Tissue Distribution and Characterization of Soluble and Membrane-bound Forms of Metallocarboxypeptidase D

doi:10.1074/jbc.271.46.28884

Tissue Distribution and Characterization of Soluble and Membrane-bound Forms of Metallocarboxypeptidase D*

Lixin Song and
Lloyd D. Fricker^‡

From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

^‡To whom correspondence should be addressed:
Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.
Tel.: 718-430-4225; Fax: 718-430-8922; E-mail: fricker{at}aecom.yu.edu.

Abstract

Metallocarboxypeptidase D (CPD) is a recently discovered 180-kDa membrane-bound carboxypeptidase E-like enzyme (Song, L. and Fricker, L. D. (1995) J. Biol. Chem. 270, 25007-25013). In the present study, a soluble CPD-like activity has been purified to homogeneity and characterized. On denaturing polyacrylamide gels, the soluble enzyme from bovine pituitary glands appears as two bands of 170 and 135 kDa which are converted to 155 and 115 kDa by endoglycosidase F. Both of the soluble forms of CPD are recognized by an antisera raised against CPD purified from rat brain membranes. The partial N-terminal amino acid sequences of the two soluble forms are identical to each other and to the predicted N terminus of duck gp180. The soluble and membrane forms of CPD have similar pH optima, inhibitor specificities, and kinetic parameters for substrate hydrolysis. CPD-like enzymatic activity is detected in all rat tissues examined, with highest levels in pituitary, brain, and adrenal. Western blot analysis indicates that both soluble and membrane forms of CPD are present in rat brain, heart, liver, and kidney. At least four distinct 100-180-kDa forms of CPD are detected on Western blots, although an antiserum raised against the C-terminal region of rat CPD recognizes only the 180-kDa membrane-bound form. The finding that CPD is widely distributed suggests a broad role for this enzyme in the processing of proteins that transit the secretory pathway.

Footnotes

↵* This work was supported in part by National Institutes of Heath Grants DK-51271 and DA-04494 and Research Scientist Development Award DA-00194 (to L. D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CPE

carboxypeptidase E

CPD

carboxypeptidase D

FPLC

fast protein liquid chromatography

dansyl

5-dimethylaminonaphthalene-1-sulfonyl

NaAc

sodium acetate

PC

prohormone convertase.
↵²F. Eng, unpublished data.
↵³Fricker, L. D., Berman, Y. L., Leiter, E. H., and Devi, L. A. (1996) J. Biol. Chem. 271, in press.
↵⁴X. Xin, O. Varlamov, R. Day, W. Dong, M. M. Bridgett, E. H. Leiter, and L. D. Fricker, submitted for publication.
↵⁵L. Song and O. Levy, unpublished data.
↵⁶O. Varlamov and L. Fricker, unpublished data.
- Received July 29, 1996.
- Revision received September 3, 1996.