Carboxypeptidase E Activity Is Deficient in Mice with the fat Mutation

doi:10.1074/jbc.271.48.30619

Carboxypeptidase E Activity Is Deficient in Mice with the fat Mutation

EFFECT ON PEPTIDE PROCESSING*

From the ^‡ Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, the
^∥ Department of Pharmacology, New York University Medical Center, New York, New York 10016, and the
^∥ The Jackson Laboratory, Bar Harbor, Maine 04609

^§ 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

Carboxypeptidase E (CPE) is involved in the biosynthesis of many peptide hormones and neurotransmitters. Mice with the fat mutation have previously been found to have a point mutation in the cpe gene, and to have greatly reduced levels of CPE-like enzyme activity in the pituitary and pancreatic islets (Naggert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). In the present report, we examined CPE-like activity and peptide processing in several tissues of C57BLKS/LtJ-Cpe^fat/Cpe^fat mutant (Cpe^fat/Cpe^fat) mice. Whereas CPE-like activity is detected in homogenates of Cpe^fat/Cpe^fat mouse tissues, the majority of this activity is not due to CPE based on the sensitivity to p-chloromercuriphenyl sulfonate. In addition, the Cpe^fat/Cpe^fat activity does not bind to a substrate affinity column under conditions that bind CPE. Furthermore, the enzyme activity and immunoreactive properties of the activity purified from Cpe^fat/Cpe^fat brain are distinct from those of CPE. Taken together, these data suggest that CPE is completely inactive in the Cpe^fat/Cpe^fat mice, and that all of the CPE-like activity is due to other carboxypeptidases such as carboxypeptidase D. Levels of Leu-enkephalin in Cpe^fat/Cpe^fat mouse brain are approximately 5-fold lower than those in control brain. Treatment of the Cpe^fat/Cpe^fat brain extract with carboxypeptidase B restores the level of Leu-enkephalin to the level in control brain. Interestingly, the large molecular weight enkephalin-containing peptides are elevated 2-3-fold in Cpe^fat/Cpe^fat mouse brain. These data indicate that CPE plays an important role in the processing of peptide hormones in various tissues, but that other carboxypeptidases also contribute to peptide processing. Furthermore, the increase in levels of high molecular weight enkephalin peptides in the Cpe^fat/Cpe^fat mouse suggests that CPE is required for efficient peptide processing by the endopeptidases.

Footnotes

↵* This work was supported in part by National Institute on Drug Abuse Grant DA-04494, National Institute on Drug Abuse Research Scientist Development Award DA-00194, a grant from the Juvenile Diabetes Foundation (to L. D. F.), a grant from the American Diabetes Foundation and National Institutes of Health Cancer Center Support Grant CA34196 (to E. H. L.), Grant T32 DA-07254 from the National Institute on Drug Abuse (to Y. L. B.), and National Institutes of Health Grant NS-26880 and National Institute of Neurological Diseases and Communicative Disorders and Stroke Research Career Development Award NS-01788 (to L. A. D.). 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

CPB

carboxypeptidase B

dansyl

5-methylaminonaphthalene-1-sulfonyl

GEMSA

guanidinoethylmercaptosuccinic acid

PCMPS

p-chloromercuriphenyl sulfonate.
↵² L. Song and L. Fricker (1996) J. Biol. Chem., in press.
↵³ L. Song and L. Fricker, submitted for publication.
- Received June 4, 1996.
- Revision received August 19, 1996.