Bovine Pancreatic Procarboxypeptidase B. II. Mechanism of Activation<sup>*</sup>

Cox, D. J.; Wintersberger, Erhard; Neurath, Hans

doi:10.1021/bi00912a018

Cited by 59 publications

(32 citation statements)

References 20 publications

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“…Analysis of the inhibitor profile, pH optimum and amino acid composition of the purified insulin-secretorygranule carboxypeptidase indicated that it is distinct from carboxypeptidase N (Plummer & Hurwitz, 1978), lysosomal carboxypeptidase B (Ninjoor et al, 1974;Lones et al, 1983), exocrine (pro)carboxypeptidase B (Folk et al, 1960;Cox et al, 1962) and urinary carboxypeptidase (kininase) (Skidgel et al, 1984). However, its properties do resemble those of the enzyme variously termed 'enkephalin convertase', 'carboxypeptidase E' and carboxypeptidase H (EC 3.4.17.10), which B A 0 0.05 k 0 J.…”

Section: Discussionmentioning

confidence: 99%

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

Davidson

Hutton

1987

Biochemical Journal

140

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A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.

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Section: Discussionmentioning

confidence: 99%

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

Davidson

Hutton

1987

Biochemical Journal

140

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“…Protein concentrations were determined from the absorbance at 280 mg, El2¿, = 21 (Cox et al, 1962). Enzymatic Activities.…”

Section: Methodsmentioning

confidence: 99%

“…It was therefore of interest to ascertain whether the zinc-binding site of bovine carboxypeptidase B shared some of the features of that of carboxypeptidase A. The presence of seven cysteic acid residues in the acid hydrolysate of the performic acid-oxidized enzyme (Cox et al, 1962) lends added interest to such an investigation, since, potentially, more than one cysteinyl residue could participate in the binding of the metal atom. The present experimental findings show, however, that six of the seven cysteic acid residues occur as disulfide-bonded cystines; this leaves only one cysteinyl residue as a potential ligand for zinc, and this is, in fact, its function in the native enzyme.…”

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confidence: 99%

A Zinc-binding Thiol Group in the Active Center of Bovine Carboxypeptidase B^*

Wintersberger¹,

Neurath²,

Tl³

et al. 1965

Biochemistry

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Bovine pancreatic carboxypeptidase B contains a single thiol group which binds zinc to the apoenzyme. This thiol group does not react with sulfhydryl reagents unless zinc is first removed. This can be accomplished either by exposure to chelating agents such as 1,10-phenanthroline or by denaturation. When zinc is removed by exposure to 1,10-phenanthroline, readdition of the metal to the apoenzyme restores both peptidase and esterase activities to the degree expected from the fraction of free thiol present in the apoenzyme. Partial blocking of the thiol group, by reaction with iodoacetamide, prevents reconstitution of Bovine pancreatic carboxypeptidases A (Neurath and Schwert, 1950) and B (Wintersberger et al., 1962) differ in substrate specificity and in amino acid composition but resemble each other in several physicochemical characteristics. In particular, bovine carboxypeptidase B (Cox et al., 1962), like the comparable enzyme from porcine pancreas (Folk et al., 1960) and bovine carboxypeptidase A (Vallee and Neurath, 1955), contains 1 g-atom of zinc per mole of enzyme (mw approximately 34,500). The metal is essential for the catalytic function of each of these enzymes. Removal of the zinc atom has resulted in the identification of the metal-binding site of carboxypeptidase A (Vallee et al., 1960a). A number of approaches all led to the conclusion that the sole free cysteine residue (Vallee et al., 1960b) and a nitrogen donor, probably the alamino group of the N-terminal residue (Coleman and Vallee, 1961;Coombs et al., 1964), bind the zinc atom to the apoenzyme of carboxypeptidases and 5. The zinc-binding cysteinyl peptide has been isolated (Walsh et al., 1962) and a preliminary report of its amino acid *

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“…CPBs from porcine and bovine pancreases have been isolated and well characterized (Folk et al, 1960;Folk et al, 1962;Wolff et al, 1962;Cox et al, 1962;Wintersberger et al 1962;Schmid & Herriott, 1976;Clauser et al, 1988;Edge et al, 1998;Ventura et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera

2006

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Carboxypeptidase B was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 34,000. The value of the specificity constant (kcat/Km) for hydrolysis of benzoyl-glycyl-L-arginine by the purified enzyme was 1.72×10 5 M -1 sec -1 . The optimal pH and the optimal temperature of the enzyme were pH 7.5 and 55 ℃, respectively. The enzyme was unstable above 50 ℃ and below pH 5.0. The enzyme was activated by Co 2+ , and inhibited by EDTA. The N-terminal amino acid sequence of the enzyme was determined as ATFDYNKYHSYQEIMDWVTN.

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Bovine Pancreatic Procarboxypeptidase B. II. Mechanism of Activation^*

Cited by 59 publications

References 20 publications

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

A Zinc-binding Thiol Group in the Active Center of Bovine Carboxypeptidase B^*

Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera

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Bovine Pancreatic Procarboxypeptidase B. II. Mechanism of Activation*

Cited by 59 publications

References 20 publications

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing

A Zinc-binding Thiol Group in the Active Center of Bovine Carboxypeptidase B*

Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera

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Bovine Pancreatic Procarboxypeptidase B. II. Mechanism of Activation^*

A Zinc-binding Thiol Group in the Active Center of Bovine Carboxypeptidase B^*