NUCLEOSIDE DIPHOSPHATE KINASE ASSOCIATED WITH PISUM-SATIVUM MICROSOMAL-MEMBRANES - APPARENT BINDING OF GTP-GAMMA-S AT NM CONCENTRATIONS

Anion exchange chromatography of microsomal membrane polypeptides prep ared from etiolated Pisum sativum hypocotyls generated 3 fractions of proteins which exhibited high affinity GTPgammaS binding. Scatchard an alysis of the major binding fraction and of recombinant P. sativum nuc leoside disphosphate kinase (NDK) indicated binding constants of 44 nM and 53 nM respectively. Analysis of proteins from the same ion-exchan ge fraction by SDS-PAGE and fluorography following incubation with [S- 35]-GTPgammaS, enabled visualisation of a 16 kDa [S-35]-thio-phosphory lated protein, whilst immunoblotting of the proteins with anti-NDK-ant ibodies also labelled a 16 kDa polypeptide. This protein was highly en riched by HiTrap Blue-Sepharose chromatography using ATP as eluent, an d could be easily recovered from Procion Red HE3B-Sepharose, eluting a t a position coincident with the apparent GTPgammaS binding peak. Thes e data indicate that in the presence of GTPgammaS at low concentration s, membrane associated NDK, will auto-thio-phosphorylate, and interfer e significantly with measurement of real, reversible binding phenomena . High affinity GTPgammaS binding measurements are therefore not good diagnostic indicators of the presence of signal transducing G-proteins in membrane preparations from plant tissues.