Affinity chromatography purification of solubilized FSH testicular membrane receptor.

Abstract

Since our present studies and those of others suggested that hCG binds weakly to specific FSH receptors, purification of FSH receptor was attempted with hCG affinity chromatography. Calf testis FSH receptor was initially isolated with differential and sucrose gradient centrifugation. Several particulate membrane fractions were isolated from sucrose density interfaces and two displayed enriched specific FSH binding. One of those fractions3 F,, was more enriched with both specific FSH binding sites and the plasma membrane markers, S’nucleotidase and oubain-sensitive Na+K+�ATPase activities, and appeared less contaminated with other subcellular fractions. Other highly purified antierior pituitary hormones and hCG competed less than 1% as effectively as highly purified FSH for specific receptors in that membrane fraction. Consequently, FSH receptor, derived from the enriched F, particulate fraction, was solubilized with 1% Triton X-100 and subjected to further purification with hCG affinity chromatography. Bound solubilized FSH receptor was eluted from the hCG-affinity bed with [125 II FSH and studied by nondissociating polyacrylamide gel electrophoresis. A major radioactive peak, consistent with [12511 -FSH-receptor complex, was observed and migrated with an Rf significantly different from that for free 112511-FSH. Moreover, when the solubilized FSH receptor was eluted from the affinitycolumn with 1 M NaCI and subjected to sodium dodecyl sulfate gel electrophoresis, one major polypeptide band with an estimated MW of 134,000 was found along with six other faintly staining bands. Those observations strongly suggest that specific FSH receptor may be purified