Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase A.Guissani and C.Portier Institut de Biologie Physico-chimique,

It is already known that modification of E. col i polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites. I N T R O D U C T I O N During the last decade, the elucidation of the reaction mechanism and protein structure of polynucleotide phosphorylase from various sources has made great progress. (1). Nevertheless, the structure-function relationship of this enzyme remains unsolved. Very recently, the quaternary structure of E. coli PNPase has been worked out (2). This enzyme can be isolated in two molecular forms A and B having very similar catalytic properties ; the function of the P subunit is as yet unknown. The A species, probably corresponding to the extensively purified enzyme studied in this laboratory up to now, carries the active center both different and distant from the "polynucleotide attachment s i te" , (subsides I I , (3) ) that we supposed to be dispersed over a large area of the enzyme surface. The existence of this attachment site seems of prime importance (3 ,4 ,5 ,6 , ) with regard to the mechanisms of action of PNPase, either progressive or synchronous. A model study was undertaken with an altered enzyme called "4°C polynucleotide phosphorylase" that shows modified catalytic properties 3015 © Information Retrieval Limited 1 Falconberg Court London W1V5FG England Nucleic Acids Research resulting from the loss of part of the protein molecule through endogenous proteolytic degradation (7). The present paper describes the structural parameters and discusses the mode of action of this proteolysed enzyme in relation to the loss of polymer attachment sites. MATERIAL AND METHODS Chemicals Biogel A 1.5 m was from Biorad ; non-labelled nucleotide ADP from Sigma ; labelled nucleotide ( C) ADP from Amersham ; and labelled orthophosphate from Commissariat al'Energie Atomique. Poly A and (pA) „ were prepared in this laboratory as well as poly (A) 14 ° U ( C) . Electrophoresis products were obtained from Canaico ; sodium dodecylsulphate and urea were products of Merck ; dimethylsuberimidate was a gi f t from Alain Expert Besangon (IBPC, Paris). Marker proteins were either from Worthington or from Boehringer. E. col i proteolysed enzyme : Native polynucleotide phosphorylase was purif ied by the standard procedure (2) ; it was then left at 4°C for seven weeks under the action of endogenous proteases ; degradation was regularly followed by electrophoresis on acrylamide gel and in situ polymerisat ion (8) . This enzyme is referred to as "polynucleotide phosphorylase 4 ° C " . Stokes Radius determination : This was performed through a calibrated column of Biogel A 1 .5 m, according to Ackers (9). Parameters and formula used have been described in a previous paper (2). Enzymic act ivi ty assays : Polymerisation has been measured by ( C) ADP incorporation into the acid insoluble precipitate. Incubations were 32 performed at 37°CPhosphorolysis in the presence of labelled ( P) inorganic orthophosphate was followed by the formation of labelled diphosphate nucleoside. For Km measurements of polynucleotides, a continuous method of phosphorolysis was used (10). Acrylamide gels : Series of gels at different acrylamide concentrations were performed according to Hedrick and Smith (11). Sodium dodecylsul-