Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid.

A group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), are the major proteins found in bovine seminal fluid. These proteins are secretory products of seminal vesicles, and they bind to spermatozoa upon ejaculation, suggesting that there are binding sites for these proteins on the spermatozoa. It was of interest to characterize these binding sites on spermatozoa which may help in the elucidation of the biological function of BSP proteins. The binding sites on spermatozoa are resistant to protease or acid treatment and are heat-stable but extractable with organic solvents. The solvent-extractable material, when coated on plastic microtitration wells, binds radiolabeled BSP proteins thus indicating the lipid nature of the BSP binding sites on spermatozoa. We investigated the specificity of interaction of BSP proteins with lipids using liposomes of phospholipids, solid-phase, and thin-layer chromatography-overlay techniques. Results showed that BSP-A1, -A2, and -A3 proteins bound specifically to those phospholipids which contain the phosphorylcholine group. In contrast, BSP-30-kDa protein preferentially bound to phospholipids containing the phosphorylcholine moiety but also interacted with phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Furthermore, of those lipids that were extracted from spermatozoa, only phospholipids which contain the phosphorylcholine moiety bound radiolabeled BSP proteins. These data suggest that the BSP protein binding sites on spermatozoa are phospholipids. We propose that this specific interaction plays an important role in the membrane modification of spermatozoa that occurs during capacitation and/or acrosome reaction.     

Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.     

Steroid-protein interactions. 40. The effect of fatty acids on progesterone binding to human serum albumin

Human serum albumin was delipidated by solvent extraction or by treatment with charcoal. Progesterone complexes formed with these albumin preparations had higher association constants than those formed with the untreated samples. The charcoal method of delipidation resulted in somewhat higher affinity constants than extraction with chloroform/methanol. Addition of 5 mol lauric acid per mol albumin reduced the association constant of the progesterone complex by approx. 50%. Studies with lauric, myristic, and palmitic acid showed that the decrease of binding affinity for progesterone was proportional to the amount of fatty acid added to albumin, and to its chain length. These results confirm and extend our previous findings of inhibition of progesterone binding to human albumin by long-chain fatty acids.     

Steroid-protein interactions. Fluorescence quenching of progesterone-binding globulin and alpha1-acid glycoprotein upon binding of steroids.

The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on α₁-acid glycoprotein was also studied. The intrinsic fluorescence of the progesterone-binding globulin and of α₁-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23 °C of 2 × 10⁹m^?1 for progesterone-binding globulin and 1 × 10⁶m^?1 for α₁-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 × 10⁹m^?1 were determined for desoxycorticosterone, 1 × 10⁸m^?1 for testosterone, and 2 × 10⁶m^?1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3β-ol-20-one, 5α-pregnanedione, and 5β-pregnanedione, steroids lacking the Δ⁴-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n → π1) absorption band of steroids containing the Δ⁴-3-keto chromophore.