Purification and partial characterization of two acid phosphatases from rat bone

Abstract

Acid phosphatase activity in homogenized tibiae and femora of suckling rats was extracted with 0.3M KCl and 0.1% Triton X-100. A high-speed supernatant was treated with protamine sulfate, dialyzed, and chromatographed on CM-52 cellulose. All of the acid phosphatase activity was eluted with a sodium acetate buffer and combined ionic strength-pH gradient into two peaks (E₁ and E₂). Both enzyme peaks were further purified with Sephadex G-200, which resulted in 700- and 1000-fold purification for E₂ and E₁, respectively. A total of 220 units (µmoles substrate/min) of E₂ with a specific activity of 160 units/mg protein has been obtained in one run by this procedure. E₁ has a high molecular weight (>100,000) and shows preference for monophosphate ester substrates, is markedly inhibited by tartrate, and has a pH optimum near 5. E₂ has a lower molecular weight (p-nitrophenyl phosphate (p-NPP)], but high activity with ADP and ATP. E₂ is unaffected by tartrate and shows a pH optimum near 6. Both enzymes are competitively inhibited by inorganic phosphate, and E₂, but not E₁, is markedly inhibited byp-chloromercuribenzoate. Withp-NPP as substrate, E₁ and E₂ have distinctly different values for K_m. E₁ appears similar to the high molecular weight acid phosphatases of soft tissues. However, E₂ appears to differ from the low molecular weight phosphatases in soft tissues with regard to substrate specificity.