The Amino Acid Composition of Bovine Pancreatic Carboxypeptidase A<sup>*</sup>

Bargetzi, Jean-Pierre; Kumar, K. Suresh; Cox, D. J.; Walsh, Kenneth A.; Neurath, Hans

doi:10.1021/bi00906a046

Cited by 123 publications

(39 citation statements)

References 27 publications

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“…The amino acids were analysed on a Beckman Unichrom automatic amino acid analyzer, with the accelerated procedure of Benson and Patterson [23]. The value for ,9-thienylalanine and a-amino-8-guanidopropionic acid were used to apply corrections for changes in instrumental sensitivity [24]. The values for aspartic acid, threonine, serine, tyrosine and ammonia were corrected by extrapolating to zero time the values obtained at the various times of hydrolysis.…”

Section: Methodsmentioning

confidence: 99%

Bovine Erythrocyte Cupro‐Zinc Protein

Bannister

Wood

1971

European Journal of Biochemistry

125

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Bovine erythrocyte cupro-zinc protein was obtained by ethanol-chloroform fractionation of red blood cells and purified by chromatography on fibrous DEAE-cellulose, gel filtration on Sephadex G-75, and rechromatography on microgranular DEAE-cellulose. The protein has a molecular weight of about 33000 by gel filtration, and contains 2 gram atoms of cupric copper and 2 gram atoms of zinc per mole. The amino acid composition of the protein has been determined. The protein has an isoelectric point a t pH 4.95 by isoelectric focussing and is resolved into two "size" isomers, one being a minor component, in polyacrylamide gel electrophoresis. The apoprotein behaves as a molecule of smaller "size" than the native protein components in polyacrylamide gel electrophoresis. The ultraviolet spectra of the native and apoproteins show unusual features. These spectra together with the ultraviolet spectra of partially reconstituted apoprotein indicate a stabilizing effect of the zinc atoms on the structure of the chromophoric system. It is suggested that the copper and zinc atoms are not mutually replaceable at their respective sites in the protein. The near ultraviolet and visible spectra of the native protein have been fitted with Gaussian curves which indicate absorption bands a t 340, 440 and 680 nm. The value of &M/Cu2+ for the 680 nm band has been found to be 120, indicating a cupric complex of high symmetry. man DE 32) were obtained from British Drug Houses Ltd. ; DEAE-Sephadex A-50 and Sephadex G-75 and G-100 from Pharmacia AB; and synthetic ampholyte solution for isoelectric fucossing (Ampholine) from LKB-Produkter AB. Chemicals, where possible, were of analytical grade, and were not purified further except guanidine hydrochloride and iodoacetic acid which were recrystauized from ethanol and n-heptane, respectively. Glass-distilled water was used in large scale preparative work. This water was deionized for analytical work by means of an Elgastat B125 deionizer (Elga Products Ltd.). Preparation of Crude Erythrocyte Cupro-Zinc ProteinFresh ox blood was obtained from a slaughterhouse. Four volumes of blood were collected in a stainless steel bucket containing 1 vol. of 3.8O/, (wlv) sodium citrate. Eight litres of blood were processed at a time. The plasma was separated after centrifuging the blood at 1800 xg for 60 min at 4". The cells were mixed with an equal volume of cold distilled water, and the haemolysate was left overnight a t 4". The haemolysate was then chilled at -20" until ice crystals started to form, and 0.5 vol. of a mixture of ethanol and chloroform (3 : I , v/v), which had been pre-chilled to -2O", was added slowly with continuous stirring. The viscous mixture was diluted with 0.1 vol. of cold 0.9O/, (w/v) NaCl and centrifuged at

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Section: Methodsmentioning

confidence: 99%

Bovine Erythrocyte Cupro‐Zinc Protein

Bannister

Wood

1971

European Journal of Biochemistry

125

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“…These were amino acid analysis [2], separation of peptides by gel filtration, by chromatography and by electrophoresis using thin-layer plates of paper. They will not be described here as experimental details are readily available in the literature.…”

Section: Standard Proceduresmentioning

confidence: 99%

The Structures of Some Peptides from Bee Venom

et al. 1978

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The sequences are given of two peptides (secapin and melittin F) whose isolation from the venom of the common European honey bee (Apis mellifera) has previously been described [Gauldie et al. (1976) Eur. J. Biochem. 61, 369--3761. Some structural studies on the known peptide 401 (MCD peptide) are described. The positions of the two disulphide bridges in peptide 401 have been determined by (an essentially) novel method.We recently described [l] a procedure for the isolation of the peptide components of the venom of the common European honey bee (Apis mell[fem also known as Apis mellifica). We found three hitherto unknown peptides: the sequence of two of these are given in this paper. We also report some structural studies on the known peptide 401 (MCD peptide). MATERIALS AND METHODS Isolation of PeptidesSecapin, melittin-F and peptide 401 were isolated in homogeneous form as previously described [l]. The amounts of these peptides present in the original venom have been estimated as 1 %, < 1 x, and 2-3 x respectively [l]. Standard ProceduresThese were amino acid analysis [2], separation of peptides by gel filtration, by chromatography and by electrophoresis using thin-layer plates of paper. They will not be described here as experimental details are readily available in the literature. Standard DeterminationA modification of the Edman method [3] was used and is described below. All reagents were carefully purified immediately prior to use.The peptides were (where appropriate) reduced and carboxymethylated. The resulting derivatives (usually in amounts in the range 10-12 mg) were dis- solved in a mixture of propan-1-01 and water (3/4, v/v) containing a buffer system consisting of dimethylallylamine (0.4 M) and enough trifluoroacetic acid to give a reading on the pH meter of 9.5. Phenyl isothiocyanate (30 pl) was then added and reaction allowed to proceed under nitrogen for 30 min at 50 "C. Excess reagent was removed by extraction with benzene and the phenylthiocarbamyl derivatives of the peptides were isolated by lyophilisation at low pressure (0.01 mm-Hg, 1.33 Pa). They were then washed with a mixture of ethylacetate and acetone (l/l, v/v) and again lyophilised. (The inclusion of this wash produces clearer chromatograms.)Cyclisation and cleavage reactions were carried out using trifluoroacetic acid as described by Edman [3]. Residual peptides were precipitated by the addition of ether and then 1,2-dichloroethane. The use of ether increases the recoveries of peptides and enables even small peptides to be recovered in good yields. The recovered peptides were washed with 1,2-dichloroethane and dried before further degradation. The thiazolinone derivatives of the cleaved N-terminal amino acids were recovered by evaporation of solvent in a stream of nitrogen. Treatment with HCl(1 M) for 10 min at 80 "C converted them into the phenylthiohydantoin derivatives. These were extracted with ethyl acetate, dried, dissolved in 1,2-dichloroethane and identified by chromatography on plates of thinlayer silica gel G (Merck AG or E...

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“…Peptide samples were hydrolysed by heating with HCl (6 M) at 105 "C for 24 or 48 h as described by Bargetzi et al [31]. Amino acid analysis was done in conventional fashion using a single-column Technicon amino acid analyzer.…”

Section: Amino Acid Analysismentioning

confidence: 99%