Purification and identification of the heat-stable factor required for pregnenolone-binding protein activity. Evidence that the factor is adenosine 3',5'-diphosphate.
This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.
Lee
Y C
YC
Section on Adrenal Cell Biology, Endocrinology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Oeda
T
T
Driscoll
W J
WJ
Fales
H M
HM
Strott
C A
CA
D000244:Adenosine Diphosphate / Q000302:isolation & purification / Q000378:metabolism*; D000302:Adrenal Cortex / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D006168:Guinea Pigs; D008297:Male; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet
D002352:Carrier Proteins; D000244:Adenosine Diphosphate; D000469:Alkaline Phosphatase