Adrenocortical pregnenolone-binding protein activity requires a small heat-stable factor: evidence that regulation by phosphorylation/dephosphorylation occurs at the level of the factor, not the protein.
The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.
Driscoll
W J
WJ
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Lee
Y C
YC
Strott
C A
CA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000302:Adrenal Cortex / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000302:isolation & purification / Q000378:metabolism*; D003956:Dialysis; D006168:Guinea Pigs; D006358:Hot Temperature; D008954:Models, Biological; D010766:Phosphorylation; D011284:Pregnenolone / Q000378:metabolism*; D011402:Pronase; D011987:Receptors, Steroid / Q000378:metabolism*; D012997:Solvents
D003067:Coenzymes; D011987:Receptors, Steroid; D012997:Solvents; D011284:Pregnenolone; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D011402:Pronase