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R E Offord

Publications and source records attributed to R E Offord.

At least 55 records · Page 3Linked to original sources
PubMed1988

Identification by fast atom bombardment mass spectrometry of insulin fragments produced by insulin proteinase.

We describe the isolation by reversed-phase h.p.l.c. of a number of products of the degradation of insulin by insulin proteinase and their direct analysis by fast atom bombardment mass spectrometry (f.a.b.-m.s.). Various semisynthetically labelled insulins were used, including [[2H2]GlyA1]insulin and [18O]LysB29]insulin. The results obtained confirm and extend the results obtained by non-mass-spectrometric methods [Davies, Muir, Rose & Offord (1988) Biochem. J. 249, 209-214, and papers cited therein]. Cleavage sites were identified between positions A13-A14, A14-A15, B9-B10, B13-B14, B24-B25 and B25-B26. The advantages and disadvantages of the application of f.a.b.-m.s. to such studies are discussed.

Amino Acid Sequence↗
PubMed1987

Semisynthetic human [[3H2]Phe1]proinsulin.

Biosynthetic human proinsulin (obtained by recombinant DNA techniques) was used as the starting material for the preparation, by semisynthetic methods, of [3H]proinsulin with the label at the N-terminal phenylalanine residue. The labelled proinsulin was characterized by its retention time on reversed-phase h.p.l.c., by polyacrylamide-gel electrophoresis, by the time course of its enzymic conversion into insulin and by chromatographic analysis after extensive proteolytic degradation. The specific radioactivity of the product was 5 Ci/mmol. Experimental details of the preparation of human [[3H]Phe1]proinsulin, the isolation of this product by isocratic h.p.l.c. and gel filtration, and further characterization of protein intermediates have been deposited as supplement SUP 50138 (12 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on prepayment [see Biochem. J. (1987) 241, 5].

Chromatography, High Pressure Liquid↗
PubMed1987

The state of the N-terminus of recombinant proteins: determination of N-terminal methionine (formylated, acetylated, or free).

The removal of N-terminal methionine from proteins produced by recombinant DNA techniques is often far from quantitative. Furthermore, a proportion of the methionylated product may be N alpha-blocked and thus not easily accessible to conventional (Edman) techniques of protein characterization. In this paper, a method for overcoming the resulting analytical problems is described. The technique is based on perdeuteroacetylation (performed only if unblocked methionine is to be determined), cleavage with cyanogen bromide, extraction of any acylhomoserine lactone into ethyl acetate, formation of a chemical derivative, and analysis by combined gas-liquid chromatography/mass spectrometry (GC/MS). The remaining cyanogen bromide fragments, insoluble in ethyl acetate, are available for further analysis by mass spectrometric or other methods if required. Using an acylhomoserine lactone labeled with a stable isotope as internal standard, the method is semiquantitative. It should be possible to develop a quantitative method if appropriate polypeptide standards are prepared. N-Terminal processing of eight recombinant-derived proteins is discussed.

Acetylation↗
PubMed1986

Identification of some cleavage sites of insulin by insulin proteinase.

In a previous study [Muir, Offord & Davies (1986) Biochem. J. 237, 631-637] the chromatographic and electrophoretic behaviour of a major labelled fragment in the degradation of tritiated insulins by insulin proteinase were used to locate the probable sites of cleavage which had produced this fragment. In order to define these cleavage sites more precisely, authentic markers for the fragments which would be produced by cleavages at, or adjacent to, the most likely sites have now been synthesized. These markers were compared with labelled fragments of the A- and B-chains of insulin produced by insulin proteinase. The results, together with those of our previous study, show that in order to produce the observed major labelled fragment, the enzyme must have cleaved the insulin A-chain between leucine-A13 and tyrosine-A14 and the insulin B-chain between serine-B9 and histidine-B10. In addition, a minor component was observed in the labelled B-chain fragment which corresponded to a cleavage either between histidine-B10 and leucine-B11 or between leucine-B11 and valine-B12.

Binding Sites↗
PubMed1986

A new non-covalent complex of semisynthetically modified tryptic fragments of cytochrome c.

