Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma.
Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.
Minelli
A
A
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, University of Perugia, Italy.
Moroni
M
M
Fabiani
R
R
Miscetti
P
P
Mezzasoma
I
I
D015720:5'-Nucleotidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D012661:Semen / Q000201:enzymology*; D013379:Substrate Specificity
D008670:Metals; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase