The Subunit Structure of Bovine Procarboxypeptidase A-S6.<sup>*</sup> Chemical Properties and Enzymatic Activities of the Products of Molecular Disaggregation

Brown, James R.; Greenshields, R. N.; Yamasaki, Makoto; Neurath, Hans

doi:10.1021/bi00904a040

Cited by 92 publications

(33 citation statements)

References 12 publications

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“…aspartate transcarbamylase (Gerhart and Schachman 1965), or zymogens, e.g. procarboxypeptidase (Brown et al 1963), there is some evidence suggesting that fraction I protein is comprised of different subunits (Rutner and Lane 1967;Sugiyama and Akazawa 1967).…”

Section: Discussionmentioning

confidence: 99%

Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

Moon¹,

Thompson²

1969

Aust. Jnl. Of Bio. Sci.

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SummaryFraction I protein isolated from spinach beet chloroplasts was purified by ammonium sulphate fractionation and Sephadex G·200 gel filtration. The isolated protein was reduced and S-carboxymethylated, and the dissociated protein resolved into two distinct subunits by gel filtration on Sephadex G-200 in an 8M urea buffer at pH 10·0.A comparison of the amino acid composition of the two subunits showed distinct differences between them. The molecular weights of the two protein subunits ""ere estimated to be 54,000 and 16,000 by comparison of their elution volume on gel filtration with elution volumes of reduced and carboxymethylated proteins of known molecular weight.

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Section: Discussionmentioning

confidence: 99%

Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

Moon¹,

Thompson²

1969

Aust. Jnl. Of Bio. Sci.

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“…To account for this phenomenon, Neurath and co-workers postulated activation mechanisms for the binary and ternary complexes by trypsin whereby CTGC is activated ®rst yielding active chymotrypsin (Brown et al, 1963;Uren & Neurath, 1972). The dissociation of the complex was observed only at highly basic pH or after extensive treatment accounting for the subsequent activation of PCPA.…”

Section: Modulation Of the Proteolytic Activation By The Arrangement mentioning

confidence: 99%

“…In contrast, PCPA2 and PCPB have been described only as monomers (Pascual et al, 1990;Avile Âs et al, 1993;Oppezzo et al, 1994). Oligomeric PCPA has been found in a binary complex with the serine proteinase zymogens chymotrypsinogens B and C or with proproteinase E in human, pig, whale and rat (for a review, see Avile Âs et al, 1993) or in a ternary complex with the latter two proenzymes in bovine, sheep, camel and goat (Brown et al, 1961(Brown et al, , 1963Kerfelec et al, 1985). The occurrence of PCPA in oligomeric complexes is therefore quite widespread.…”

Section: Role Of the Ternary Complexmentioning

confidence: 99%

“…This complex was initially termed procarboxypeptidase A-S6, because this proenzyme is the major and ®rst identi®ed component of the complex and because it has a sedimentation coef®cient of 6 S (Brown et al, 1963). The three zymogens of the complex were named as subunit I, II and III, although the latter, proproteinase E, was initially isolated and studied as an N-terminally truncated form.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of an oligomer of proteolytic zymogens: detailed conformational analysis of the bovine ternary complex and implications for their activation 1 1Edited by I. A. Wilson

Gomis-Rüth

Gomez-Ortiz

Vendrell

et al. 1997

Journal of Molecular Biology

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“…Bovine 6S procarboxypeptidase A is known to be formed by the noncovalent association of three subunits (1). Subunit I is the immediate precursor of carboxypeptidase A.…”

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confidence: 99%

Dissociation of bovine 6S procarboxypeptidase A by reversible condensation with 2,3-dimethyl maleic anhydride: application to the partial characterization of subunit III.