We have prepared a semisynthetic analogue of fully acetimidylated horse cytochrome c, a complex in which the peptide bond between residues glycine-37 and arginine-38 is lacking. In contrast with the complex that we have previously described [Harris & Offord (1977) Biochem. J. 161, 12-25], in which the break in continuity is between residues arginine-38 and lysine-39, the new analogue has a nearly normal redox potential, and can more fully restore succinate oxidation to mitochondria depleted of cytochrome c. Studies of this and other analogues lead us to propose an explanation for the low biological activity of complex (1-38)-(39-104) and a role for the invariance of arginine-38.

Cytochrome c Group↗
PubMed1986

The identification of a major product of the degradation of insulin by 'insulin proteinase' (EC 3.4.22.11).

We have studied a major product in the degradation of insulin by insulin proteinase (EC 3.4.22.11). Semisynthetic [[3H]PheB1]insulin and [[3H]GlyA1]insulin were used in the experiments. The structure of the fragment was deduced by observing the chromatographic and electrophoretic migration of the label both before and after further digestion of the fragment with proteinases of known specificity, with and without additional treatment by performic acid. Ambiguities were resolved by studying the behaviour of authentic fragments of known structure, isolated and characterized after digestion of intact insulin by proteinases of known specificity. We conclude that a major product in the degradation of insulin by insulin proteinase consists of a truncated section of the A chain, joined by the disulphide bridge B7-A7 to a truncated section of the B chain. The A-chain fragment consists most probably of residues A1-A13, and the B-chain fragment consists most probably of residues B1-B9. The similarity between this fragment and that found by other workers when insulin is degraded by intact hepatocytes is significant in the light of proposals that insulin proteinase is a possible participant in the physiological degradation of insulin by target cells.

Binding Sites↗
PubMed1985

The preparation of tritiated insulin specifically labelled by semisynthesis at glycine-A1.

We have prepared and characterized semisynthetic [GlyA1-3H]insulin. The preparation was carried out at specific radioactivities ranging from 1Ci/mmol to 44Ci/mmol. The largest quantity prepared in any one synthesis was 3.5 mCi. Chemical degradation showed that the label was in its expected position in the molecule. The semisynthetic product behaved authentically on reversed-phase h.p.l.c. and radioimmunoassay. It gave the expected profiles of biological activity as regards depression of blood sugar concentration in rats and stimulation of conversion of glucose into lipid in isolated rat fat-cells. We discuss some applications for which this tracer would be particularly suited. An expanded version of this paper, containing full experimental details of the semisynthesis and characterization of [GlyA1-3H]insulin, has been deposited as Supplementary Publication SUP 50129 (30 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.

Animals↗
PubMed1985

Similarity of pituitary and pineal human oxytocin neurophysins indicated by peptide mapping after radioactive alkylation and proteolysis.

We have compared the immunoreactive oxytocin neurophysin from the human pineal gland with human neurohypophyseal neurophysin. The two proteins were reduced and carboxymethylated with [14C]-iodoacetic acid. Since the pineal protein was available only in a very small amount, we were obliged to use [12C]-carboxymethyl apomyoglobin as a nonalkylatable carrier. The trypsin and subtilisin digests of the two labelled proteins were compared by high-voltage electrophoresis on paper, partition paper chromatography, and high-performance liquid chromatography. The distribution of radioactivity in the above peptide separations suggests a great similarity between the two proteins and few significant differences. As far as we are aware, this study is the first attempt to analyze an extrahypophyseal neurophysin at the molecular level.

Alkylation↗
PubMed1984

A case of spurious product formation during attempted resynthesis of proteins by reverse proteolysis. Some batches of 'pure' glycerol contain cross-linking agents.

In cases where enzyme-catalysed synthesis of a peptide bond is being used to re-form a protein from two large peptide fragments, the organic co-solvent chosen has so far been glycerol, for most solvents in use in small-molecule systems are potent protein denaturants. We have found, however, that impurities contaminating certain batches of glycerol are effective in cross-linking the complexes formed by these peptide fragments, thus mimicking the enzyme-catalysed process. In one such case, the reported re-formation of cytochrome c from a two-fragment complex system, cytochrome c-T, the extent and rate of conjugate formation duplicates that reported for enzymic resynthesis. We observed no difference between mixtures containing or lacking enzyme. We warn of the danger of confusion possible to those engaged in studies of resynthesis, and suggest a simple control of purchased glycerol to avoid it. We recommend similar caution to those (X-ray crystallographers and others) who seek to stabilize protein solutions by adding large quantities of glycerol.

