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H G Gattner

Publications and source records attributed to H G Gattner.

At least 37 records · Page 2Linked to original sources
PubMed1983

The synthesis of [A 19-3-iodotyrosine] and [A 19-3,5-diiodotyrosine]insulin (porcine).

The chemical synthesis of [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), using the amino-acid derivatives 3-iodotyrosine and 3,5-diiodotyrosine is described. The synthesis of the iodinated A-chains were performed by segment condensation in solution using acid labile protecting groups. The hydroxyl groups of Tyr(I) and Tyr(I2) were unprotected. For the temporary protection of the alpha-amino groups of the A-chain segments containing iodinated tyrosines, the 1-(4-biphenylyl)-1-methylethoxycarbonyl group was selected. After deprotection and sulphitolysis the iodinated A-chain tetra-S-sulphonates were purified by ion exchange chromatography on DEAE cellulose at pH 5.6. Reduction to the sulphhydryl form and the combination with native porcine B-chain yielded [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), respectively. Purification of the first product was achieved by gel filtration and of the later by ion exchange chromatography on CM-cellulose at pH 4.5 and gel filtration. The monoiodinated insulin had a biological activity of 24 +/- 2% and the diiodinated analogue 2.6 +/- 0.2% as determined in an in vitro lipogenesis assay with epididymal adipocytes.

Animals↗
PubMed1983

[B17-D-leucine]insulin and [B17-norleucine]insulin: synthesis and biological properties.

The chemical synthesis of two porcine insulin analogues is described. Leucine in position B17 of the native molecule was substituted by its D-enantiomer and by L-norleucine, respectively. Both B-chain derivatives were synthesized by fragment condensation and purified as di-S-sulphonates by gel filtration followed by ion exchange chromatography on SP-Sephadex at pH3. Combination with native sulphhydryl A-chain yielded [DLeuB17]insulin and [NleB17]insulin. Both insulin analogues were isolated by gel filtration followed by ion exchange chromatography on CM-cellulose at pH 4.0. Biological activities of the analogues were determined relative to native pork insulin: 1) glucose oxidation in rat epididymal adipocytes was 6% for [DLeuB17]insulin and 16% for [NleB17]insulin, 2) receptor-binding affinity tested with cultured human fibroblasts and with rat adipocytes was 3% for [DLeuB17]insulin and 26% for [NleB17]insulin, and 3) thymidine incorporation into DNA of human fibroblasts was 35% for [DLeuB17]insulin and 100% for [NleB17]insulin.

Adipose Tissue↗
PubMed1983

[Increased yields of insulin by recycling of non-combined insulin chains].

The yield of insulin by the combination of reduced A and B chains is increased from 15% to 33-34% if the non-insulin-containing fractions of the gel filtration are isolated and used for further combinations (recycling). Crude insulin recovered by gel filtration can be highly purified by preparative high-performance liquid chromatography. Lower yields of insulin were obtained on recombination of chains which had been recycled five times or more. Sulfur analyses show a loss of cystine sulfur. The occurrance of lanthionine and lysinoalanine indicates an alkaline destruction of cystine/cysteine during the repeated combination steps.

Amino Acids↗
PubMed1982

Xid-defective male (CBA/N X C57BL/6)F1 accessory cells present bovine insulin to long-term cultured F1-restricted T-cells.

The reactivity of H-2b-restricted murine T cells towards bovine insulin was reported to depend on the expression of Ia. W39, a private specificity of I-Ab, on antigen-presenting cells. Cells of male (CBA/N X B6)F1 mice carrying the mutation xid on the X chromosome lack IA. W39 on the cell surface. These cells are unable to present bovine insulin to primed T cells derived from female (CBA/N X B6)F1 mice. We show here that spleen cells of male (CBA/N X B6)F1 hybrids served perfectly as accessory cells for the insulin-dependent induction of a proliferative response of long-term cultured T cells with (B10 X B10.BR)F1 genotype, restricted to recognizing insulin in the context of F1-unique I-A determinants.l The epitope on the insulin molecule essential for stimulation was determined to depend on the glutamic acid residue in position 4 of the A chain of insulin. This contracts with the H-2b-restricted response of B6 mice to bovine insulin, which appears to be directed at the A chain loop determinant (amino acids A8 and A10). These data suggest that distinct I-Ab-encoded structures, the expression of which is regulated independently, may serve as components of restriction elements for H-2b and (H-2b X H-2k)F1 restricted T cells, which are specific for different epitopes of bovine insulin.