Puigserver¹,

Desnuelle²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Bovine 6S procarboxypeptidase A can be dissociated into its three subunits by acylation with dimethyl maleic anhydride. The deacylated subunits are obtained in a largely native form due to instability of the bonds to dimethyl maleate at pH values near neutrality. The seven first residues of subunit III are identical to residues 18-24 of bovine chymotrypsinogen B and very similar with the same residues in bovine chymotrypsinogen A and C (subunit II) and also in proelastase A of the African lungfish. Therefore, this subunit is likely to be a chymotrypsinogen or proelastase-A-like zymogen which has lost the ability to be activated on account of a deletion of the N-terminal residues from half-cystine 1 to valine 17. Like other pancreatic zymogens, subunit III appears to possess a weakly functional active site.Bovine 6S procarboxypeptidase A is known to be formed by the noncovalent association of three subunits (1). Subunit I is the immediate precursor of carboxypeptidase A. Upon tryptic activation, subunit II yields a serine endopeptidase (2) resembling porcine chymotrypsin C (3, 4). The apparently inactive subunit III has been assumed to derive from subunit II by autolysis or amino acid replacement in the region of the active site (2).The three subunits are so firmly bound that it has not yet been possible to achieve their dissociation without altering their structure. Dissociation at high pH and room temperature (1) yields a completely denatured subunit I and a still activatable but modified subunit II (5). Acylation of the proteins' NH2 groups by negatively charged succinate groups (6) leads to an activatable subunit I, though the succinates cannot be removed under conditions preserving protein integrity. No information about the nature and structure of subunit III is presently available.Ready dissociation of 6S procarboxypeptidase under very mild conditions has now been obtained by condensation with 2,3-dimethyl maleic anhydride (DMMA), which is known to yield unstable derivatives at pHs near neutrality (7). Subunit III was prepared by this technique in practically pure form and likely is a pancreatic serine zymogen modified by a deletion in the N-terminal region that prevents its activation. MATERIALS AND METHODS 6SProcarboxypeptidase A was purified to homogeneity from pancreas acetone powder (8, 5). Subunit II, either free or as part of the trimer, was activated through incubation with trypsin for 90 min at 00 (enzyme-substrate weight ratio, 1:100) in 50 mM Tris-HCl buffer, pH 8.0, and the resulting chymotryptic activity was evaluated potentiometrically on acetyl-i,-tyrosine ethyl ester (ATEE). Subunit I was activated through incubation for 3 hr at 370 with trypsin (1: 5 w/w or 1: 50 w/w for the trimer or free subunit, respectively) in the same buffer as above made 100 mM in CaCl2. Carboxypeptidase activity was measured spectrophotometrically at 233 nm, using carbobenzoxyglycyl-iphenylalanine (Cbz-Gly-Phe) as substrate. An absorbance (A"m) at 280 nm of 19 was used for the determination of all protein c...

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The Subunit Structure of Bovine Procarboxypeptidase A-S6.^* Chemical Properties and Enzymatic Activities of the Products of Molecular Disaggregation

Cited by 92 publications

References 12 publications

Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

Crystal structure of an oligomer of proteolytic zymogens: detailed conformational analysis of the bovine ternary complex and implications for their activation 1 1Edited by I. A. Wilson

Dissociation of bovine 6S procarboxypeptidase A by reversible condensation with 2,3-dimethyl maleic anhydride: application to the partial characterization of subunit III.

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The Subunit Structure of Bovine Procarboxypeptidase A-S6.* Chemical Properties and Enzymatic Activities of the Products of Molecular Disaggregation

Cited by 92 publications

References 12 publications

Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

Subunits From Reduced and S-Carboxymethylated Ribulose Diphosphate Carboxylase (Fraction I Protein)

Crystal structure of an oligomer of proteolytic zymogens: detailed conformational analysis of the bovine ternary complex and implications for their activation 1 1Edited by I. A. Wilson

Dissociation of bovine 6S procarboxypeptidase A by reversible condensation with 2,3-dimethyl maleic anhydride: application to the partial characterization of subunit III.

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The Subunit Structure of Bovine Procarboxypeptidase A-S6.^* Chemical Properties and Enzymatic Activities of the Products of Molecular Disaggregation