Kinetics and optical spectroscopic studies on the purple acid phosphatase from beef spleen.
A new purification scheme has been developed for the purple acid phosphatase from beef spleen; typical yields are 8 mg of homogeneous enzyme per kg of spleen in only five steps. Kinetics studies have shown that the enzyme is strongly inhibited by fluoride, phosphate, and [p-(acetylamino)-benzyl]phosphonate, a nonhydrolyzable substrate analogue; the last two of these show simple competitive inhibition. In contrast, cyanide, azide, tartrate, and p-nitrophenol show no inhibition at concentrations up to 10 mM. Molecular weight estimations by gel electrophoresis and gel permeation chromatography give a value of 40 000 for the native enzyme, which is shown to consist of two subunits of apparent molecular weight 24 000 and 15 000. Careful metal analyses indicate the presence of 2.1 +/- 0.1 iron atoms per enzyme molecule, and less than 0.1 copper, zinc, nickel, or manganese atom per enzyme. The purple enzyme (lambda max 550 nm) is reversibly converted to a pink, active form (lambda max 505 nm) upon treatment with mild reducing agents (dithioerythritol or ascorbate). Addition of competitive inhibitors to the pink form causes rapid reversion to the purple form. Electron paramagnetic resonance spectroscopy at several temperatures showed only weak g = 4.3 signals (less than 0.1 spin/molecule) for the native, reduced, and inhibited forms of the enzyme.
D000135:Acid Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D005459:Fluorides / Q000494:pharmacology; D007700:Kinetics; D063065:Organophosphonates / Q000494:pharmacology; D009943:Organophosphorus Compounds; D010710:Phosphates / Q000494:pharmacology; D013053:Spectrophotometry; D013154:Spleen / Q000201:enzymology*
D063065:Organophosphonates; D009943:Organophosphorus Compounds; D010710:Phosphates; C032163:4-(acetylamino)benzylphosphonate; D000135:Acid Phosphatase; D005459:Fluorides