MICROSOMAL AND LYSOSOMAL ACID PHOSPHATASE ISOENZYMES OF MOUSE KIDNEY CHARACTERIZATION AND SEPARATION

Abstract

Biochemical methods have been employed to characterize and separate acid phosphatases from lysosomal and microsomal fractions in order to decide whether different isoenzymes reside in these subcellular locations. Microsomal and lysosomal fractions of mouse kidney homogenate were isolated by differential centrifugation. Acid phosphatase of lysosomal fraction goes into solution after lysosomes have been repeatedly frozen and thawed, whereas acid phosphatase of microsomal fraction is firmly bound to the membrane and is freed of contamination by lysosomal enzyme after ultrasonication and centrifugation. The membrane-bound microsomal acid phosphatase is labile at 37°C, pH 4.9, more active toward phenolic substrates (phenyl phosphate and p-nitrophenyl phosphate) than β-glycerophosphate, α-naphthol phosphate or naphthol AS-BI phosphate. It also has a higher pH optimum (6.3), is resistant to l-tartrate and oxalate inhibition and has a slower electrophoretic migration rate in Triton X-100-impregnated polyacrylamide gels. The free lysosomal acid phosphatase is relatively heat-stable, is less active against phenolic substrates, is sensitive to l-tartrate and oxalate inhibition, has a lower pH optimum (5.6) and has a faster migration rate in electrophoresis. These two acid phosphatases can also be separated by diethylaminoethyl-cellulose chromatography. This study thus demonstrated the existence of an acid phosphatase isoenzyme in the microsomal membrane with different biochemical properties from the lysosomal isoenzyme of acid phosphatase.