A NEW-TYPE OF CA2-DEPENDENT, MG2+-STIMULATED ATPASE OF RAT-LIVER PLASMA-MEMBRANE()

Incubation of a glycoprotein fraction obtained from rat liver plasma m embrane which has been previously well characterized using [gamma-P-32 ]ATP results in the phosphorylation of a 230-kDa glycoprotein (pgp230) . It is composed of a 120-kDa subunit (pgp120) and a 110-kDa subunit ( pgp110) linked by interchain disulfide bonds. Peptide maps of pgp120 a nd pgp110 suggest extensive similarity in their polypeptide chains. Gl ycan analysis reveals between four and six hybrid-type oligosaccharide chains for both phosphoproteins. Immunoblotting using monoclonal anti bodies and endoglycosidase digestion exclude an identity of pgp120 or pgp110 with the hepatocyte plasma membrane glycoproteins dipeptidylpep tidase IV or the taurocholate transport protein, which co-purify and c omigrate in SDS/PAGE. Protein phosphorylation is Ca2+-dependent (K-0.5 (Ca2+) = 0.35 mu M, in the absence of Mg2+). In the presence of Mg2+, the glycoprotein undergoes rapid cycles of phosphorylation and dephosp horylation, resulting in ATPase activity. Analysis of phosphorylated a mino acids identifies phosphothreonine as the major one. Photoaffinity labeling with 8-azido-[alpha-P-32]ATP demonstrates the presence of on e or more ATP binding site(s). Preincubation of pgp230 with various pu rine or pyrimidine nucleotides (ATP, UTP, TTP ADP, GDP, AMP, CMP) or k nown P-2-purinoceptor agonists or antagonists (adenosine 5'-[alpha,bet a-methylene] triphosphate, 2-methyl-thio-adenosine 5'-triphosphate, su ramin) inhibits its phosphorylation by [gamma-P-32]ATP. The biological function of pgp230 is unknown at present. Several findings of the pre sent study are compatible with the idea that pgp230 may be involved in a P-2-purinoceptor function of the hepatocyte. Following this concept , a mechanism is discussed where a cytosolically exposed high-affinity Ca2+-binding site of pgp230 would allow for receptor feedback control , via phosphorylation and dephosphorylation, by sensing changes in cyt osolic Ca2+ concentration.