Carboxypeptidases A and B have been isolated individually from aqueous extracts of mammalian pancreatic acetone powders by affinity chromatography on [N-(epsilon-aminocaproyl)-p-aminobenzyl]succinyl-Sepharose 4B (CABS-Sepharose). The affinity ligand was synthesized from DL-benzylsuccinic acid, purified, and characterized by UV absorption and NMR spectroscopy. Both enzymes from the various species were homogeneous by NaDodSO4-polyacrylamide gel electrophoresis and displayed high specific activities. No cross contamination of one enzyme species with the other was found. The ease of synthesis of the ligand from its commercially available precursor, its stability, and the mild elution conditions render CABS-Sepharose an excellent affinity support for the single-column isolation of both carboxypeptidases A and B. The procedures extend the utility of this resin previously demonstrated for carboxypeptidase A from human pancreatic juice [Peterson, L. M., Sokolovsky, M., & Vallee, B. L. (1976) Biochemistry, 15, 2501]. The use of CABS-Sepharose as a general affinity matrix for the isolation of metallocarboxypeptidases is suggested.
The nmr spectra of 1,3-thiazolidine, 2-fer/-butyl-l,3-thiazolidine, V,S-diacetylcysteamine, N,N,S-triacetylcysteamine, cysteamine hydrochloride, sodium 2-aminoethylmercaptide, and bisaminoethyl disulfide have been obtained and completely analyzed. On the basis of the coupling constants the thiazolidines are considered to exist in conformations close to envelopes with either C-4 or C-5 as the flap atom and with the 2 substituent anti to the flap (2 or 3a). Conformational analysis of five-membered rings by nmr methods is often made difficult by the profusion of nearly equienergy conformers which must be considered and by the inadequacy of the Karplus equation for determination of reliable rotational angles. In spite of these difficulties, considerable progress has been made with five-membered rings having an ethylene group isolated by heteroatoms in the 1 and 3 positions. These include 1,3-dioxolanes,2-8 1,3-dithiolanes,9 and 1,3-oxathiolanes.9-11 Research in these laboratories directed toward analyzing preferred conformations of coenzyme A necessitated study of the nmr spectra of acyclic cysteamine derivatives possessing an ethylene bridge separating sulfur and nitrogen atoms. A study of 1,3-thiazolidine and its 2-teri-butyl derivative was also needed as a means to define model coupling constants for gauche-and trans-related vicinal protons in the SCH2CHoN unit. The results of these investigations of model systems permit us to discuss the preferred conformation of the thiazolidines studied and to compare the rotational angles of thiazolidines to those of previously studied heterocycles. ResultsjV.S-Diacetylcysteamine (I) and N,N, S-triacetylcysteamine (II) together with N-acetylcysteamine (III) were all obtained by acetylation of cysteamine using an (1) Taken from the Ph.D. Thesis of T. J. B. submitted in partial fulfillment of the requirements for the Ph.D. degree in chemistry of the Polytechnic Institute of Brooklyn.(2) F. Alderweireldt and M. Anteunis, Bull Soc. Chim. Belg., 74, 488 (1965).
Bovine procarboxypeptidase A exhibits intrinsic hydrolytic activity toward haloacyl amino acids (Behnke and Vallee, 1972), as well as toward conventional peptide and ester substrates for carboxypeptidase A (Bezzone, 1974; Uren and Neurath, 1974). The kinetics of hydrolysis of a series of such substrates by native procarboxypeptidase has now been examined in detail in order to ascertain the extent to which the binding and catalytic sites of carboxypeptidase preexist inthe zymogen. Distinct differences in the substrate binding sites of the zymogen compared with the enzyme are apparent from their respective kinetic profiles as well as from the effects of modifiers on their activities. Substrate activation with the dipeptides BzGly-L-Phe and CbzGly-L-Phe, well known for carboxypeptidase, is exhibited also by the zymogen, but the corresponding substrate inhibition by CbzGly-L-Phe and BzGly-Ophe is absent. Moreover, the substrate inhibition of carboxypeptidase by CbzGlyGly-L-Phe and BzGly-Ophe is replaced by substrate activation in the zymogen...
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