A gonadotropin-releasing hormone-binding inhibitor from bovine ovaries. Purification and identification as histone H2A.
Bovine, ovine, rat, and human ovaries contain a protein defined as gonadotropin-releasing hormone (GnRH)-like because it reversibly inhibits the high affinity binding of GnRH to rat ovarian membranes, but these same tissues contain little, if any, detectable GnRH. In the present study this GnRH-binding inhibitor (GnRH-BI) was purified from bovine ovaries by a combination of reversed-phase high pressure liquid chromatography, cation-exchange, and gel filtration chromatography. Purification was monitored by analysis of GnRH-like activity in the highly specific rat ovarian membrane receptor assay. Amino acid composition and partial sequence analysis indicated that the purified ovarian GnRH-BI is histone H2A. Calf thymus histone H2A and the purified ovarian protein showed identical dose-dependent (ID50 = 2 microM), competitive, and reversible inhibitory effects on GnRH binding to rat ovarian membranes. The inhibitory effects could not be explained by charge interactions alone since spermine and spermidine at 10-fold higher concentrations did not inhibit GnRH binding. Furthermore, the effects showed specificity since EGF binding to rat ovarian membranes was not inhibited. It is possible that the inhibition of GnRH binding by the purified ovarian fraction was due to low level contamination by a highly active binding inhibitor. However, based on the variety of different purification procedures, the identical effects seen with histone H2A, and the absence of other proteins on gel electrophoresis, we conclude that the ovarian GnRH-BI is probably histone H2A.
Aten
R F
RF
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06510.
Behrman
H R
HR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006657:Histones / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010053:Ovary / Q000032:analysis* / Q000378:metabolism; D011869:Radioligand Assay
D000596:Amino Acids; D006657:Histones; D007987:Gonadotropin-Releasing Hormone