Cholesterol limits estrogen uptake by liposomes and erythrocyte membranes.

Multilamellar vesicles (MLV) were prepared from phospholipids with and without cholesterol in equimolar amounts and [4-14C]estradiol. Unincorporated estrogen was removed by petroleum ether extraction or by aqueous buffer washes. In either case, cholesterol-containing vesicles incorporated one-half the estradiol as vesicles without sterol. Addition of estradiol to preformed vesicles followed by buffer washes showed that vesicles without cholesterol invariably retained more estradiol than those with the sterol. Reduction of the cholesterol content to one-half increased estradiol incorporation. The pattern of estradiol removal from MLV with successive buffer washes indicated that much of the steroid associated with cholesterol-containing vesicles was superficially bound to the membrane but vesicles without cholesterol incorporated the estrogen into the bilayer structure. To test the role of cholesterol in limiting the uptake of an estrogen by cells, right-side out resealed ghosts of ox erythrocytes were prepared. They were partially depleted of cholesterol by exposure to small unilamellar vesicles of dioleoylphosphatidyl choline. A decrease in cholesterol content correlates with an increase in estradiol uptake by red cell ghosts. The experiments described point to a central role of cholesterol in limiting the uptake of steroids. The loss of cholesterol of steroid producing cells caused by tropic hormones may be key to their mode of action in promoting secretion of steroid hormones. Likewise, the long-term genomic responses of steroid target cells may depend upon their cholesterol content and the ease by which the steroid can penetrate the membrane barrier.

Activity of mushroom tyrosinase on catechol and on a catechol estrogen in an organic solvent.

A suspension of tyrosinase-coated glass beads in butanol effectively oxidizes catechol substrate. The enzyme is not soluble in the organic solvent and activity can be stopped by removal of the solid state enzyme after low-speed centrifugation or decantation. The product was assayed by HPLC and by its reactivity towards Besthorn's reagent, which gave a reaction typical for o-quinones. Addition of water to the extent of 0.5 to 4% raised the rate of substrate utilization but the accumulation of quinone first increased and then began to decrease. It is suggested that the product in dry butanol is prevented from reacting further by lack of water, which is necessary to promote secondary reactions causing free radical formation and leading ultimately to polymerization to melanin. Successive washes of the solid state enzyme with butanol increased enzyme activity, indicating presence of a butanol extractable inhibitor in the tyrosinase preparation. The enzyme on glass beads in butanol suspension was stabilized by the presence of substrate. 2-Hydroxyestradiol acted as an inhibitor of the tyrosinase-catalyzed oxidation of catechol. The data obtained can be interpreted to mean that the oxidation of the estrogen in the presence of tyrosinase, as previously reported, may be dependent upon the enzyme-catalyzed oxidation of catechol. The oxidation product of catechol, the o-quinone, is likely to function as oxidant towards 2-hydroxyestradiol.

Cell membranes and multilamellar vesicles: influence of pH on solvent induced damage.

Pigment leakage from sheep and horse erythrocytes and from red beet tissue induced by non-polar solvents was determined as a function of pH. The results were compared to disruption of multilamellar vesicles (MLV) composed of phospholipids with equimolar cholesterol under identical conditions of solvent exposure and pH. Solvent access to cholesterol was used to measure vesicle disruption. MLV were made from 1,2-dioleoyl phosphatidylethanolamine, sphingomyelin (SP) and various phosphatidylcholines to simulate the major lipid components of membranes. Pigment leakage from erythrocytes caused by petroleum hydrocarbon (b.p. 60-80 degrees C) was maximal at pH 2-4 and at pH 10, but minimal at pH 6.8; alcohols caused less pigment leakage than petroleum hydrocarbon. Beta-cyanin leakage from beet tissue induced by petroleum hydrocarbon was maximal at pH 2, with very little leakage at pH 4, 6.6 and pH 10. Alcohols caused minimal damage to beet tissue above pH 2. Cholesterol removal by petroleum hydrocarbon from MLV of mixed lipid composition was maximal at pH 2-4, reduced at pH 6.8 and minimal at pH 10. Lipid mixtures in which fatty acyl side chains of one phospholipid were of a different length than the other lost more sterol than mixtures in which the acyl side chains were of identical chain length. MLV with more than 25% SP lost more sterol than those with less or no SP. Results show that in mixtures of phospholipids, SP exposes the hydrocarbon phase of a bilayer to solvent extraction, a property that was also observed in native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases.

The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.

Isolation of estradiol-2,3-quinone and its intermediary role in melanin formation.

