Albumin, a biologically active protein acting on Leydig cells.
The objective of the present study was to characterize further the albumin fraction of rat testicular fluid (rTF), which can enhance luteinizing hormone (LH)-stimulated pregnenolone production by immature Leydig cells in vitro. Testicular fluid, obtained from 300 rat testes was fractionated by sequential ammonium sulfate precipitation, dye-affinity, anion-exchange chromatography, chromatofocussing and an additional heat treatment. The final fraction showed a single band when analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel and isoelectric focussing gel. The protein had a molecular weight of 67 kDa, an isoelectric point of 5.0 and was identified as albumin after Western blotting using an antibody against rat serum albumin. Albumin in this fraction gave a dose-dependent (0.1-2% protein, w/v) increase in LH-induced pregnenolone production, up to 4-fold, and the increase in specific bioactivity when compared to rTF was 1.4-fold. Selective depletion of albumin from testicular fluid was used as another approach to confirm that albumin itself is the main biologically active component in rTF. rTF from mutant analbuminemic rats (albumin content 0.02 mg/ml) did not stimulate LH-induced steroid production in our assay, in contrast to rTF from normal rats (albumin content 40 mg/ml). Albumin fractions obtained from rat, bovine and human sera were also effective in stimulation of steroid production in the presence of LH, in contrast to chicken serum albumin which gave no stimulation. The stimulatory effect of albumin is not caused by bound fatty acids, nor by the presence of modified forms of albumin such as testibumin or the albumin-bilirubin complex. Our results indicate that Leydig cells are more active in steroid production when surrounded by high but physiological concentrations of albumin.
Melsert
R
R
Department of Biochemistry (Division of Chemical Endocrinology), Erasmus University Rotterdam, The Netherlands.
Hoogerbrugge
J W
JW
Rommerts
F F
FF
D000418:Albumins / Q000302:isolation & purification / Q000502:physiology*; D000818:Animals; D001826:Body Fluids / Q000032:analysis; D002417:Cattle; D011232:Chemical Precipitation; D002645:Chickens; D002846:Chromatography, Affinity; D002849:Chromatography, Gas; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007985:Leydig Cells / Q000502:physiology*; D008297:Male; D011506:Proteins / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D049231:Saposins
D000418:Albumins; D006023:Glycoproteins; C483345:PSAP protein, human; D011506:Proteins; C483347:Psap protein, rat; D049231:Saposins