Degradation products of insulin generated by hepatocytes and by insulin protease

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages. Insulin-sized products were obtained by Sephadex G-50 filtration. These insulin-sized products were injected on reverse-phase HPLC, and the peaks of radioactivity were identified. The product patterns generated by the enzyme and by hepatocytes were essentially identical with both isomers. The products were also sulfitolized to prepare the S-sulfonate derivatives of the B-chain and B-chain peptides. Again, the patterns on HPLC generated by the enzyme and by hepatocytes with both isomers were identical. Each of the original product peaks was also sulfitolized and injected separately on HPLC to relate B-chain peptides with product peaks. Again, the peptide compositions of the product peaks for both enzyme and hepatocytes were essentially identical. To identify the cleavage sites in the B-chain of insulin produced by insulin protease, the peptides from the degradation of [125I]iodo(B-26)insulin were purified and submitted to automated Edman degradation to identify the cycle in which radioactivity appeared. Seven peptides with cleavages on the amino side of the B26 residue were identified, and the cleavage sites were determined. Cleavages were found between B-9 and B-10 (Ser-His), B-10 and B-11 (His-Leu), B-14 and B-15 (Ala-Leu), B-13 and B-14 (Glu-Ala), B-16 and B-17 (Tyr-Leu), B-24 and B-25 (Phe-Phe), and B-25 and B-26 (Phe-Tyr). Peptides were also isolated from [125I]iodoinsulin incubated with isolated hepatocytes, and the cleavage sites in several of these were determined. These agreed exactly with the cleavage sites identified generated by the enzyme. The major peptides generated by the degradation of [125I]iodo(B-16)insulin were also isolated and sequenced, again showing identical cleavage sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Processing and presentation of insulin. I. Analysis of immunogenic peptides and processing requirements for insulin A loop-specific T cells

The processing and presentation of insulin by B hybridoma cells to insulin A loop-specific T cell hybridomas was investigated. We found that the activation of these T cells requires insulin to be processed in a manner that permits unfolding of the molecule and prevents extensive proteolysis. An analysis of insulin peptides formed by either enzymatic digestion in vitro or solid phase synthesis revealed that a conformational determinant comprised of residues A1-A14 disulfide-linked to B7-B15 is most immunogenic to these T cells. Reduction and/or proteolysis of this peptide markedly decreases its immunogenicity. The pork insulin A1-A14/B7-B15 peptide differs only at residue A4 from its mouse insulin homolog. Thus, Glu A4 forms part of the antigenic site recognized by a pork insulin/I-Ad-specific mouse T cell. This insulin peptide can be induced to assume an alpha-helical configuration in a hydrophobic environment. In addition, virtually all of the residues of this peptide are identical with those predicted to be situated in amphipathic regions of the native insulin molecule. N-Ethylmaleimide and bacitracin, which inhibit the activity of two cytosolic enzymes that cleave insulin, enhance the antigen presentation of insulin. This suggests that these enzymes may participate in the nonlysosomal antigen processing of insulin by a B lymphocyte. A comparison of the relative avidity of several T cell hybridomas, which have the same apparent specificity for this insulin peptide, showed that an increase in their avidity was associated with a degeneracy in their fine specificity. Our data demonstrate that the efficiency of processing and presentation of a given antigenic determinant is related to the conformation of the determinant and the specificity and avidity of the T cell.     

Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. Sequencing of the B chain fragments demonstrated that the Drosophila enzyme cleaves the B chain of insulin at four sites between residues B10 and B11, B14 and B15, B16 and B17, and B25 and B26. These cleavage sites correspond to four of the seven cleavage sites generated by the mammalian insulin protease. These results demonstrate that all the insulin cleavage sites generated by the Drosophila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function.     

Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes. Insulin degradation products were purified by HPLC and sequenced by automated Edman degradation. Only two A chain cleavage sites were identified, one the previously reported A13-A14 and the other between A14 tyrosine and A15 glutamine. These data thus identify the second A chain cleavage site and further support the role of insulin protease in hepatic metabolism of insulin.     

