Km And Vmax Determination Of P-Npb Case Study

Superior Essays
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),

Related Documents

  • Improved Essays

    Discussion In this study, the Catechol enzyme was studied under the conditions of varying pH, temperature, substrate concentration, and enzyme concentration. In Figure 1, the data suggested that the trend was neither directly nor inversely proportional, but the highest activity rate was at 24°C. Most enzymes denatured at higher temperatures of approximately 40°C, which led to the inability to see any color change (Helms et al., 1998). At lower temperatures, the enzyme was somewhat efficient because molecules move slower at lower temperatures, so enzymes lost productivity.…

    • 753 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    Consequently, five test tubes were prepared at different temperatures to observe which test tube's temperature is the optimal temperature of the enzyme. These are the test tube and their temperature; Tube 1- was blank, Tube 2- room temperature, Tube 3- 35° C, Tube 4- 45° C and Tube 5- 55° C. These tubes had 1 mL of the substrate mixture and 1 mL of pH 6 buffer. These tubes were placed in a spectrophotometer to measure their reaction.…

    • 1134 Words
    • 5 Pages
    Improved Essays
  • Decent Essays

    Sweta Soni 2/22/15 Biochem Lab Experiment 5 Introduction The purpose of Experiment 5-1 is to separate a mixture of three molecules—blue dextran, cytochrome c, and potassium chromate—through the use of gel-filtration chromatography. The volume at which each molecule elutes will be recorded in order to determine the accuracy of separation. The purpose of Experiment 5-2 is to purify the E. coli enzyme, β-Galactosidase through the use of affinity chromatography on P-aminobenzyl-1-thio-β-D-galactopyranoside agarose, which is a good substrate for the enzyme.…

    • 363 Words
    • 2 Pages
    Decent Essays
  • Superior Essays

    1.Introduction: Background/Aim. 1.1 Nature of the microorganism: Enterococcus faecalis was placed under group D streptococci because of similar lysis characters; Identified as Gram positive, non-motile, anaerobic fermentative, lactic acid bacteria; are commensal inhabitant of intestinal bacteria and opportunistic pathogens. They occur singly or in pairs or chains. 1.2 Economic importance: Enterococcus faecalis is also used as a probiotic for production of Mediterranean cheese and meat products that is normally responsible for organoleptic nature of final product in fermentation reactions. 1.3 Habitat: Enterococci are extremely hardy microbes such that they are able to inhabit at wide range of temperatures (10°and 45°C), pH values (4•6 and…

    • 1058 Words
    • 5 Pages
    Superior Essays
  • Improved Essays

    Similar symptoms are seen in other different types of leukodystrophies. Current…

    • 547 Words
    • 3 Pages
    Improved Essays
  • Great Essays

    Peroxidase Lab Report

    • 1615 Words
    • 7 Pages

    The effect that temperatures at 4°C, 23°C, 32°C, and 48°C , pH 3, 5, 7, and 9, the boiled extract, and hydroxylamine had on the peroxidase enzyme extracted from Brassica rapa. Emma O’Donoghue Fundamentals of Biology I Lab Professor William Olsen October 8, 2015 Abstract The purpose of this lab was to determine the effects that a number of factors had on enzyme activity. The enzyme used during this lab was peroxidase, which was extracted from the organism Brassica rapa.…

    • 1615 Words
    • 7 Pages
    Great Essays
  • Superior Essays

    Gene Expression Lab Report

    • 2568 Words
    • 11 Pages

    Gene Expression Paper RESULTS The hypothesis is that as the amount of lactose increases in a treatment, the higher the enzyme production and activity. A total of six treatments were done in order to determine whether the amount of lactose in a substance correlates with the amount and production of b-galactosidase. Experiment 1: The use of OD600 as an indicator of bacterial population size. In addition, the use of Abs 420 as an indicator of protein production.…

    • 2568 Words
    • 11 Pages
    Superior Essays
  • Improved Essays

    Enzyme Lab Report

    • 537 Words
    • 3 Pages

    EXPERIMENT: The effect of an acidic fluid on the activity of an enzyme INTRODUCTION: There are several factors that may influence the activity of an enzyme to include temperature and the pH levels. In order to better understand how the pH level affects the actions of an enzyme, the experiment will introduce different foods with acids and bases through mixture of direct contact. OBJECTIVE:…

    • 537 Words
    • 3 Pages
    Improved Essays
  • Great Essays

    UGT1A4 Research Paper

    • 1008 Words
    • 5 Pages

    et al. Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A. The Journal of biological chemistry 282, 36514-36524, doi:10.1074/jbc. M703107200…

