Measurement of caprine plasma beta-mannosidase with a p-nitrophenyl substrate.
Experimental conditions for measuring caprine plasma beta-D-mannosidase activity are described with p-nitrophenyl-beta-D-mannopyranoside as a substrate. The plasma enzyme was stable for 3 months at -20 C or 1 week at 4 C. The optimal pH for activity was 5.0 in citrate-phosphate or acetate buffer. Enzyme activity was linear with time up to 24 hours at 37 C, but incubation of plasma at 56 C for 5 minutes resulted in loss of all activity. The apparent Michaelis-Menten constant (Km) for the plasma enzyme was 10.0 mM. Plasma beta-mannosidase from clinically normal and beta-mannosidosis carrier goats did not differ with respect to pH optimum, heat stability, or Km. The coefficient of variation for the assay, determined by assaying a plasma pool over a 3-month period, was 10.7% (mean: 115 nmole of p-nitrophenol formed/hour/ml of plasma). The assay described can be used to evaluate plasma beta-mannosidase measurements as a test for detecting carriers of caprine beta-mannosidosis, a newly described lysosomal storage disease.
D000818:Animals; D006580:Genetic Carrier Screening / Q000379:methods; D006023:Glycoproteins / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D006041:Goats / Q000097:blood*; D007700:Kinetics; D008361:Mannosidases / Q000097:blood* / Q000378:metabolism; D008362:Mannosides / Q000378:metabolism; D008661:Metabolism, Inborn Errors / Q000235:genetics / Q000662:veterinary; D044902:beta-Mannosidase
D006023:Glycoproteins; D006027:Glycosides; D008362:Mannosides; C036119:4-nitrophenyl-beta-D-mannopyranoside; D008361:Mannosidases; D044902:beta-Mannosidase