Evidence for the existence of a GTP-dependent factor in hepatic cytosol that stimulates cyclic AMP binding: possible role in the modulation of cyclic AMP action.
GTP, in physiologic concentration (10(-4) mol/L), enhances cAMP binding to an Mr 57,000 binding protein (BP) in hepatic cytosol, which probably is the phosphorylated receptor subunit of protein kinase II (PK II). When we attempted to separate PK II from other hepatic cytosol proteins by DEAE-cellulose chromatography, we observed that GTP caused little stimulation of [3H]cAMP binding in an eluate fraction (110 to 170 mmol/L KCI), which was rich in PK II but did stimulate cAMP binding to the 210 to 325 mmol/L KCI fraction, which also contains PK II. This suggested that the latter fraction might contain a cofactor necessary for GTP stimulation of cAMP binding, which was lacking in the 110 to 170 mmol/L KCI fraction. Cyclic AMP BP in the 210 to 325 mmol/L fraction was removed by absorption onto cAMP agarose in the presence of 325 mmol/L KCI. When an aliquot of the BP-poor fraction, containing the putative cofactor, was added to the 110 to 170 mmol/L fraction containing PK II, the addition of 10(-4) mol/L GTP to the mixture increased [3H]cAMP binding by more than 80%. Cofactor activity could be extracted from the 210 to 325 mmol/L eluate by adsorption onto cAMP agarose in the presence of 10 mmol/L K phosphate, and eluted with 100 mmol/L KCI, suggesting that the cofactor may bind to the cAMP BP under appropriate circumstances. Addition of this eluted cofactor fraction to the 110 to 170 mmol/L fraction in the presence of 10(-4) mol/L GTP, increased the specific binding of [3H] cAMP more than twofold. Pretreatment of the cofactor fraction with trypsin eliminated this effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Rosenberg
E M
EM
Department of Medicine, Albany Medical College, NY 12208.
Goodman
A D
AD
Lipinski
T L
TL
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism
D014361:Trypsin Inhibitors; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D014357:Trypsin