Purification of a candidate gonadotrophin surge attenuating factor from human follicular fluid.
Gonadotrophin surge attenuating factor (GnSAF) is a new non-steroidal ovarian substance, different from inhibin, which attenuates the pre-ovulatory luteinizing hormone (LH) surge in superovulated women. Human follicular fluid (FF) was used as a source for the isolation of GnSAF, the activity of which was monitored in an in-vitro pituitary bioassay. Primary rat pituitary cells were incubated with test substances for 48 h and subsequently washed and incubated with 0.1 micromol/l gonadotrophin releasing hormone (GnRH) plus test substances for 4 h. GnSAF activity was expressed as the reduction of GnRH-induced LH secretion in the 4 h incubation. GnSAF was purified from 250 ml of FF which was heat-treated at 80 degrees C for 5 min. Heparin-sepharose chromatography, Con-A sepharose chromatography, reversed-phase high-performance liquid chromatography (HPLC) and preparative native gel electrophoresis were used for GnSAF fractionation. Using these purification steps, we have obtained an apparently homogeneous preparation that stains as a single band on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. GnSAF has an apparent molecular weight of 12.5 kDa and was identified by amino acid sequence (mass spectrometry) to be the C-terminal fragment of human serum albumin.
Pappa
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A
Department of Pharmacology and Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece.
Seferiadis
K
K
Fotsis
T
T
Shevchenko
A
A
Marselos
M
M
Tsolas
O
O
Messinis
I E
IE
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015571:Follicular Fluid / Q000737:chemistry*; D006031:Glycosylation; D042341:Gonadal Hormones; D012739:Gonadal Steroid Hormones / Q000302:isolation & purification; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D007986:Luteinizing Hormone / Q000378:metabolism; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000187:drug effects; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D017385:Sequence Homology
D042341:Gonadal Hormones; D012739:Gonadal Steroid Hormones; D011506:Proteins; C080538:attenuin; D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone