Hormonal modulation of riboflavin carrier protein secretion by immature rat Sertoli cells in culture

We report here that a protein species with biochemical and immunological similarity with chicken egg riboflavin carrier protein (RCP) is synthesized and secreted by immature rat Sertoli cells in culture. When quantitated by a specific heterologous radioimmunoassay, optimal concentrations of FSH (25 ng/ml) brought about 3-fold stimulation of RCP secretion. FSH, in the presence of testosterone (10−6 M) brought about 6-fold stimulation of secretion of RCP over the control cultures which were maintained in the absence of these two factors. The aromatase inhibitor (1,4,6-androstatrien-3,17-dione) curtailed 85% of the enhanced secretion of RCP, suggesting that the hormonal stimulation is mediated through in situ synthesized estrogen and this could be confirmed with exogenous estradiol-17 β which brought about 3 — fold enhancement of secretion of RCP at a concentration of 10−6 M. When tamoxifen (10 μM) was added along with FSH and testosterone, there was 75% decrease in the enhanced secretion of RCP. Addition of this anti-estrogen together with exogenous estradiol resulted in 55% decrease in elevated levels of RCP. Cholera toxin (1 μg/ml) and 8-bromo-cyclic AMP (0.5 mM) mimicked the action of FSH on the secretion of RCP thus suggesting that FSH stimulation of RCP production may be mediated through cyclic AMP. These findings suggest that estrogen mediates RCP induction in hormonally stimulated sertoli cells presumably to function as the carrier of riboflavin to the developing germ cells through blood-testis barrier in rodents

Nature of the thiamin-binding protein from chicken egg yolk

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin-binding protein of pregnant rat serum was immunol. similar to rat serum albumin as assessed by a sensitive RIA. In RIA for rat biotin-binding protein, the binding of [125I] rat biotin-binding protein to anti-chicken egg yolk biotin-binding protein antibodies was displaced by both rat serum (10-100 nL) and purified rat serum albumin (0.1-10 ng). Similarly, in RIA for rat serum albumin the binding of [125I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin-binding protein antibodies was displaced by unlabeled rat biotin binding protein at comparable concn. (0.5-10 ng). Significant fractions of radioiodinated rat biotin-binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin-binding protein. In immature rats, the circulating half-lives of rat biotin-binding protein and rat serum albumin were 12 and 17 h, resp. The rat biotin-binding protein and rat serum albumin were analyzed by techniques that exploit their physicochem. properties. They displayed similar electrophoretic mobilities in alk. as well as denaturing SDS-PAGE. However, in nonequil. pH gradient PAGE they resolved clearly. In 2-dimensional tryptic peptide map anal., the 2 proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. Thus, the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunol. crossreactivity