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.     

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver

The cleavage of insulin by the partially purified insulin protease was studied using the four [¹²⁵I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 > A-14 > A-19 > B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the ‘insulin’ peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the ‘insulin’ peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g. containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).     

FTIR studies of secondary structures of bovine insulin and its derivatives

The amide I bands of the deconvolved FTIR spectrum of bovine insulin, despentapeptide (B26-B30) insulin and desoctapeptide (B23-B30) insulin in D2O solution have been assigned to alpha-helix, the 3(10) helix, irregular helix, extended chains, beta-turns and other secondary structures. From the peak areas the relative contents of these structures obtained are in general agreement with those calculated from the known structures of porcine insulin and DPI in the crystalline state. The main difference in the structure of DOI with those of insulin and DPI is the shortening of the helix segment and an extended chain for the C terminal segment in the B chain.     

Transpeptidation during the analytical proteolysis of proteins

Since peptide mapping with proteolytic enzymes such as trypsin and Staphylococcus aureus V8 protease is a powerful tool for the characterization of proteins, investigators should be cognizant of possible artifacts due to the technique itself. This article describes the identification of minor peaks found in the maps of recombinant human relaxin and insulin-like growth factor I as transpeptidation products. Both proteins have some homology to insulin with relaxin being composed of two chains designated A and B, while insulin-like growth factor I is composed of a single polypeptide chain. Digestion of relaxin with trypsin at pH 7.2 yields two peptides, T2,3(A10-18) and T7(B10-13), linked together by a disulfide bond. An unexpected component at a 10% level was identified to be the T2-T7 peptide pair where T3(ArgA18) has formed a peptide bond with the amino-terminal LeuB10 of the T7 peptide. It was also observed that the digestion of insulin-like growth factor I with V8 protease normally yields two peptides V4(13-20) and V9(59-70) linked by a disulfide bridge. A minor peak at a 1 to 2% level was identified to be a single polypeptide resulting from the formation of a peptide bond between the amino-terminal Met59 of V9 and the carboxyl-terminal Asp20 of V4, with the disulfide bond intact. These transpeptidation products were isolated by reversed-phase HPLC and identified using amino-terminal sequence and mass spectrometric analyses.     

Isolation of insulin degradation products from endosomes derived from intact rat liver

Rats were injected with [125I]iodoinsulin labeled at either the A14 or B26 tyrosine, and the animals were killed and livers subcellularly fractionated to yield light (early or neutral) endosomes and heavy (late or acidic) endosomes. 125I-Labeled material was extracted from endosomes and analyzed by Sephadex G-50 filtration and high performance liquid chromatography (HPLC). Radiolabeled material in both types of endosomes is comprised of high molecular weight, insulin-sized, and low molecular weight components, with B chain-labeled small molecular weight material in two peaks, one corresponding to iodotyrosine and one to small peptides (Mr less than 1500). As compared with A chain label, however, less of the B chain material appears in the degradation components (both high and low molecular weight fractions) suggesting that a fragment of B chain containing the B26 residue is lost from the endosomes. Analysis on HPLC shows that significant amounts of the insulin-sized and high molecular weight material have proteolytic cleavage(s) in the B chain with an intact A chain. The B chain-derived labeled peptides elute from HPLC identically with products generated by insulin protease. These results therefore show substantial insulin degradation occurring in light endosomes prior to endosomal acidification and to receptor dissociation, suggesting receptor-bound insulin is a substrate for insulin protease.     

Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

A novel metalloprotease from Bacillus cereus for protein fibre processing

A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 °C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn³, Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴, Glu²¹, after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P₁ position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a K_m values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool.     

Activity of purified hepatitis C virus protease NS3 on peptide substrates.

The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.     

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.     

