Protein Science, Vol 1, Issue 6 710-721, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase

L. L. SHEKELS, A. J. SMITH, R. L. VAN-ETTEN and D. A. BERNLOHR
Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (K(inact) = 10 {mu}M), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (K(react) = 23 {mu}M), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP{alpha} and HAAP{beta} (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP{alpha} and HAAP{beta} share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP{beta} has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP{beta} was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (K(i) = 17 {mu}M). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.
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