Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.
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) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), 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 (Ki = 17 microM). 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.
Shekels
L L
LL
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Smith
A J
AJ
Van Etten
R L
RL
Bernlohr
D A
DA
D016475:3T3 Cells; D000135:Acid Phosphatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D001152:Arsenicals; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; C029341:oxophenylarsine; D005982:Glutathione Transferase; D000135:Acid Phosphatase; D017027:Protein Tyrosine Phosphatases