Animals↗
PubMed1982

Preparation of partially protected des-alanineB30-insulin-B-chain-disulphide and its use in semisynthesis.

The preparation of N alpha 1 N epsilon 29-bis-methylsulphonylethyloxycarbonyl-des-alanineB30-B-chain-disulphide-O gamma 13 O gamma 21-dimethyl ester is described. Starting with the di-S-sulphonate-B-chain it was prepared by i) removal of the C-terminal amino acid by digestion with carboxypeptidase A, ii) reduction and oxidation to form the cyclic disulphide, iii) esterification of the three carboxyl groups (alpha COOH = LysB29, gamma COOH - GluB13 and GluB21) followed by selective tryptic hydrolysis of the alpha-carboxyl ester, and iv) the protection of both amino groups (alpha NH2 = PheB1, epsilon NH2 = LysB29). This B-chain derivative was activated selectively at the C-terminal carboxyl group by formation of a mixed anhydride, and condensed with the dipeptide Thr-Arg. All protecting groups were removed by treatment with alkali. The human-B-chain C-terminal elongated with Arg was obtained in a yield of 30% based on the protected des-AlaB30-B-chain derivative.

Animals↗
PubMed1982

[LeuB24]- and [LeuB25]insulins are not antagonists of lipogenesis in adipocytes.

Semisynthetic human [LeuB24]-and [LeuB25]insulins were investigated to determine whether they show antagonistic properties towards insulin-stimulated lipogenesis in isolated fat cells. In contrast to other reports, we could detect only an additive agonistic effect when constant concentrations (e.g. 0.3 ng/ml) of the analogues were mixed with varying concentrations of insulin, or when constant concentrations (e.g. 0.3 ng/ml, 0.6 ng/ml) of insulin were mixed with varying concentrations of the analogues. Similar results were obtained with mixtures of insulin and NA2-acetyl- or NA2-propionyl-des-GlyA1-insulins. These results do not support the contention that a diabetic state could be caused by either of these mutant human insulins.

Adipose Tissue↗
PubMed1982

Preparation and properties of citraconylinsulins.

Acylation of insulin with citraconic anhydride was studied at different pH values. The controlled acylation at pH 8.5 and 7 yielded mainly A1-citraconylinsulin (41%) and A1,B1-dicitraconylinsulin (39%), respectively. Acylation with excess reagent at pH 5.6, followed by partial deblocking at pH 5 for 30 min and 17 h, led mainly to A1,B1-dicitraconylinsulin (51%) and B1-citraconylinsulin (40%), respectively. The order of the deblocking rates for the three citraconyl groups at pH 3.5 and 5 was B29 much greater than A1 greater than B1. The biological activity of citraconyl derivatives: A1-citraconyl, B1-citraconyl and A1,B1-dicitraconylinsulin were found to be 15%, 100% and 15% (in vitro fat cell assay) and 46%, 78% and 48% (in mouse convulsion assay), respectively. In a double insulin immunoassay these derivatives had 40%, 75% and 30% immunoreactivity, respectively.

Adipose Tissue↗
PubMed1982

[B22-D-arginine]insulin: synthesis and biological properties.

