A chain and despentapeptide (26-30) and desoctapeptide (23-30) S-(thiomethyl)-B chains of insulin at pH 10.8 and a molar ratio of A/B = 1.5, difference spectra of the mixed against the separated chains with negative peaks at 245 and 295 nm and a weak positive peak at 278 nm indicate interaction of the chains leading to Tyr environmental changes as in the case for the intact chains. With the shortened B chains, freshly dissolved from lyophilized powders, it takes some 2 h for the difference spectra to approach completion whereas with the solutions of the shortened B chains left standing overnight at pH 10.8 and 4 degrees C the difference spectra, similar in shape to that described above, appear almost immediately after mixing. Solvent perturbation with 20% ethylene glycol suggests some ordered structure for the despentapeptide but not for the desoctapeptide B chain. The interactions of the A chain with the shortened B chains appear to be weaker as compared to that with the intact B chain as shown by decreasing reconstitution yields for the intact, despentapeptide, and desoctapeptide B chains respectively with the A chain. The above results indicate that the C-terminal portion of the B chain is important not only for the activity of insulin but also for the correct pairing of the chains.     

Number of ways of joining SH groups to form multi-peptide chain proteins

The expression for the total number of isomeric structures formed upon oxidation of SH groups has previously been correctly obtained for single chain proteins only. Expressions have now been obtained for molecules consisting of 2 and 4 peptide chains of the types A2 and A2B2. The latter type is particularly important as exemplified by the immunoglobulins and the insulin receptor. For the oxidation of insulin A chain with 4 SH groups, the total number of isomeric A2 structures is 59--this is different to all the values previously reported. Lack of consideration of symmetry problems probably accounts for the erroneous results obtained by earlier workers for the number of ways of randomly joining two identical chains. The total number of isomeric structures formed from the oxidation of two light and two heavy chains with 5 and 11 SH groups respectively of the human immunoglobulin GI has been found to be 4.8 X 10(16).     

Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.     

Post-translational acquisition of insulin binding activity by the insulin proreceptor. Correlation to recognition by autoimmune antibody

The post-translational acquisition of ligand binding activity by the insulin receptor was examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S] methionine, labeled receptor species were separated into "active" and "inactive" forms by affinity chromatography on insulin-agarose and then were characterized and quantitated. It was found that the newly translated high molecular weight proreceptor lacks the capacity to bind insulin. The acquisition of binding activity is relatively slow (t1/2 = 45 min) and occurs prior to conversion of the proreceptor to the mature alpha- and beta-subunits by proteolytic cleavage and maturation of its N-linked oligosaccharide chains (t1/2 = 3 h). Glycosylation appears to be required for this activation since the aglycoproreceptor, synthesized in the presence of tunicamycin, does not acquire insulin binding activity. However, once the proreceptor has acquired ligand binding activity, removal of its N-linked oligosaccharide chains with endoglycosidase H has no effect on the ability of the proreceptor to bind insulin. The modification of the proreceptor to bind insulin. The modification of the proreceptor that gives rise to insulin binding activity most likely involves a conformational change in the binding domain. A human autoimmune antibody that recognizes only the active insulin binding site does not interact with the inactive proreceptor, whereas a rabbit polyclonal antireceptor antibody recognizes all forms. Thus, the autoimmune antibody must recognize a new epitope created during conversion of the inactive proreceptor to the active form.     

Implications of invariant residue LeuB6 in insulin-receptor interactions

By the chemical synthesis of modified insulin B chains and the combination of the synthetic B chains with natural insulin A chains, we have prepared insulin analogs with natural and unnatural amino acid replacements of invariant residue LeuB6. Analogs have been investigated by reference to their potencies for interaction with the insulin receptor (as assessed by competition for 125I-labeled binding to isolated canine hepatocytes) and to their abilities to undergo the structural transitions that are characteristic of insulin self-aggregation (as assessed by the spectroscopic analysis of analog complexes with cobalt). Our results identify that (a) replacement of LeuB6 by glycine has nearly the equivalent effect as deletion of residues B1-B6 in decreasing receptor binding potency of the analog to only about 0.05% of that of insulin; (b) relative to the GlyB6 derivative, replacements that increase the relative hydrophobicity of the residue B6 side chain also increase the relative receptor binding potencies of the resulting analogs; (c) negative steric effects resulting from substitutions by valine, phenylalanine, and gamma-ethylnorleucine limit the potential for enhancing potency as the result of increased hydrophobicity; and (d) two analogs with disparate potency for receptor interaction (those with alanine and gamma-ethylnorleucine at position B6, analogs exhibiting about 1 and 48% of the potency of insulin, respectively) undergo the T6----R6 structural transition in the presence of Co2+ and phenol which is typical of insulin but result in hexameric complexes with greatly reduced stability. We conclude that leucine provides a closely determined best fit at insulin position B6, and we discuss our findings in terms of insulin conformations that may apply to the receptor-bound state of the hormone.     

A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis

A procedure is described which provides high yields of pyridylethylated cysteine during gas-phase sequencing of peptides. The method decreases transfer losses by reducing the number of transfer steps required for reduction, alkylation, prior to sequencing a peptide. Proteins bound to polybrene-coated glass-fiber filters used in a gas-phase sequenator may be reduced, pyridylethylated, and sequenced directly on the filter. Reduction of cystine-containing peptides is performed using the nonnucleophilic reductant, tributylphosphine [U. T. Rüegg and J. Rudinger (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 47, pp. 111-116, Academic Press, New York] with concomitant alkylation by 4-vinylpyridine in a buffer soluble in the organic phase. Excess reagents and the buffer are removed by drying the filter and briefly washing with chlorobutane. No N-alkylation is apparent under the conditions described, nor are any amino acid side chains modified. The procedure affords high yields and is particularly useful when subnanomole levels of material must be reduced and alkylated.     

In vitro processing of insulin for recognition by murine T cells results in the generation of A chains with free CysSH.

Studies on the processing of insulin as an Ag for the presentation to MHC class II-restricted T cells revealed that the amino acid residues 1-14 of the insulin A chain are recognized by insulin-specific T cells. An A1-14 peptide containing three cys-residues that were protected by S-sulfonate groups still needed processing by APC for efficient presentation similar to native insulin. We suspected that reductive deblocking or opening of disulfide bonds that generates CysSH-residues may be an essential processing step for these Ag. Due to the instability of SH-groups it was not possible to test A chain peptides with free SH-groups in the usual way for processing-independent presentation by fixed APC. However, under acidic conditions (pH 5) during APC pulsing with the Ag we could demonstrate that the freshly reduced A1-14 fragment as well as reduced insulin are able to bind to Ia Ag and to stimulate appropriate T cells without further processing. Various substitutions of cys-residues by Ser within this peptide revealed that only CysA7 is critical for Ia binding and/or T cell recognition. In intact insulin, this residue links the A chain containing the T cell epitope to the B chain. Therefore, we propose that insulin processing is not dependent on proteolysis or on the generation of a conformational determinant but on the separation of A and B chains resulting in A chains whose cys-residues are converted into CysSH.     

Low resolution crystal structure of hagfish insulin

Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P4₁2₁2 with a monomer in the asymmetric unit. The application of the Rossmann &; Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 Å resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.