Partial characterization of a new nucleotide binding glycoprotein of hepatocyte plasma membrane.
Hepatocyte plasma membranes contain a glycosylated 230-kDa Ca(2+) -dependent, Mg(2+)-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 germ lectin-Sepharose, and 5'-AMP-Sepharose. It has a high-affinity Ca2+ binding site. In the presence of Ca2+, it forms a phosphorylated intermediate by autocatalytic transfer of the terminal phosphate residue from ATP. Maximal Ca(2+)-dependent autophosphorylation is observed at pH 5-6. Photoaffinity labeling using 8-azido-[alpha-32P]ATP or [y-32P]ATP confirms the presence of ATP binding sites. Incubation with [alpha-32P]ATP leads to a rapid but transient labeling of pgp230. Various nucleotides, nucleotide receptor agonists, or antagonists inhibit Ca(2+)-dependent phosphorylation by [y-32P]ATP. The concentrations of half-maximal inhibition range from 10(-7) M to 10(-3) M. The rank order of inhibitory potency is: ATP > alpha,beta-methylene-ATP > CTP = TTP > y-4-amino-phenyl-ATP = 2-methyl-thio-ATP > UTP = GTP > GDP = ADP = beta,y-methylene-ATP = beta, y-methylene-TTP = beta,y-methylene-GTP = adenosine-5'-O-2-thiodiphosphate = CMP = AMP > adenosine > cytidine > guanosine = suramin > Reactive blue 2 > iso-butyl-methyl-xanthine > thymidine > uridine. These data suggest a nucleotide binding capacity of this new hepatocyte membrane glycoprotein. Further investigations should be carried out to reveal its biological function.
Kreisel
W
W
Klinikum Der Albert-Ludwigs-Universität, Medizinische Klinik, Abteilung Gastroenterologie, Hepatologie und Endokrinologie, Freiburg, Germany.
Spamer
C
C
Heilmann
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D006023:Glycoproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
D006023:Glycoproteins; D000255:Adenosine Triphosphate