An analogue of porcine insulin which differs from the native molecule in that the amino-acid residue B22-L-arginine is replaced by its D-enantiomer has been synthesized. The [D ArgB22]B-chain was synthesized by the segment condensation method and purified as the di-S-sulfonate by ion exchange chromatoggraphy on SP-Sephadex at pH 3.5. Combination with native porcine sulfhydryl A-chain gave [DArgB22]insulin which was purified by ion exchange chromatography on SP-Sephadex at pH 4.5 with a linear NaCl gradient. The biological activity of this analogue as measured by glucose oxidation in rat epididymal adipocytes was 2%. Thymidine incorporation into DNA of human fibroblast was 16%. The immunoreactivity using antipork insulin antibody in a double antibody immunoassay was 4%. The receptor-binding affinity as measured by radioreceptor assays was 2% with cultured human fibroblasts and 1% with rat adipocytes. These results suggest that the L-configuration at B22-arginine is essential for retaining the biological, immunological and receptor-binding properties of the hormone.

Adipose Tissue↗
PubMed1981

[Protein semisynthesis with the help of mixed anhydrides and enzymes: chemistry and synthesis of insulins].

Proteins play a prominent role in nature and their biosynthesis occurs via stepwise combination of amino acids. One can imitate this method in laboratory or synthesize the polypeptide chain by combining smaller preformed fragments (fragment condensation). Reversible protection of reactive groups and solubility problems arising are the most important features in this regard. Semisynthesis, i.e., coupling of amino acids or peptides to natural material may help to overcome these difficulties. The preparation of hybrid preproinsulin by mixed anhydride synthesis and the conversion of pork insulin to human insulin by enzyme-catalyzed peptide synthesis are two examples of the semisynthesis of proteins. In both cases optimal reaction conditions are essential for maximal yield of the product desired. In spite of the rapid improvement of gene technology, chemical peptide synthesis will retain its value for the preparation of biologically and pharmacologically interesting substances.

Anhydrides↗
PubMed1981

Two mutant forms of human insulin. Structural consequences of the substitution of invariant B24- or B25-phenylalanine by leucine.

In 1979 the first abnormal human insulin was discovered. With the minute samples from the patient a Phe leads to Leu replacement could be established in either position B24 or B25. For the unequivocal localization of the substitution both the Leu analogues had to be prepared by semisynthesis. While another laboratory did this with the sequence of porcine insulin, here we are dealing with the true analogues of human insulin. In the present paper the structural consequences of the substitutions are investigated. Human insulin obtained by total synthesis served as a reference. Its CD spectral properties are herewith documented. According to the substantial deviations of the CD spectrum of [Leu B24]insulin, the introduction of the new side-chain forces conformational changes to occur not only in its immediate surrounding but also in the peptide chain. The failure to give the typical CD spectral response to variations of protein and zinc concentration indicates that the ability to form quaternary structure is impaired. Though dimerization was confirmed by gel chromatography to be largely reduced, it is concluded that, in addition, interactions normally responsible for the increase in tyrosine-CD with association are weakened. [Leu B25]insulin, on the other hand, does exhibit all CD spectral effects characteristic of the native hormone, though quantitatively somewhat reduced. The CD spectroscopic results are in full agreement with the computergraphic analysis of the sterical consequences of the substitutions. For B24-leucine an acceptable packing without movements of the mainchain and/or B15-leucine and without affecting dimerization is impossible, whereas B25-leucine can be accommodated without causing bad contacts either in the monomer or in the dimer. The structural results do not explain why [Leu B25]-insulin should have a lower biological activity than the B24 analogue, 2.1 +/- 0.3% versus 20.9 +/- 2.8%, in the fat cell test. They suggest, however, an important but not critical stereospecific role for the B25-phenylalanine in binding.

Circular Dichroism↗
PubMed1981

Semisynthesis of human proinsulin, I. Preparation of arginyl-A-chain cyclic bis-disulfide.