T cell autoreactivity to insulin in diabetic and related non-diabetic individuals

T cell autoreactivity to insulin in type I diabetic and related non-diabetic individuals was analyzed. Peripheral T lymphocytes from both insulin-treated diabetic and untreated non-diabetic members of four families were found to proliferate in vitro in response to human insulin. T cell autoreactivity to insulin therefore does not appear to be diagnostic of the onset of type I diabetes. Highest T cell responses to human insulin were usually detected in insulin-dependent type I diabetes patients treated with a mixture of beef and pork insulin than with self insulin, the greater the dose of animal insulin the higher the T cell response. The T cell repertoires for self insulin appear to be similar in diabetics and non-diabetics based on their patterns of T cell reactivity to beef insulin, port insulin, human insulin, and various peptide of human insulin. The autoreactive T cells analyzed recognize two conformational epitopes of human insulin formed by interactions between A chain and B chain residues. One epitope is associated with the A chain loop and is present in the A1-A14/B1-B16 peptide, and the other epitope consists mainly of B chain residues located in the A16-A21/B10-B25 peptide. These two epitopes are present in amphipathic alpha-helical regions of insulin. HLA-DR (DR3, DR4, and DR5) and HLA-DQ (DQw2/DQw3) Ag can restrict these T cell responses to human insulin epitopes. The ability to detect insulin-specific autoreactive T cells in healthy non-diabetic individuals supports the hypothesis that autoreactive lymphocytes do not necessarily elicit autoimmune disease if present in an environment in which their activity is immunoregulated.     

Exogenous adenosine 3': 5'-monophosphate can release yeast from catabolite repression.

A new simple fast and reproducible purification procedure for the proteinase from rat liver mitochondria has been worked out. The specificity of cleavage of peptide bonds in glucagon, oxidized A and B chains of insulin and yeast proteinase B inhibitor by the proteinase of the inner mitochondrial membrane has been studied. The proteinase hydrolyzed three peptide bonds in glucagon, Tyr (13) - Leu (14), Trp (25) - Leu (26) and Phe (22) - Val (23) (minor cleavage site); none in the insulin A chain; one in the B chain of insulin, Tyr (16) - Leu (17); and three in the yeast proteinase B inhibitor, Phe (4) - Ile (5), Phe (20) - Leu (21) and Tyr (41) - Thr (42) (minor cleavage site).Thus, the mitochondrial proteinase cleaves peptide bonds at the carboxyl site of an aromatic amino acid and the amino site of a leucine, isoleucine, threonine or valine. The comparison with chymotrypsin A shows that the mitochondrial proteinase cleaves peptide bonds in a more restricted manner.     

Sequence of plasmin proteolysis at the NH2-terminus of the b beta-chain of human fibrinogen

Employing high-performance liquid chromatography (HPLC), we have isolated and quantified the peptides that are released from the NH2-terminus of human fibrinogen B beta-chains by plasmin proteolysis. The peptides were identified by amino acid composition and by a radioimmunoassay developed for fibrinopeptide B detection. B beta 1-42 was the earliest fragment released during limited plasmin proteolysis. The level of this peptide reached a maximum and then began to decline during the course of the digestion. In addition, increasing levels of B beta 1-21 and of FPB followed the production of B beta 1-42. Using purified B beta 1-42 as a substrate, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components which were separated by HPLC: B beta 1-14 (FPB) and beta 22-42. The rate of cleavage at the 14-15 bond, which is the customary site of thrombin proteolysis, was not affected by the addition of hirudin indicating that this was not the result of trace contamination with thrombin. We have also examined plasmin proteolysis at the NH2-terminal region of the B beta-chains of a variety of fibrinogen derivatives and have found similar patterns of B beta 1-42 release. Using HPLC data, we have estimated the Km for plasmic cleavage of the beta 21-22 bond to be 1.8 X 10(-5) M and of the beta 14-15 bond to be 2.8 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

An insulin analogue with gamma-amino butyric acid substitution for A13Leu-A14Tyr.

An insulin A chain analogue, [A13-14 GABA, A21 Ala]A chain, for which the dipeptide Leu-Try at A13-A14 was substituted by a non-coded amino acid, gamma-amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid-phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13-14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50% in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C-N peptide bond by an unnatural C-C bond may have general meaning in structure and function studies of other proteins.