Mechanistic studies of carboxypeptidase Y from <i>Saccharomyces cerevisiae</i>. pH and pD profiles and inactivation at low pH (pD) values

Chang, Wei‐Der; Douglas, Kenneth T.

doi:10.1042/bj1870843

Cited by 5 publications

(3 citation statements)

References 24 publications

(23 reference statements)

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“…The substractive method is designed to obtain the quantitative recovery of DABTHSer and DABTH-Thr which is crucial to assign the correct amino acid sequence when peptide mixture is sequenced. DABTH-Ser and DABTH-Thr are destroyed in the direct DABITCjPITC method because the use of trifluoroacetic acid [24]. In the subtractive DABITCjPITC method, peptide is degraded only by PITC according to the similar procedure described [21].…”

Section: Amino Acid Sequence Determinationmentioning

confidence: 99%

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Thrombin specificity

Chang

1985

European Journal of Biochemistry

202

108

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a-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative aminoterminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of a-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by a-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit a-thrombin in 20 pl of 50 mM ammonium bicarbonate at 25 "C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for a-thrombin has the structures of (a) P4-P3-Pro-Arg-P 1'-P2', where P3 and P4 are hydrophobic amino acid and PI', P2' are nonacidic amino acids and (b) P2-Arg-P l', where P 2 or P 1' are Gly. The requirement for hydrophobic P 3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P 3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P 1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by a-thrombin can be SedSOnably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage. a-Thrombin is a trypsin-like serine proteinase which preferentially cleaves polypeptide substrates at Arg/Lys-Xaa bonds [I -31. Unlike trypsin, a-thrombin displays highly restricted specificity toward protein substrates. This specificity is best known by its selective cleavage of two Arg-Gly bonds from amongst 181 Arg/Lys-Xaa bonds in fibrinogen [4]. Attempt to delineate the structural element in fibrinogen which accounts for restricted thrombin cleavage has mostly focussed on the fibrinogen Aa chain [5 -81 and has led to the findings that (a) the Aa (1-23) fragment contains a fundamental structure element for thrombin binding Table 1 for numbering of residues.) The recognization of the importance of hydrophobic P2 and P9 residues has further led to the development of a series of useful tripeptide substrates [9 -131 and inhibitors [I 4, 151. Another source of understanding of thrombin specificity comes from thrombin cleavage at non-fibrinogen polypeptides. A number of peptides and proteins have been Abbreviations. DABS-GI, dimethylaminoazobenzene sulfonyl chloride;...

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Section: Amino Acid Sequence Determinationmentioning

confidence: 99%

“…Their relative cleavage rates were compared and measured by quantitative aminoterminal analysis using DABITC [16,171.…”

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confidence: 99%

Thrombin specificity

Chang

1985

European Journal of Biochemistry

202

108

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“…The destruction of DABTH-Ser and DABTH-Thr was found to be initiated by side-chain dehydration (Ilse & Edman, 1963), which mainly occurred in anhydrous acid solution (Chang, 1979a). However, in the single N-terminal determination (Chang, 1980), the yield of DABTH-Ser and DABTH-Thr can be as high as 80-98% when aqueous acid is used directly for cleavage and conversion reaction. The parent DABTH-Ser could also be largely saved by using automatic solid-phase DABITC/PITC degradation (Hughes et al, 1979), as the cleaved thiazolinone derivative eluted by trifluoroacetic acid is immediately collected in aqueous solution, thus avoiding long exposure to anhydrous acid.…”

Section: Discussionmentioning

confidence: 99%

N-terminal sequence analysis of polypeptide at the picomole level

Chang¹

1981

Biochemical Journal

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This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the byproducts of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.

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Enzyme Handbook

Schomburg¹,

Salzmann²

1991

Enzyme Handbook

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Mechanistic studies of carboxypeptidase Y from Saccharomyces cerevisiae. pH and pD profiles and inactivation at low pH (pD) values

Cited by 5 publications

References 24 publications

Thrombin specificity

Thrombin specificity

N-terminal sequence analysis of polypeptide at the picomole level

Enzyme Handbook

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