The major specific aim of this project will be to isolate and characterize inhibin from porcine follicular fluid. Our data indicate inhibin is a large molecular complex (gel exclusion and irradiation inactivation) of approximately 200,000 daltons. Our approach will thus be designed to isolate this type of large molecule and several initial approaches have been applied. These include ethanol precipitation, molecular exclusion chromatography, lithium salt fractionation, matrix gel affinity chromatography, and ion exchange chromatography. Several labs have reported inhibin activity to be associated with fractions of 15,000 to 35,000 daltons but agreement on the specific properties is lacking, and activity recovery is routinely very low. We would examine whether an isolated high molecular weight complex could be suitably dissociated to produce high yields of a lower molecular weight inhibin. In our preliminary studies we have established a laboratory reference preparation which has shown excellent stability over 3 years. This will be used to evaluate activity recovery at each step of fractionation. Only effective steps (greater than 66% recovery) will be retained. Some of our studies have used in vivo asays. These are too cumbersome and costly. We will use the pituitary cell culture assay (both secretion and cell content versions) to follow the activity in the series proposed. We observed (1976) that some fractions from pseudopregnant rat ovaries inhibited LH binding in a radioligand assay. The corpora lutea were shown to be the principle source of this activity. We have shown that the effect is observed with FSH and hCG binding, also. Thus, we do not ascribe great physiological significance to the observation. However, we have shown the activity is localized within specific (but several) fractions from a DEAE-cellulose chromatogram. As a less important specific aim (in terms of effort and resources) we propose to characterize these fractions further as potentially interesting probes for hormone binding.
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