title: Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes.
abstract: Human red cell lysates contain at least seven electrophoretically distinct isoenzymes of purine-nucleoside phosphorylase (PNPase); the proportion of more anodal bands increases as the erythrocyte ages, suggesting that the native enzyme is subjected to progressive post-translational modifications. The age dependent electrophoretic changes observed in the hemolysate are associated with the downward curvature of the Lineweaver-Burk double reciprocal plot at high inosine-substrate concentrations unlike the single-banded PNPase from tissue cultures of rapidly dividing cells. Thanks to the high resolution power of the ion-exchange HPLC technique utilized we have been able to fully separate all the seven isoenzymes and correlate structural to functional modifications in PNPase from human erythrocytes. Our results indicate that the downward curvature of Lineweaver-Burk plot is not due to a mixture of isoforms with low and high Km for inosine but that the allosteric activation by the inosine substrate is the direct consequence of structural modification(s) on the "primary" form of the enzyme.
authors.affiliation: Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studi di Urbino, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Accorsi
authors.forename: M P
authors.initials: MP
authors.lastname: Piacentini
authors.forename: E
authors.initials: E
authors.lastname: Piatti
authors.forename: A
authors.initials: A
authors.lastname: Fazi
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood* / Q000302:isolation & purification
chemical_list: D007527:Isoenzymes; D011683:Purine-Nucleoside Phosphorylase