Determination of acid hydrolases in human platelets

Abstract

Most acid hydrolases in platelets are determined by methods developed for other tissues. Therefore, we have established the optimal conditions for the measurements of 13 acid glycosidases, aryl phosphatase, aryl sulfatase, and β-glycerophosphatase in crude and dialyzed lysates of human platelets. The substrates used were the glycosides, phosphates and sulfates of p-nitrophenol and 4-methylumbelliferone, phenolphthalein-β-glucuronide, p-nitrocatechol sulfate and β-glycerophosphate. The pH optimum for each enzyme activity did not depend on the type of substrate, whereas K m and V could vary as much as 100-fold. For most of the acid hydrolases, V was higher than the enzyme level determined in the usual way, with the highest substrate concentration obtainable, because this concentration was close to K m . The possible existence of low molecular weight enzyme inhibitors in the lysates was tested by comparing K m at two different lysate concentrations and by comparing the enzyme level in crude lysate with that obtained after dialysis. Only inhibitors for α- and β-galactosidase and aryl sulfatase were found. α-Glucosidase had two pH optima, one at pH 4.1 and the other at 6.0. p-Nitrocatechol sulfatase had a nonlinear time course, similar to the enzyme present in liver. Where possible, K m and V were determined in both citrate-phosphate and acetate buffers, and differences were found for β-fucosidase, α-galactosidase, β-glucosidase, β- N-acetylglucosaminidase, and α-mannosidase. The sensitivity and accuracy of the fluorophor-based assays with 4-methylumbelliferone-containing substrates were markedly better than the chromophore-based assays. In contrast, the determination of β-glycerophosphatase was particularly inaccurate due to formation of large amounts of phosphate from endogenous sources in the lysates. The following acid hydrolases activities were found in human platelet lysates (listed in descending order of total levels): Aryl phosphatase, β- N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, α-mannosidase, aryl sulfatase ( p-nitrocatechol substrate), α-arabinosidase, β- N-acetylgalactosaminidase, α-galactosidase, α-fucosidase, β-fucosidase, β-glucosidase, and α-glucosidase (pH 4.1). β-Cellubiosidase and α-xylosidase were not present in the lysates.