Purification and Biochemical Characterization of a Protein-palmitoyl Acyltransferase from Human Erythrocytes

doi:10.1074/jbc.272.17.11021

Purification and Biochemical Characterization of a Protein-palmitoyl Acyltransferase from Human Erythrocytes*

From the Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India

^¶To whom correspondence should be addressed. Fax: 91-33-3506790.

Abstract

Protein palmitoylation involves the post-translational attachment of palmitate in thioester linkage to cysteine residues of proteins. The labile nature of the thioester linkage makes possible the palmitoylation-depalmitoylation cycles that have emerged in recent times as additions to the repertoire of cellular control mechanisms. However, detailed understanding of these cycles has been limited by the lack of knowledge of the transferases and thioesterases likely to be involved. Here, we describe the purification of a protein-palmitoyl acyltransferase (PAT) from human erythrocytes. PAT behaved as a peripheral membrane protein and catalyzed the attachment of palmitate in thioester linkage to the β-subunit of spectrin. On SDS-polyacrylamide gel electrophoresis, PAT appeared as a 70-kDa polypeptide. Antibody against this polypeptide could immunodeplete PAT activity from the crude extract, confirming the assignment of the 70-kDa polypeptide as PAT. PAT-mediated spectrin palmitoylation could be inhibited by nonradioactive palmitoyl-, myristoyl-, or stearoyl-CoA. The apparent K_m for palmitoyl-CoA was 16 μM.

Footnotes

↵^‡ Research Associate of the Council of Scientific and Industrial Research.
↵^§ Senior Research Fellow of the Council of Scientific and Industrial Research.
↵* This work was supported in part by grants (to J. B.) from the Departments of Biotechnology and of Science and Technology, Government of India. 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:

PAT

protein-palmitoyl acyltransferase

PAGE

polyacrylamide gel electrophoresis.
- Received April 22, 1996.
- Revision received February 11, 1997.