The semisynthesis of Arg-A-(SS)2 is described by the following chemical and enzymatic procedures: 1) A-(SSO theta 3)4 was acylated with arginine N-carboxyanhydride by the known method. Arg-A-(SSO theta 3)4 was purified, reduced and oxidized to Arg-A(SS)2. When A(SS)2 was acylated under identical conditions a gel-like product was obtained which could be purified after oxidative sulfitolysis. This was then converted to Arg-A-(SS)2 as described above. Both the pathways gave the desired product in 20-25% yield. 2) Boc-Orn(Msc) was quantitatively attached to A-(SS)2 by the mixed anhydride method, the Msc group was then removed and the ensuing N delta-free amino function was amidinated. Traces of unconverted Orn derivative (less than 6%) were still present. 3) Boc-Arg(HBr) was attached to A-(SS)2 by the mixed anhydride method and Arg-A(SS)2 was isolated after removal of the Boc group in 25% yield. 4) Boc-Arg(DHCH) was attached to A-(SS)2 by the mixed anhydride method. DHCH and Boc groups were removed in two steps and Arg-A-(SS)2 was isolated in 29% yield. 5) Boc-Arg was condensed with A-(SS)2 by trypsin-catalyzed synthesis. The removal of Boc group and purification yielded the desired product in 40-42% yield. This procedure was the most efficient and proceeded stereospecifically.

Arginine↗
PubMed1981

[An improved method for the combination of modified insulin chains (author's transl)].

A procedure is described in which the yields of the combination of insulin A and B chains, especially of modified chains, are increased. The chains are reduced simultaneously in 8M urea buffer at pH 8.6 with 2-mercaptoethanol. The thiol chains are not isolated but transferred to the oxidation buffer at pH 10.6 by means of gel chromatography on Sephadex G-25. During the chromatography about 50% of the tetrathiol A chain is oxidized specifically to a dithiol A chain disulphide, in which presumably the 6,11-disulphide ring is closen. Generally we observed that the combination yields of modified A or B chains are lower than the yields of combinations of unmodified A or B chains. It seems that there is a correlation between the biological activity of the analogue and the yield obtained by the recombination of the chains.

Chemical Phenomena↗
PubMed1980

(A1-phenylalanine) insulin: a new synthetic analogue.

An analogue of porcine insulin which differs from the native molecule in that the tyrosine residue in Pos. A19 is replaced by phenylalanine has been synthesized. The [PheA19]A chain was synthesized by the fragment condensation and purified as tetra-S-sulfonate by ion-exchange chromatography on DEAE-cellulose at pH 3.5. Conversion of the latter into the sulfhydryl form and combination with native sulfhydryl B chain yielded the [PheA19]insulin, which was purified by ion-exchange chromatography on CM-cellulose at pH 4.0 with an linear NaCl gradient. The biological activity of this analogue was 22.6% as measured by the rat epididymal adipocytes. It was suggested that in the insulin molecule the hydroxyl function of A19-tyrosine participates in an hydrogen-bond with the carbonyl function of A1-glycine. That hydrogen bond formation is critical for the stability of the hormone-receptor complex. The low biological activity found by us supports this hypothesis. The circular dichroism data in far UV suggest that the introduction of phenylalanine leaves the molecule structure essentially undisturbed, however the change observed in near UV could be accounted for the exchange.

Amino Acid Sequence↗
PubMed1980

(A14-phenylalanine)insulin: a new synthetic analogue.

An analogue of porcine insulin which differs from the native molecule in that the amino acid residue A14-tyrosine is replaced by phenylalanine has been synthesized. The [PheA14]A chain was synthesized by the fragment condensation method and purified as tetra-S-sulfonate by ion-exchange chromatography on DEAE-cellulose at pH 5.6. Conversion of the tetra-S-sulfonate A chain to the sulfhydryl form and combination with native porcine sulfhydryl B chain gave the [PheA14]insulin, which was purified by gel filtration and ion-exchange chromatography on DEAE-cellulose. The biological activity of this analogue was 96 +/- 6% as measured by the rat epididymal adipocytes. This shows that A14-tyrosine is not essential for the biological activity of the hormone.

Amino Acid Sequence↗