PURIFICATION AND PARTIAL CHARACTERIZATION OF 2 ACID-PHOSPHATASES FROM RAT BONE

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 (<40,000) and shows negligible activity with monophosphate esters [except with 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 by p-chloromercuribenzoate. With p-NPP as substrate, E1 and E2 have distinctly different values for Km. 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. © 1979 Springer-Verlag.