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Km And Vmax Determination Of P-Npb Case Study

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Km and Vmax determination of glucocerebrosidase towards p-NPG
The Michaelis constant (Km) is a measure of the affinity of the enzyme towards the substrate, with smaller values representing greater affinity. Km and the maximum rate (Vmax) of leukocyte GCase were obtained through the Lineweaver-Burk plot (Figure 4) with artificial substrate p-NPG in concentrations from 0.71 mM to2.50 mM. Km and Vmax values for leukocyte GCase using p-NPG as substrate was found to be 12.6 mM and 333 U/mg respectively.

Fig. 4. Determination of Michaelis constant of GCase for different p-NPG substrate concentrations (0.71 mM-2.50 mM) in sodium acetate buffer (50mM, pH 5.0). All the samples were assayed in the presence of 6 mg/ml sodium taurocholate and 70 μl of …show more content…
Daniels, L.B., Glew, R.H., Diven, W.F., Lee, R.E., and Radin, N.S. (1981) An improved fluorometric leukocyte β-glucosidase assay for Gaucher’s disease. Clin. Chim. Acta, 115 (3), 369–375.
13. Wenger, D.A., Clark, C., Sattler, M., and Wharton, C. (1978) Synthetic substrate \s s-glucosidase activity in leukocytes: A reproducible method for the identification of patients and carriers of Gaucher’s disease. Clin. Genet., 13 (2), 145–153.
14. Beutler, E., Kuhl, W., Trinidad, F., Teplitz, R., and Nadler, H. (1971) Beta-glucosidase activity in fibroblasts from homozygotes and heterozygotes for Gaucher’s disease. Am. J. Hum. Genet., 23 (1), 62.
15. Ho, M.W., Seck, J., Schmidt, D., Veath, M.L., Johnson, W., Brady, R.O., and O’Brien, J.S. (1972) Adult Gaucher’s disease: kindred studies and demonstration of a deficiency of acid beta-glucosidase in cultured fibroblasts. Am. J. Hum. Genet., 24 (1), 37.
16. Magalhaes, J., SáMiranda, M.C., Pinto, R., Lemos, M., and Poenaru, L. (1984) Sodium taurocholate effect on β-glucosidase activity: a new approach for identification of Gaucher disease using the synthetic substrate and leucocytes. Clin. Chim. Acta, 141 (2–3), …show more content…
Michelin, K., Wajner, A., Goulart, L. da S., Fachel, Â.A., Pereira, M.L.S., de Mello, A.S., Souza, F.T., Pires, R.F., Giugliani, R., and Coelho, J.C. (2004) Biochemical study on β-glucosidase in individuals with Gaucher’s disease and normal subjects. Clin. Chim. Acta, 343 (1), 145–153.
35. Strasberg, P.M., and Lowden, J.A. (1982) The assay of glucocerebrosidase activity using the natural substrate. Clin. Chim. Acta, 118 (1), 9–20.
36. Grabowski, G.A., and Dagan, A. (1984) Human lysosomal β-glucosidase: Purification by affinity chromatography. Anal. Biochem., 141 (1), 267–279.
37. Maret, A., Salvayre, R., Potier, M., Legler, G., Beauregard, G., and Douste-Blazy, L. (1988) Comparison of Human Membrane-Bound β-Glucosidases: Lysosomal Glucosylceramide-β-Glucosidase and Non-Specific β-Glucosidase, in Lipid Storage Disorders, Springer, pp. 57–61.
38. Fishman, W.H., Springer, B., and Brunetti, R. (1948) Application of an improved glucuronidase assay method to the study of human blood β-glucuronidase. J. Biol. Chem., 173 (2), 449–456.
39. Kato, K., Yoshida, K., Tsukamoto, H., Nobunaga, M., Masuya, T., and Sawada, T. (1960) Synthesisof p-Nitrophenyl β-D-Glucopyranosiduronic Acid and Its Utilization as a Substrate for the Assay of β-Glucuronidase Activity. Chem. Pharm. Bull. (Tokyo), 8 (3),

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