Purification and Characterization of Carboxypeptidase D, a Novel Carboxypeptidase E-like Enzyme, from Bovine Pituitary (*)
- From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
- § To whom correspondence should be addressed. Tel.: 718-430-4225; Fax: 718-430-8922.
Abstract
Carboxypeptidase E (CPE) is involved in the biosynthesis of most neuropeptides and peptide hormones. Until recently, CPE was
the only intracellular carboxypeptidase thought to be involved in neuroendocrine peptide processing. However, the finding
that fat/fat mice, which have a mutation within the CPE gene that inactivates the enzyme, are capable of a reduced amount of insulin processing
suggests that another carboxypeptidase is present within the secretory pathway. We have detected a CPE-like enzyme, designated
CPD, which has many properties in common with those of CPE. Like CPE, CPD is a metallocarboxypeptidase that has a pH optimum
of 5.5-6. The K
and K
values for a series of short peptide substrates show only minor differences between CPD and CPE. Several active site-directed
inhibitors also show generally similar potency toward the two enzymes, although guanidinoethylmercaptosuccinic acid is approximately
10-fold more potent, and hippuryl-Arg is approximately 100-fold more potent as an inhibitor of CPD than of CPE. A major difference
between the two enzymes is the molecular masses; CPE is 50,000-56,000, whereas CPD is approximately 180,000. Also, CPD does
not elute from a substrate affinity column when the pH is raised to 8, which elutes CPE, although CPD can subsequently be
eluted by arginine. Both CPE and CPD are present in purified bovine anterior pituitary secretory vesicles, but the tissue
distribution of CPD is more uniform than that of CPE. Antisera to the N- and C-terminal regions of CPE do not recognize CPD.
The partial N-terminal amino acid sequence of bovine CPD shows 30-40% homology with an N-terminal region of bovine and rat
CPE and 70% homology with a duck protein known as gp180, a hepatitis B virus particle binding protein that shows 47% homology
to CPE. Taken together, these results suggest that CPD is a novel secretory pathway enzyme that may be the bovine homologue
of gp180.
Footnotes
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↵* This work was supported by National Institute on Drug Abuse Grants R01 DA-04494 and K02 DA-00194 and by an Irma T. Hirschl Career Scientist Award (to L. D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- CP
-
carboxypeptidase
- FPLC
-
fast protein liquid chromatography
- bis-Tris
-
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
- GEMSA
-
guanidinoethylmercaptosuccinic acid
- MGTA
-
2-mercaptomethyl-3-guanidinoethylthiopropanoic acid
- APMSA
-
aminopropylmercaptosuccinic acid
- TPCK
-
tosylphenylalanyl chloromethyl ketone
- TLCK
-
N
-p-tosyl-L-lysine chloromethyl ketone.
-
↵2L. D. Fricker and E. H. Leiter, unpublished data.
-
↵3F. Eng and D. Ganem, personal communication.
-
↵4L. Song and L. D. Fricker, manuscript in preparation.
-
↵5R. Skidgel, personal communication.
-
- Received June 28, 1995.
- Revision received August 3, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
