Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa alpha beta-B dimer.
Conditioned medium of cultured Sertoli cells from 21-day-old rats was used as starting material for the isolation of inhibin. Inhibin activity was monitored by the dose dependent suppression of the follicle-stimulating hormone release of cultured rat pituitary cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the highly purified inhibin preparation revealed a 32 kDa protein after silver staining, which could be separated in subunits of 18 kDa and 12 kDa after reduction. Western blot analysis with an antibody recognizing the 22 N-terminal amino acids of the alpha-subunit of 32 kDa bovine inhibin confirmed the presence of a 32 kDa inhibin molecule under non-reducing conditions, whereas an 18 kDa alpha-subunit was found after reduction. An antibody recognizing the beta-A subunit of inhibin did not yield a signal after Western blotting. N-terminal amino acid sequence analysis of two highly purified preparations of inhibin obtained using different methods yielded the sequence predicted for a 32 kDa alpha beta-B dimer on basis of cDNA nucleotide sequence. This result is in agreement with the large excess of beta-B over beta-A mRNA in the rat testis.
Grootenhuis
A J
AJ
Department of Biochemistry, Erasmus University Rotterdam, The Netherlands.
Timmerman
M A
MA
Hordijk
P L
PL
de Jong
F H
FH
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D007265:Inhibins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012708:Sertoli Cells / Q000378:metabolism*
D000596:Amino Acids; D003470:Culture Media; D007265:Inhibins; D005640:Follicle Stimulating Hormone