We have reported previously that 2-hydroxyestradiol can be oxidized in the presence of catechol by mushroom tyrosinase, with a stoichiometric requirement of molecular oxygen (Jacobsohn, G.M. and Jacobsohn, M.K. (1984) Arch. Biochem. Biophys. 232, 189-196). It is then incorporated into melanin (Jacobsohn et al. (1988) J. Steroid Biochem. 31, 377-385). We now report on the isolation and characterization of the o-quinone as a product of the enzyme reaction from 2-hydroxyestradiol. The o-quinone was isolated from incubates and identified by its FTIR spectrum, in particular, by the appearance of a new band at 1652 cm-1, its migration in HPLC systems, its ultraviolet spectrum, its derivatization with phenylenediamine and comparison of these properties with the periodate oxidation product of the same substrate. The enzyme oxidation of the catechol estrogen was performed at 37 degrees C and did not require an activator; dopa at concentrations higher than 5 microM was inhibitory. At concentrations lower than 5 microM, dopa acted catalytically and was not consumed during the course of reaction. Ascorbic acid inhibited the reaction. The quinone exhibited both reversible and irreversible binding to performed melanin and to melanin actively synthesized by the enzyme. Incubation of 18 microM newly synthesized [4-14C]estradiol-2,3- quinone with mushroom tyrosinase for 45 min at 37 degrees C in presence of 400 microM dopa showed incorporation (irreversible binding) of 6.3 +/- 0.3% of label into melanin produced during the course of reaction. Similar incubations for 45 min of pre-formed melanin prepared from 400 microM dopa showed incorporation of 4.4 +/- 0.2% of the label. Reversible binding was 10-times greater than incorporation for both actively synthesized and preformed melanins. In the absence of dopa or catechol, enzyme incubations of either 2-hydroxy-estradiol or its quinone did not yield melanin. Data suggest that estradiol-2,3-quinone is an intermediate in the incorporation of the catechol estrogen into melanin by tyrosinase.

Effect of pH on the affinity of phospholipids for cholesterol.

The ability of multilamellar vesicles of phosphatidylcholine and phosphatidylethanolamine in aqueous phase to prevent access to cholesterol by a nonpolar solvent was examined. Phosphatidylethanolamine vesicles. In mixed vesicles, cholesterol was retained in proportion to the amount of phosphatidylcholine. To alter the charge and hydration of head groups, pH was adjusted from 1.2 to 12.5. Above pH 8, both phosphatidylethanolamine and phosphatidylcholine retained sterol in a 1:1 molar ratio of phospholipid to cholesterol, regardless of acyl side chain composition. Between pH 2.0 and pH 8.0, sterol retention varied with type of head group and side chain. Lipids with 16-carbon saturated side chains retained more sterol than 18-carbon unsaturated or 12-carbon saturated side chains. Between pH 1.1 and 2.0, none of the phosphatidylethanolamines retained sterol, but long chain phosphatidylcholines, saturated or unsaturated, retained sterol in a 1:1 molar ratio of phospholipid to sterol. Short chain phosphatidylethanolamines and phosphatidylcholines retained 0 to 20% at the low- to mid-pH range. Size of multilamellar vesicles, measured by Doppler effect light scattering analysis, had no bearing on sterol retention. Sonication of vesicles, which increases surface curvature, increases the retention of sterol. Fluorescence polarization indicated that cholesterol does not interact with DPPC or DLPC side chains. The observations can be interpreted in terms of space requirements of head groups, including charge repulsion and hydration. Other factors, such as monovalent cation replacement by protons, juxtaposition of charged groups on vesicle surfaces and length and unsaturation of acyl side chains affect the affinity of phospholipids for cholesterol.

Oxidation of 2-hydroxyestradiol and its incorporation into melanin by mushroom tyrosinase.

The presence of catechol in a reaction mixture has been shown previously to promote oxidation of 2-hydroxyestradiol by mushroom tyrosinase. It was now found that the oxidized products of the catecholesterogen are incorporated into melanin under the influence of the enzyme. Whether the oxidation is restricted to tyrosinase or to enzymes with specific steroid oxidizing properties was examined by separating tyrosinase on agarose gel followed by hydroxylapatite. The effectiveness of separation was monitored electrophoretically. Two bands of enzyme activity of 127 kDa were found. One of these bands could be cleanly separated from the other. The fraction which contained the single band, as well as the one which contained both bands, had similar apparent Km values; i.e. 1.5 x 10(-4) and 2.1 x 10(-4) M. They both catalyzed oxidation of 2-hydroxyestradiol but only in the presence of catechol. All enzyme fractions showed the same pattern of activity towards the estrogen. HPLC analysis of reaction products of catechol indicated that not all of the substrate was consumed during the reaction. About 26% remained unreacted at an initial concentration of 100-400 microM of catechol. This remaining catechol, rather than its reaction products, appears to function as activator of the steroid reaction. The data are consistent with the presence on the enzyme of an allosteric activator site specific for catechol and an active site with a much lower structural specificity occupied by the catecholestrogen.

