Rabbit erythrocyte purine nucleoside phosphorylase. Differential-inactivation studies

Abstract

1. Qualitative studies on the stability of rabbit erythrocyte purine nucleoside phosphorylase showed a marked decrease in the susceptibility of the enzyme to thermal inactivation and digestion by proteinases of different specificities in response to certain of its substrates. 2. The extent to which inosine stabilizes the enzyme against thermal and proteolytic inactivation is related in a quantitative manner to the concentration of this substrate; it is proposed that differences in the rates of inactivation of the enzyme may reflect substrate-induced conformational changes in the enzyme structure that could alter the binding properties of the enzyme in a kinetically significant way. 3. A synergistic effect in the stabilization of the enzyme is observed in response to both substrates, inosine and phosphate, when the enzyme is inactivated with Pronase. 4. In the presence of substrate an increased rate of inactivation after reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) is reported. 5. Differential-inactivation studies were also carried out with calf spleen purine nucleoside phosphorylase, and the results are discussed in relation to the kinetic properties displayed by this enzyme.

Rabbit erythrocyte purine nucleoside phosphorylase. Initial-velocity studies

“…Kinetic studies on human erythrocyte 1979 purine nucleoside phosphorylase have also indicated that the phosphorolytic reaction proceeds via an ordered, sequential reaction sequence, and moreover, binding studies showed that guanosine, but not orthophosphate, readily binds to the native enzyme (Kimetal., 1968). Also, in thefollowingpaper (Savage & Spencer, 1979) and elsewhere (Savage, 1978), evidence is presented that indicates that inosine and guanosine readily bind to the native rabbit enzyme, whereas orthophosphate under similar conditions will apparently only bind to the enzyme that already has the nucleoside bound. It may be pertinent at this point to mention that a random reaction sequence may give rise to non-linear double-reciprocal plots (Dalziel, 1958), since such a mechanism provides the possibility of more than one enzymic form with which the substrate may react on the way to products.…”