Amino Acids↗
PubMed1984

A mass-spectrometric investigation of the mechanism of the semisynthetic transformation of pig insulin into an ester of insulin of human sequence.

In the trypsin-mediated semisynthetic transformation of pig insulin into an ester of insulin of human sequence, the B30 alanine residue of the pig hormone is replaced by an ester of threonine. The mechanism of this reaction was investigated by carrying out the transformation in a medium containing water enriched with 18O. Subsequent analysis by combined g.l.c.-mass spectrometry demonstrated that the oxygen isotope, is incorporated into the B29 carbonyl group of the insulin ester product. This result, together with those of appropriate controls, supports the conclusion that the transformation occurs, in the system studied, by a mechanism involving hydrolysis followed by coupling, and not by direct transpeptidation as has been previously found the case for another such system [Markussen & Schaumberg (1983) in Peptides 1982 (Bláha & Malon, eds.), pp. 387-394, W. de Gruyter, Berlin and New York].

Amino Acid Sequence↗
PubMed1983

The use of corticosteroids encapsulated in erythrocytes in the treatment of adjuvant induced arthritis in the rat.

Corticosteroid esters have been encapsulated into intact erythrocytes and used as an intravenous treatment for adjuvant induced arthritis in the rat. The treatment consisted of injections of the encapsulated steroids with the effects monitored for up to 14 days. On an equivalent weight basis both encapsulated cortisol-21-phosphate and prednisolone-21-sodium hemisuccinate proved superior to the free steroid esters administered in solution by injection.

Acute Disease↗
PubMed1983

Encapsulation of drugs in intact erythrocytes: an intravenous delivery system.

A number of drugs and the plasma antiprotease alpha 1-antitrypsin has been encapsulated in intact erythrocytes after hypotonic swelling, using a technique designed to preserve the viability of the cells. By labelling the cells with fluorescein isothiocyanate it has been shown that the cells survive exceptionally well when returned to the animal's circulation. Cell survival has been demonstrated in the rat, rabbit and guinea-pig. With encapsulation of cortisol-21-phosphate and methotrexate it was found that blood levels of the drug were maintained for a longer period than when the free drug was administered. Cortisol-21-phosphate was hydrolysed enzymatically by acid phosphatase located primarily in the erythrocyte membrane. An in vitro test involving the interaction or erythrocytes with phagocytes was developed to determine the viability or erythrocytes after being subjected to the encapsulation process. Preparations which did not interact with phagocytes survived when returned to the animal's circulation. The encapsulation procedure increased the fragility of the cell membrane compared to that of normal cells as measured by the leakage of haemoglobin after thermal treatment but it was found that encapsulated cortisol-21-phosphate in cells actually stabilized the membrane. The electrical charge on the membrane of encapsulating cells was the same as that of the normal cells. The charge on reformed ghosts was lower than that of normal cells. Reformed ghosts were rapidly removed when introduced into the circulation. The encapsulation procedure and its possible applications are discussed.

Animals↗
PubMed1983

Amino acid sequence determination by g.l.c.--mass spectrometry of permethylated peptides. Optimization of the formation of chemical derivatives at the 2-10 nmol level.

A new technique is described that permits the permethylation of acylated peptides at the 2-10 nmol level. The presence of up to 400 micrograms of sodium dodecyl sulphate per sample does not affect the reaction yields. The technique, which is a miniaturization of the widely used methyl iodide/dimethylsulphinyl carbanion procedure, employs a layer of hexane to exclude moisture and oxygen from the reaction mixture. Analysis of the peptide derivatives by combined g.l.c.--mass spectrometry permits amino acid sequence information to be obtained. In addition to studies of digests of a model substrate (glucagon), the new permethylation technique has been used to identify a peptide of interest from a digest of a cytochrome and to define the N-termini of two proteins at the 5 nmol level.

Acetylation↗
PubMed1983

A new mass-spectrometric C-terminal sequencing technique finds a similarity between gamma-interferon and alpha 2-interferon and identifies a proteolytically clipped gamma-interferon that retains full antiviral activity.

A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity.

Amino Acid Sequence↗
PubMed1983

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione.

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Evidence for the vitamin K-dependent gamma-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence.

The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated.

Animals↗