The Differentiation of Acid Phosphatases of Human Blood Platelets

Summary

The Acid Phosphatase was tested in human platelets and in the rat liver mitochondrial-lysosomal fraction with p-nitrophenylphosphate and β-glycerophosphate as substrates. In the platelets the following differences were found between the hydrolysis of these two substrates, whereas in rat liver no such differences were observed. 1. The relative rates of hydrolysis and the pH optima for both substrates are different (pH 4.6 for the β-glycerophosphatase, pH 6.0 for the p-nitrophenylphosphatase in the platelets). 2. The p-nitrophenylphosphatase of the platelets is inhibited by p-chlormercuribenzoate and N-ethylmaleimide, but not by fluoride or L + tartrate, whereas the contrary is true for the platelet β-glycerophosphatase and the rat liver activities. 3. The platelet p-nitrophenylphosphatase is rapidly inactivated by preincubation at 40-45° C for 15 min, the other phosphatases are much more heat-resistant. 4. Sucrose density gradient centrifugation of platelet homgenates showed a separation of the two platelet phosphatase activities, the p-nitrophenylphosphatase with its maximum at lower densities than the β-glycerophosphatase.

It is concluded that in human platelets there are at least two different Acid Phosphatases. The β-glycerophosphatase probably represents the lysosomal (as compared to the rat liver enzyme) phosphatase whereas the p-nitrophenylphosphatase of the platelets is a different enzyme whose subcellular localization and functions are as yet unknown.

• References

• 1 Baggiolini M, Hirsch J. G, de Duve G. h. Resolution of Granules from Rabbit Heterophil Leucocytes into Distinct Populations by Zonal Centrifugation. J. Cell Biol 40: 529 1969;
• 2 Baggiolini M, Hirsch J. G, de Duve C. h. Further Biochemical and Morphological Studies of Granule Fractions from Rabbit Heterophil Leucocytes. J. Cell Biol 45: 586 1970;
• 3 Barber A. J, Jamieson G. A. Isolation and Characterisation of Plasma Membranes from Human Blood Platelets. J. Biol. Chem 245: 6357 1970;
• 4 Blank M. L, Snyder F. Specifities of Alkaline and Acid Phosphatases in the rephosphorylat- ing of Phospholipids. Biochemistry 09: 5034 1970;
• 5 Chen T. T, Toribara T. Y, Warner H. Microdetermination of Phosphorus. Anal. Chem 28: 1756 1956;
• 6 Cohn Z. A, Hirsch J. G. The Isolation and Properties of the specific cytoplasmic Granules of Rabbit polymorphonuclear Leucocytes. J. Exp. Med 112: 983 1960;
• 7 Day H. J, Holmsen H, Hovig T. Subcellular Particles of Human Blood Platelets. Scand. J. Haemat., Suppl 7 1969;
• 8 de Duve C. h, Wattiaux R. Functions of Lysosomes. Ann. Rev. Physiol 28: 435 1966;
• 9 Gianetto R, de Duve C. h. Tissue Fraction Studies 4. Biochem. J 59: 433 1955;
• 10 Henning R, Kaulen H. D, Stoffel W. Isolation and Chemical Composition of the Lysosomal and the Plasma Membrane of the Rat Liver Cell. Hoppe-Seyler’s Ztschr. f. physiol. Chem 351: 1191 1970;
• 11 Hirschhorn R, Hirschhorn K, Weissmann G. Appearance of hydrolase rich Granules in Human Lymphocytes induced by PHA and Antigen. Blood 30: 84 1967;
• 12 Holmsen H, Day H. J. The Selectivity of the Thrombin-induced Platelet Release Reaction : Subcellular Localization of released and retained Constituents. J. Lab. Clin. Med 75: 840 1970;
• 13 Holmsen H, Day H. J, Pimentel M. A. Adenine Nucleotide Metabolism of Human Platelets V. Biochim. Biophys. Acta 186: 244 1969;
• 14 Kaulen H. D, Henning R, Stoffel W. Comparison of some Enzymes of the Lysosomal and the Plasma Membrane of the Rat Liver Cell. Hoppe-Seyler’s Ztschr. f. physiol. Chem 351: 1555 1970;
• 15 Kowalski E, Kopec M, Wegrzynowicz Z, Hurwic M, Budzynski A. Z. A lysosomal Concept of the Platelet Release Reaction and Viscous Metamorphosis. Thrombos. Diathes. Hämorrh. (Stuttg) 16: 134 1966;
• 16 Linneweh F, Löhr G. W, Waller H. D, Gross R. Über die Diagnose von Glykogenosen aus der Glucose-6-phosphatase Aktivität und dem Glykogen-Gehalt der Thrombozyten. Enzym, biol. clin 02: 188 (1962/63).
• 17 Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. Protein Measurement with the Folin Phenolreagent. J. Biol. Chem 193: 265 1951;
• 18 Marcus A. J, Zucker-Franklin D, Safier L. B, Ullman H. L. Studies on Human Platelet Granules and Membranes. J. Clin. Invest 45: 14 1966;
• 19 Moake J. L, Ahmed K, Bacher N. R, Gutfreund D. E. Mg-dependent, Na-K-stimulated ATP’ase of Human Platelets. Biochim. Biophys. Acta 02: 337 1970;
• 20 Nadler H. L, Egan ThJ. Deficiency of lysosomal Acid Phosphatase. New Engl. J. Med 282: 302 1970;
• 21 Nelson B. D. Rat Liver Acid Phosphatase: Differences in Lysosomal and Cytoplasmic Forms. Proc. Soc. Exp. Biol. Med 121: 998 1966;
• 22 Paigen K, Griffiths S. K. The intracellular Location of Phosphoprotein Phosphatase Activity. J. Biol. Chem 234: 299 1959;
• 23 Siegel A, Lüscher F. Non-Identity of α-Granules of Human Blood Platelets with typical Lysosomes. Nature 215: 745 1967;
• 24 Straus W. Concentration of Acid Phosphatase, Ribonuclease, Desoxyribonuclease, β-Glucu-ronidase and Cathepsin in “Droplets” isolated from the Kidney Cells of normal Rats. J. Biophys. Biochem. Cytol 02: 513 1956;
• 25 Wagner R, Yourke A. Alkaline and Acid Phosphatases in White Blood Cells and Blood Platelets. Arch. Biochem 54: 174 1955;