Purification and physicochemical characterization of a human placental acid phosphatase possessing phosphotyrosyl protein phosphatase activity.
A 17-kilodalton (kDa) human placental acid phosphatase was purified 21,400-fold to homogeneity. The enzyme has an isoelectric point of pH 7.2 and a specific activity of 106 mumol min-1 mg-1 using p-nitrophenyl phosphate as a substrate at pH 5 and 37 degrees C. This placental acid phosphatase showed activity toward phosphotyrosine and toward phosphotyrosyl proteins. The pH optima of the enzyme with phosphotyrosine and with phosphotyrosyl band 3 (from human red cells) were between pH 5 and 6 and pH 5 and 7, respectively. The Km for phosphotyrosine was 1.6 mM at pH 5 and 37 degrees C. Phosphotyrosine phosphatase activity was not inhibited by tartrate or fluoride, but vanadate, molybdate, and zinc ions acted as strong inhibitors. Enzyme activity was also inhibited by DNA, but RNA was not inhibitory. It is a hydrophobic nonglycoprotein containing approximately 20% hydrophobic amino acids. The average hydrophobicity was calculated to be 903 cal/mol. The absorption coefficient at 280 nm, E1% 1cm, was determined to be 5.7. The optical ellipticity of the enzyme at 222 nm was -5200 deg cm2 dmol-1, which would correspond to a low helical content. Free sulfhydryl and histidine residues were necessary for the enzyme activity. The enzyme contained four reactive sulfhydryl groups. Chemical modification of the sulfhydryls with iodoacetate resulted in unfolding of the protein molecule as detected by fluorescence emission spectroscopy. Antisera against both the native and the denatured protein were able to immunoprecipitate the native enzyme. However, upon denaturation, the acid phosphatase lost about 70% of the antigenic determinants. Both antisera cross-reacted with a single 17-kDa polypeptide on immunoblotting.
Waheed
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A
Chemistry Department, Purdue University, West Lafayette, Indiana 47907.
Laidler
P M
PM
Wo
Y Y
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Van Etten
R L
RL
D000135:Acid Phosphatase / Q000032:analysis / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007120:Immunochemistry; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D017027:Protein Tyrosine Phosphatases; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D010749:Phosphoprotein Phosphatases; D000135:Acid Phosphatase; D017027:Protein Tyrosine Phosphatases