Purification of high-molecular-weight follicle-stimulating hormone binding inhibitor in porcine follicular fluids.
We performed the purification of high-molecular-weight follicle-stimulating hormone binding inhibitor (FSHBI) from porcine follicular fluids. The FSHBI activities of high-molecular-weight fractions acquired by ultrafiltration of follicular fluids from small, medium and large follicles with Centriflo CF25 membrane cone were 277.2 +/- 24.6, 176.7 +/- 3.0 and 141.3 +/- 3.6 U, respectively. By affinity chromatography of CF25 retentate with a column of Blue Sepharose CL6B, 94.1 +/- 5.3% of FSHBI activity was recovered in the unretained fraction. The FSHBI in the unretained fraction was purified by anion-exchange chromatography with a column of Mono Q and gel filtration on Sephacryl S300HR. As a result of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified fraction, a single silver-stained band was observed at 310 kD under the non-reducing conditions. On the other hand, under the reducing conditions, SDS-PAGE revealed three bands at 178, 101 and 55 kD. A double reciprocal plot analysis of this substance showed competitive inhibition in FSH binding. The results of the present study suggest the existence of a 310 kD FSHBI composed of three subunits of 178, 101 and 55 kD in porcine follicular fluids.
Furukawa
K
K
Department of Obstetrics and Gynecology, Wakayama Medical College, Japan.
Yamoto
M
M
Kokawa
N
N
Nakano
R
R
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005640:Follicle Stimulating Hormone / Q000037:antagonists & inhibitors*; D015571:Follicular Fluid / Q000737:chemistry*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification*; D006107:Granulosa Cells / Q000378:metabolism; D008970:Molecular Weight; D006080:Ovarian Follicle / Q000502:physiology; D013552:Swine
D006020:Glycopeptides; D005640:Follicle Stimulating Hormone