Role of estradiol and 2-hydroxyestradiol in melanin formation in vitro.

Melanin formation from 3,4-dihydroxyphenylalanine (dopa) was studied in the presence of estradiol and 2-hydroxyestradiol by use of a tyrosinase isolated from B16-F10 melanoma cells grown in C57 black female mice. Both steroids were found incorporated into melanin, but the 2-hydroxy compound was incorporated to a higher extent. The melanin was also able to bind substantial amounts of the two steroids, and the more highly oxidized compound showed higher binding. Melanin isolated from incubates of dopa with mushroom tyrosinase has the ability to bind the steroids and to incorporate small amounts into its structure. It is suggested that melanin in mammalian tissues may function as a depository for estrogens, particularly for those which are more highly oxidized.

Correlation of side chain mobility with cholesterol retention by phospholipid vesicles.

Multilamellar vesicles were prepared from choline phospholipids with various fatty acyl chains, singly and in mixtures, with and without cholesterol. Mobility of acyl side chains for each type of vesicle was measured by fluorescence polarization with diphenylhexatriene, and the amounts of cholesterol and phospholipid retained by them after extraction with a nonpolar solvent were determined. The data suggest that structures of acyl chains determine the extractability of cholesterol. Phosphatidylcholines with unsaturated or short saturated side chains above transition temperature retain less cholesterol upon extraction with petroleum ether than phosphatidylcholines with saturated side chains below transition temperature. Correlation of cholesterol retention with side chain mobility showed that cholesterol is more easily removed from vesicles with mobile acyl side chains than from vesicles with rigid side chains. The presence of cholesterol also alters extractability of phospholipids from vesicles and suggests that sterol affects the polarity rather than spacing of headgroups on vesicle surfaces.

The catalytic effect of tyrosinase upon oxidation of 2-hydroxyestradiol in presence of catechol.

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding o-quinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X 10(-4) M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10(-4) M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/mol of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.

Annual Variation in the Sterol Content of Digitalis purpurea L. Seedlings.

Seedings from a single lot of Digitalis purpurea L. seeds were germinated in batches over a period of 13 months. A total lipid extract was made which was resolved into esterified and unconjugated plus glycosylated sterol fractions. The amounts of sterol in each fraction and in the total were compared for seedlings germinated at different times of the year. The amount of esterified sterols reached a maximum value from March until June, and a low value from July until January. In January, a sharp increase began which lasted until March. Amounts of unconjugated and glycosylated sterols were elevated from March until June, low from July until October, and on the rise from November until March. These data correlate with an annual cycle in seed germination. The phase of maximum sterol content of seedlings is followed by a period of null germination.

17 Beta-estradiol transport and metabolism in human red blood cells.

Attempts to measure the function of red blood cells as steroid metabolizing tissue showed that the membrane plays an important role. Of the 17beta-estradiol associated with red cells, 2/3 was bound to the membrane while 1/3 was in the soluble fraction. The binding appears to be nonspecific and linearly related to the amount incubated. Binding was more extensive at 2 C than at 26 C. Successive washes of red cells with isotonic sodium chloride solution increased their ability to bind 17beta-estradiol. Red cells incubated in isotonic salt media removed 85.1 plus or minus 3.2% (SD) of the total estrogens present and this value remained within narrow limits for incubation times as short as 15 s and as long as 19 h at 37 C. When incubated in homologous plasma, only 8-12% of estrogens in the media became associated with cells. The eight-fold difference in cell binding between salt solution and plasma can be attributed to competition with plasm aproteins. Presence of plasma proteins diminished but does not prevent activity of 17beta-hydroxysteroid dehydrogenase of red cells. Cells produce four times more estrone when incubated in saline media than when incubated in their own plasma. The enzyme is in the soluble portion of the cells and the steroids have to traverse the membrane in both directions. In the transfer of estrogens from plasma of cytoplasm, the membrane needs to be considered as a separate compartment.