Steroid hormone signalling system and fungi.

Three components of the steroid hormone signalling system, 17 beta-hydroxysteroid dehydrogenase, androgen binding proteins and steroid hormone signalling molecule testosterone were determined in the filamentous fungus Cochliobolus lunatus for the first time in a fungus. Their possible role in C. lunatus is discussed in comparison with their role in mammalian steroid hormone signalling system. The results are in accordance with the hypothesis, that the elements of primordial signal transduction system should exist in present day eukaryotic microorganisms.

Isolation and identification of testosterone and androstenedione in the fungus Cochliobolus lunatus.

The cytosol of the filamentous fungus Cochliobolus lunatus was found to contain binding proteins for testosterone and androst-4-ene-3,17-dione. Both of these steroids were also found to be good exogenous substrates for the constitutive 17 beta-hydroxysteroid dehydrogenase, also found in this fungus. We were looking for the possible endogenous substrate. The procedures for isolation and identification of the endogenous steroid molecules in the fungus C. lunatus are described in this paper. The lipids were extracted from the cells and from the growth medium and purified by column and thin-layer chromatography. Analysis of the steroids, isolated from the cells of C. lunatus by gas chromatography and combination of gas chromatography-mass spectrometry, revealed the presence of testosterone and androst-4-ene-3,17-dione. Their structures were confirmed by comparison with the standards on the basis of chromatographic behavior and mass spectra. No such structures were found in the growth medium. Endogenous synthesis of androgens in this fungus was independently confirmed by the detection of radioactively labeled testosterone when the growing cells of C. lunatus were labeled with radioactive precursor molecule [5-3H] mevalonate.

Androgen binding proteins in Cochliobolus lunatus.

Testosterone and 4-androstene-3,17-dione (androstenedione) binding proteins were found in the cytosol of Cochliobolus lunatus, a filamentous fungus. The maximal androgen binding occurred by 120 min at 0 degrees C. Androgen binding proteins were degraded by protease treatment and at high temperature. The mean Kd determined for testosterone binding protein (TBP) by Scatchard analysis was 4.5 nM and the maximum capacity Bmax was 120 fmol/mg protein; for androstenedione binding protein (ABP) the Kd was 8 nM and Bmax was 390 fmol/mg protein. Steroid binding specificity for TBP, in order of decreasing affinity, was the following: androstenedione, progesterone, dihydrotestosterone, 17 beta-estradiol and estrone. The same order was found for ABP.

Involvement of the cell wall ofNocardia restricta on the bioconversions of steroid sapogenins.

The bioconversion of tigogenin, (25R)-5α-spirostan-3ß-ol byNocardia restricta to (25R)-4-spirosten-3-one and (25R)-1,4-spirostadien-3-one was strongly increased using protoplasts instead of intact cells. The same effect could be achieved by addingß-cyclodextrin to the medium. Since 5ß-steroid sapogenins pass through the cell wall without hindrance, it can be concluded that the cell wall ofNocardia restricta is a rate-limiting factor only for 5α-isomers.

Hydroxylation of steroids with nonpolar side chains with 11 alpha-hydroxylase of Rhizopus nigricans.

Steroids with nonpolar side chains with 2, 4 and 8 C atoms were used as substrates for the 11 alpha-hydroxylase of Rhizopus nigricans. Their bioconversion was compared to that of progesterone, which was found to be far the best substrate giving the highest total bioconversion. 3-keto-4-ene steroids with nonpolar side chains were converted to their hydroxylated products in a small yield or not at all. The absence of an oxygen function in the side chain did not affect the regio-specificity of the hydroxylation, but resulted in a much lower total bioconversion. The strong effect of the oxygen function and of the length of the side chain on hydroxylation with the 11 alpha-hydroxylase of Rhizopus nigricans was demonstrated.

Bioconversion of steroid glycosides by Nocardia restricta.

The bioconversion of steroid alkaloid tomatine by Nocardia restricta yields the conjugate with lactic acid. We studied the bioconversion of some steroid glycosides without a nitrogen atom in the molecule to determine the effect of the nitrogen atom. The glycosides were of three different types: sterol glycosides, bufadienolide rhamnoside and steroid saponine. The results of bioconversions showed that Nocardia restricta converts steroid glycosides differently according to the sugar bound to the steroid aglycone. It can be concluded that in the absence of a nitrogen atom in the steroid molecule no conjugation with lactic acid by Nocardia restricta occurs.

Cholesteryl glucoside in Candida bogoriensis.

Extraction of lipids with CHCl3/CH3OH/1 M HCl from fresh and frozen cells of Candida bogoriensis showed the presence of a steryl glucoside. The product was purified and identified as a beta-cholesteryl glucoside. It hydrolyzes in methanolic HCl and therefore it is a steryl glucoside. The quantitation of the extracted cholesteryl glucoside was pursued with high-pressure liquid chromatography. The chemical ionization mass spectra of the isolated and the synthesized beta-cholesteryl glucosides were compared and found identical. The cell wall of Candida bogoriensis was weakened with enzyme (glusulase) and after homogenisation and sucrose density gradient centrifugation, five layers separated. Only the pellets of one layer showed the presence of cholesteryl glucoside in thin-layer chromatography after extraction with solvents. In the same layer, the activity of sterol glucosyl transferase was found.