title: Purine nucleoside phosphorylase from bovine lens: purification and properties.
abstract: Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38,750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97,000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15,250 and 14,650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.
authors.affiliation: Dipartimento di Fisiologia e Biochimica, Università di Pisa, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Barsacchi
authors.forename: M
authors.initials: M
authors.lastname: Cappiello
authors.forename: M G
authors.initials: MG
authors.lastname: Tozzi
authors.forename: A
authors.initials: A
authors.lastname: Del Corso
authors.forename: M
authors.initials: M
authors.lastname: Peccatori
authors.forename: M
authors.initials: M
authors.lastname: Camici
authors.forename: P L
authors.initials: PL
authors.lastname: Ipata
authors.forename: U
authors.initials: U
authors.lastname: Mura
mesh_terms: D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008970:Molecular Weight; D010777:Photochemistry; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity
chemical_list: D011683:Purine-Nucleoside Phosphorylase