Characteristics of membrane-bound 5'-nucleotidase on human prostasomes.
Examination of prostasomes, isolated from human seminal plasma, showed that there was very little remaining paranitrophenylphosphatase activity when assayed in the presence of 10 mmol/l of tartrate and 2 mmol/l of levamisole. Under these conditions it was possible to study the prostasome membrane-bound 5'-nucleotidase activity, which was unaffected by these two inhibitors. The activity was considered to be located at the external surface of the prostasome membrane and a 50-60% increase in activity was obtained by the addition of 0.05% Triton X-100. The prostasome membrane-linked 5'-nucleotidase readily hydrolysed 5'-AMP. Two other 5'-nucleoside monophosphates, 5'-IMP and 5'-GMP, were also hydrolysed, but more slowly; 2'- or 3'-AMP were practically not attacked. The prostasome membrane-linked 5'-nucleotidase obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 11.2 +/- 2.1 mumol/l and Vmax 64.7 +/- 11.4 nmol/mg protein/min. These figures were somewhat changed in presence of 0.05% Triton X-100, the Km value being reduced by 30% and the Vmax value increased by 60%. Adenosine 5' (alpha, beta methylene) diphosphate (100 mumol/l), Ni2+ (10 mmol/l) and concanavalin A (20 micrograms/ml) were all potent inhibitors of the prostasome membrane-linked 5'-nucleotidase.
Fabiani
R
R
Department of Clinical Chemistry, University Hospital, University of Uppsala, Sweden.
Ronquist
G
G
D015720:5'-Nucleotidase / Q000032:analysis* / Q000037:antagonists & inhibitors; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000328:Adult; D003208:Concanavalin A / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology; D008297:Male; D009708:Nucleotidases / Q000032:analysis; D017830:Octoxynol / Q000494:pharmacology; D015388:Organelles / Q000187:drug effects / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D012661:Semen / Q000187:drug effects / Q000201:enzymology*; D013644:Tartrates / Q000378:metabolism / Q000494:pharmacology
D013644:Tartrates; C523965:alpha,beta-methyleneadenosine 5'-diphosphate; D003208:Concanavalin A; D007978:Levamisole; D000244:Adenosine Diphosphate; D017830:Octoxynol; D009708:Nucleotidases; C018775:adenosine monophosphatase; D010744:Phosphoric Monoester Hydrolases; D015720:5'-Nucleotidase