Bovine Pancreatic Procarboxypeptidase B. I. Isolation, Properties, and Activation<sup>*</sup>

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

doi:10.1021/bi00912a017

Cited by 103 publications

(42 citation statements)

References 20 publications

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“…The A. pectinifera CPB isolated in this study showed a molecular weight of about 34,000 on SDS-PAGE, being similar to those in other animal species (33,000-35,000) (Folk et al, 1960;Wintersberger et al, 1962;Prahl & Neurath, 1966;Reeck & Neurath, 1972;Marinkovicet al, 1977;Cohen et al, 1981;Yoshinaka et al, 1984a;Bradley et al, 1996). However, lower molecular weights of CPB were reported for cod (26,000) (Overnell, 1973).…”

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confidence: 81%

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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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confidence: 81%

“…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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“…However, this activation segment does not bind to the complementary CPB, as deduced by the observed independent eiect'rophoresis of both proteins along the whole urea-gradient when they are run as a 1: 1 molar ratio mixture ( fig.2c). The inability of asB to bind to its active enzyme, in contrast to the strong binding of asA to its enzyme, could help to explain the great differences in the proteolytic activation process of both proenzymes, which requires a few minutes in the former case and several hours in the latter [3,5,8,9]. Thus, during the action of trypsin asB would be quickly released from CPB, the activity of which would be expressed very early.…”

Section: Resultsmentioning

confidence: 99%

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Vilanova¹,

Burgos²,

Cuchillo³

et al. 1985

FEBS Letters

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Monomeric procarboxypeptidase A (PCPA) and isolated proproteinase E (PPE), both from pig pancreas, were shown by means of electrophoresis on transverse urea gradients (0–9 M) to form a very stable complex, identical to their natural binary complex. Although the complex is maintained by the interaction of both active regions, the activation segment of PCPA participates directly in the binding. Procarboxypeptidase B (PCPB) also associates with PPE, but in this case the complex shows low stability. In contrast with carb‐oxypeptidases A that strongly bind to their corresponding severed activation segments, no interaction was observed between carboxypeptidase B and its severed activation segment. The above results give some insight into several characteristics of the structure and activation properties of pancreatic PCPA and PCPB.

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“…The esterase activity toward hippurylphenyllactate a t concentrations higher than 1 mM was followed by pH-stat titration [12].…”

Section: Enzymatic Activitymentioning

confidence: 99%

Porcine Carboxypeptidase B

Sokolovsky

Eisenbach

1972

European Journal of Biochemistry

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Coupling of porcine carboxypeptidase B with diazotized arsanilic acid labels an active-site tyrosine residue of the enzyme. Tyrosine modification is accompanied by a change in the activities of carboxypeptidase B towards both basic and non-basic substrates. The major arsanilazotyrosine-containing peptide, Thr-Ile-Tyr-Pro-Ma, isolated by affinity chromatography is homologous with the peptide containing tyrosine-248 from the active center of bovine carboxypeptidase A.Formation of the arsanilazotyrosine residue generates extrinsic Cotton effects above-300 nm. The dichroic spectrum of the azochromophore constitutes a sensitive probe of conformational changes accompanying the interaction of arsanilazocarboxypeptidase B with inhibitors, as monitored by changes in the circular dichroic spectrum. The changes are consistent with the hypothesis that the tyrosine residue is a component of the active-site region.The development of suitable reagents for the chemical modification of tyrosine residues has provided evidence that these residues play an important role in the mechanism of action of porcine carboxypeptidase B [l -31. This enzyme possesses, in addition to its known specificity toward basic substrates, and intrinsic activity towards non-basic substrates [a]. I n an effort to differentiate between these two specificities we have studied p-azobenzenearsonate derivatives of porcine carboxypeptidase B. This chromophoric derivative has proved useful as a spectral probe of enzyme conformation [5-71. The data presented here indicate that the modification a t the tyrosine residue (which corresponds to tyrosine-248 in carboxypeptidase A) is responsible for activity changes, and, in addition, generates circular dichroic bands that are altered by inhibitors. MATERIALS AND METHODS MaterialsCarboxypeptidase B (Code : COBC) prepared essentially according to the method of Folk et al. [8] was purchased from the Worthington Biochemical Corp. (Freehold, N.J.). p-Arsanilic acid was a product of B.D.H. ; hippuryl-1;-arginine and hippuryl-Lphenyllactate were obtained from Cyclo Corp., and carbobenzoxy-L-alanyl-L-L-alanyl-L-alanine, Z-(Ala),, from hEles-Yeda (Rehovot). Hippuryl-L-argininic acid was prepared as described previously [4]. The arsanilazo antibody-Sepharose conjugate was a gift of Dr. David Givol (The Weizmann Institute, Rehovot, Israel). All other chemicals were of the best grade available. Buffers were extracted with 0.1 dithizone in carbon tetrachloride to avoid contamination by adventitious metal ions. METHODS Protein ConcentrationsThese were determined by absorbance a t 278 nm using a molar absorptivity of 7.3 x lo4 M-l cm-l [81 * Spectrophotometry An Hitachi-Perkin-Elmer 124 spectrophotometer was used for measurements of absorbance a t single wavelenghts and a Cary-14 recording spectrophotometer was employed for determination of absorption spectra. pH and Inorganic AnalysesThe yH of solutions was measured on a Radiometer model-26 pH meter equipped with a GK2312C glass-calomel combination electrode. Zinc analyses were per...

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Bovine Pancreatic Procarboxypeptidase B. I. Isolation, Properties, and Activation^*

Cited by 103 publications

References 20 publications

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

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

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Porcine Carboxypeptidase B

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Bovine Pancreatic Procarboxypeptidase B. I. Isolation, Properties, and Activation*

Cited by 103 publications

References 20 publications

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

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

Urea‐gradient gel electrophoresis studies on the association of procarboxypeptidases A and B, proproteinase E, and their tryptic activation products

Porcine Carboxypeptidase B

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Bovine Pancreatic Procarboxypeptidase B. I. Isolation, Properties, and Activation^*