The final goal of this project is to describe the complex sequence of events triggered by pituitary polypeptide hormone binding to receptors on the plasma membrane of steroidogenic endocrine cells. The initial event is cAMP synthesis and the end result is the stimulation of steroid hormone synthesis by these cells. This acute (short time) stimulation occurs because of the increased velocity of the initial and rate-limiting stem of the steroidogenic pathway, the synthesis of pregnenolone from cholesterol catalyzed by ctyochrone P-450scc in the inner mitochondria membrane. The mechanism producing this rate enhancement is unknown, but protein synthesis inhibitors block stimulation. Using two dimensional electrophoresis, we detected in adrenal cells a rapidly induced protein i whose synthesis correlates with the time course and ACTH dose response of stimulated steroidogenesis. The proteolytic ploypeptide map of this protein resembles that of another protein p produced only in quiscent cells. These proteins will be isolated and the effect of their addition to quiscent (unstimulated) cells and/or mitochondria assessed. Subsequent studies will determined the exact processes by which stimulatin is produced i may remove an inhibitory phospholipid or steroid from the P-450scc or adds a stimulatory molecule to it. This will be investigated by binding and reconstitution studies. Studies will be carried out to detect these proteins in other types of endocrine tissue, e.g. corpus luteum and Leydig cells, to assess how generally the proteins occur. These proteins will be characterized to determine the modification that causes i to differ from p in pI; prelinimary evidence indicated that i is a glycoprotein. Lectin binding and treatment of the proteins with enzymes which remove the carbohydrate moiety should give insight into this. An additional focus will be on the end result of the stimulation, how the mitochondrion is changed. A cholesterol pool, produced in adrenals in response to stimulation, can be detected by EPR spectroscopy. The size and occurrence in other tissues of this pool will be studied using EPR spectroscopy coupled to biochemical measurements. Additionally, the influence of this cholesterol on the reduction potential of steriod complexed P-450scc and the rate of reduction of this cytochrome will be measured in stimulated and quiescent mitochondria using EPR spectroscopy.
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