    • 1008 Words
    • 5 Pages
    Great Essays
  • Improved Essays

    Pompe disease is an autosomal recessive disorder caused by partial or complete deficiency of acid alpha-glucosidase (GAA), an enzyme involved in the breakdown of glycogen into simple sugars. Mutations in the gene coding for GAA, prevent enzymatic activity of α-glucosidase, which results in toxic build up of polysaccharide inside lysosomes, and leads to damage and debilitation of organs and tissues, primarily skeletal and cardiac muscles. Individuals with both copies of the altered gene are diagnosed with the disease, while individuals with only one copy of the mutated gene are considered carriers and remain asymptomatic throughout their lifespan. Three categories of Pompe disease have been identified depending in the severity of the mutation,…

    • 629 Words
    • 3 Pages
    Improved Essays
  • Improved Essays

    Introduction Ascorbic acid is a form of vitamin C that is used by the body as a means of promoting healing of cuts and wounds, as well as boosting the immune system and as such aiding in the fight against infections. Ascorbic acid does not occur naturally and is formed by carrying out a variety of chemical reactions on corn syrup. The chemical formula for ascorbic acid is C6H8O6 and is similar to glucose which has a formula of C6H1206, this closeness in structure allows most animals (excluding humans) to convert glucose vitamin C (James Howenstine,2006). The Glut-1 receptor that activates when insulin is released in the body allows glucose and vitamin C (or in this case ascorbic acid) to enter white blood cells. White blood cells contain more…

    • 345 Words
    • 2 Pages
    Improved Essays
  • Improved Essays

    Abstract Introduction A 2-year and 7-month-old girl is undergoing several treatments to attenuate the effects of type 2 Gaucher disease on her health. She has been diagnosed with type 2 Gaucher disease since she was three months old and several symptoms have appeared during that short lap of time. Case presentation In a type 2 Gaucher disease patient, the lysosomal enzyme β-glucocerebrosidase undergoes mutation and is therefore unable to break down glucocerebroside into ceramide and glucose.…

    • 528 Words
    • 3 Pages
    Improved Essays
  • Improved Essays

    The enzyme acts on the substrate glucocerebroside which is a component of the cell membrane. In the normal lysosome, protein saposin C presents glucocerebroside to GBA which activates the enzyme. This enzyme is responsible for hydrolytic breakdown of glucosylceramide to glucose and ceramide. Deficiency of the enzyme leads to accumulation of glucosylceramide and other glycolipids in the lysosomes of macrophages, primarily in the spleen, liver, bone marrow, brain, osteoclasts and less often the lungs, skin, kidneys, conjunctivae and heart. The decimated form of glucosylceramide, glucosylsphingosine, is elevated in neuropathic disease and correlates more with phenotype severity compared to…

    • 1211 Words
    • 5 Pages
    Improved Essays
  • Decent Essays

    Type-1 Dm Case Studies

    • 322 Words
    • 2 Pages

    Background: Type-1 diabetes mellitus (T1-DM) is the commonest endocrine-metabolic disease in childhood. The prevalence of CD in type-1 DM ranges from 0.6 to 16.4% compared with 0.01–0.03% in the general population. The mechanism of association between the two diseases involves a shared genetic background of HLA genotype. Serum tissue transglutaminase IgA antibodies (tTG IgA) are considered specific and sensitive markers for screening of Celiac disease in more than 95 % of patients. Objective: Screening for the presence of serum tissue transglutaminase IgA antibodies (tTG ab) as a specific and sensitive biochemical marker for Celiac disease in patients with type-1DM and its relation to the clinical manifestations of those patients.…

    • 322 Words
    • 2 Pages
    Decent Essays
  • Improved Essays

    Lactate dehydrogenase (LDH) is an oxidoreductase found in both eukaryotes and prokaryotes. LDH catalyses the formation of lactate and NAD+ from pyruvate and NADH, during the last step of anaerobic glycolysis. The reaction is also catalysed in the other direction by LDH during the Cori cycle. This reaction can be assayed using spectrophotometric techniques; peak absorbance of NADH is at 340nm, but the peak absorbance of NAD+ is 259nm (Powers, et al., 2007). Enzyme assays are performed to measure the rate of an enzyme-catalysed reaction, known as the enzyme activity.…

    • 1627 Words
    • 7 Pages
    Improved Essays