Sadao Watanabe

Effects of free fatty acids on the binding of bovine and human serum albumin with steroid hormones

Recent studies have shown that, in addition to free steroid hormones, those bound to albumin in plasma may also be available to tissues. In this report, the effects of free fatty acids (FFA) on the binding of steroids to albumin were compared for the cases of bovine serum albumin (BSA) and human serum albumin (HSA). The apparent association constant, Ka, was estimated from the changes in the equilibrium partition coefficient of steroids between the aqueous/hexane phases caused by the addition of albumin to the aqueous phase. In the case of BSA, Ka for progesterone and testosterone increased upon binding of FFA (myristic, palmitic and stearic acid) to BSA and the maximum value of Ka for these steroids could be attained by 3--4 mol of FFA bound per mol BSA. Furthermore, the elution profiles of gel-filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the steroid/BSA complexes and that FFA potentiates the binding of these steroids to BSA. In the case of HSA, the binding affinities of progesterone and testosterone were not greatly affected by bound FFA. On the other hand, the affinities of ethynylestradiol to both BSA and HSA were unaffected below 4 mol of FFA binding per mol.

Effects of free fatty acids on the binding of steroid hormones to bovine serum albumin

The effects of binding of free fatty acids (FFA) to bovine serum albumin (BSA) on steroid hormone binding to BSA were examined. The FFA studied included myristic, palmitic, stearic, oleic and linoleic acids. The binding coefficient K was estimated from the changes in the equilibrium partition coefficient between the aqueous and the hexane phase caused by the addition to BSA to the aqueous phase. A noticeable effect of FFA binding (molar ratio FFA/BSA, 2∶1) on the affinities of α-estradiol, ethynylestradiol and dehydroisoandrosterone to BSA was not observed: however, the affinities of progesterone, androsterone and testosterone were distinctly enhanced by FFA binding. Furthermore, the elution profiles of gel filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the hormone/BSA complexes and that stronger binding of these hormones to BSA is caused by binding of FFA to BSA. The affinity of ethynylestradiol to BSA is stronger than that of progesterone and testosterone and is not affected by palmitic acid binding to BSA.

Transport of steroid hormones facilitated by serum proteins

The affinities with steroid hormones (alpha-estradiol, ethynylestradiol, progesterone, androsterone, dehydroisoandrosterone and testosterone) were observed for Cohns fraction IV-1 and V (albumin). It was estimated from the comparison with the binding coefficient K (protein-bound form/free form of hormone) in a 3.5% (w/v) bovine serum albumin (BSA) solution that 40-80% of bound hormone in bovine serum is the BSA-bound form. It becomes clear in a liquid membrane system consisting of a hexane source phase (I), a water phase and a hexane receiving phase (II) that the transport flux of hormone is governed primarily by the partition coefficients between the water/hexane phases. In the case of a hormone with a lower partition coefficient, the uptake process from the hexane phase (I) to the water phase is a rate-determining step in the transport system and the serum proteins accelerate the transport of hormones, while with an increase in the partition coefficient the rate-determining step changes from the uptake step to the release step from the water phase to the hexane phase (II) and the hormone transport is decelerated owing to the significant decrease of free hormone concentration in the aqueous phase by the associated with serum proteins for the system having the restricted amount of hormone in the hexane source phase.

Effect of gramicidin on the ion permeability of liposomes in the presence of mixed electrolytes

Abstract The membrane permeabilities of three anionic species (Cl - , Br - and NO 3 - ) and a cation (K + or Na + ) entrapped simultaneously within reverse-phase evaporation vesicles (REV) were measured with an ion chromatograph and an atomic absorption spectrophotometer. Anion and cation permeabilities of vesicle membranes conform to first-order kinetics. K + or Na + was scarcely detectable in the liposomes after removing the extravesicular electrolytes by means of a Sephadex column when gramicidin was added at a concentration exceeding 2 dimers of gramicidin per vesicle in the liposomal preparation, whereas the concentration of entrapped anions decreased by approximately first-order kinetics. When gramicidin was added to the liposome solution at an estimated level of 20 dimers per vesicle, only 30-40% of the entrapped K + or Na + permeated rapidly out of the vesicles. Measurement with a potassium ion electrode revealed that rapid release of K + occurs within five minutes after the addition of gramicidin. The K + efflux is partly compensated by H + influx. The membrane permeabilities of entrapped Cl - , Br - and NO 3 - were enhanced 2-3 times compared with those for liposomes without gramicidin.