PARTIAL CHARACTERIZATION OF A NEW NUCLEOTIDE-BINDING GLYCOPROTEIN OF HEPATOCYTE PLASMA-MEMBRANE

Hepatocyte plasma membranes contain a glycosylated 230-kDa Ca2+-depend ent, Mg2+-stimulated ATPase (pgp230), which consists of two subunits, one of 120 kDa and the other of 110 kDa. pgp230 can be enriched by the use of affinity chromatography on Concanavalin A-Sepharose, wheat ger m lectin-Sepharose, and 5'-AMP-Sepharose. It has a high affinity Ca2binding site. In the presence of Ca2+, it forms a phosphorylated inter mediate by autocatalytic transfer of the terminal phosphate residue fr om ATP. Maximal Ca2+-dependent autophosphorylation is observed at pH 5 -6. Photoaffinity labeling using 8-azido-[alpha-P-32]ATP or [y-P-32]AT P confirms the presence of ATP binding sites. Incubation with [alpha-P -32]ATP leads to a rapid but transient labeling of pgp230. Various nuc leotides, nucleotide receptor agonists, or antagonists inhibit Ca(2+)d ependent phosphorylation by [y-P-32]ATP. The concentrations of half-ma ximal inhibition range from 10(-7) M to 10(-3) M. The rank order of in hibitory potency is: ATP pha,beta-methylene-ATP>CTP=TTP>y-4-aminopheny l-ATP eta,y-methylene-TTP=beta,y-methylene-GTP=adenosine iodiphosphate =CMP=AMP>adenosine>cytidine>guanosine =suramin>Reactive blue 2 >iso-bu tyl-methyl-xanthine>thymidine>uridine. These data suggest a nucleotide binding capacity of this new hepatocyte membrane glycoprotein. Furthe r investigations should be carried out to reveal its biological functi on.