New synthesis of 2β-hydroxy-19-oxoandrost-4-ene-3,17-dione and its 2β-O analog

Treatment of 19-[oxygenated]-androst-4-ene-3,17-dione with Mn(AcO)₃ and ClCH₂COOH in benzene gave epimeric mixtures of the corresponding 2ξ-chloroacetates and 2ξ-acetates. The products were processed to give the title compound. For the synthesis of the 2-¹⁸O analog, ClCH₂C¹⁸OOH was used, which was prepared from ClCH₂COCl.     

Energy-linked transhydrogenase. Effects of valinomycin and nigericin on the ATP-driven transhydrogenase reaction catalyzed by reconstituted transhydrogenase-ATPase vesicles

Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Cytochrome c oxidase from bakers' yeast. V. Arrangement of the subunits in the isolated and membrane-bound enzyme.

Cytochrome c oxidase from baker's yeast contains three mitochondrially made subunits (I to III) which are relatively hydrophobic and four cytoplasmically made subunits (IV to VII) which are relatively hydrophilic (Mason, T. L., Poyton, R. O., Wharton, D.C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354 and Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761). In order to explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of "surface probes" was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound cytochrome c oxidase. The surface probes included iodination with lactoperoxidase and coupling with p-diazonium benzenesulfonate. In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound isocyanate group. In the membrane-bound enzyme, Subunit I was almost completely inaccessible and Subunit II was partly inaccessible to all surface probes. All of the other subunits were accessible. Similar results were obtained with the solubilized enzyme, except that the differences in reactivity between the individual subunits were less clear-cut. The results obtained with liposome-bound cytochrome c oxidase resembled those obtained with the mitochondrially bound enzyme. These data suggest that the two largest mitochondrially made subunits are localized in the interior of the enzyme and that they are genuine components of cytochrome c oxidase.     

Characterization of the major steroids present in amniotic fluid obtained between the 15th and 17th weeks of gestation

In pooled amniotic fluid obtained between the 15th and 17th weeks of gestation the concentration of free steroids and steroid glucuronides was found to be 40 micrograms/dl. The concentration of steroid monosulfates and disulfates was 19 micrograms/dl. About half of the characterized steroids are progesterone metabolites. The "fetal type" 3 beta-hydroxy-5-ene steroids were found exclusively in the sulfoconjugated form. Their concentration represents 20% of the total steroid content. The identification of two 15 beta-hydroxylated C21 steroids, 3 beta,15 beta,17 alpha-tridoxy-5-pregnen-20-one and 5-pregnene-3 beta,15 beta,17 alpha,20 alpha-tetrol isolated from mid-pregnancy amniotic fluid is reported here. Metabolites of cortisol and 17-deoxycorticosteroid metabolites had similar quantitative importance, 8.6 and 9.4%, respectively.     

Cationic amphiphiles induce fusion of acidic liposomes

Decylamine, dodecylamine and tetradecylamine induced aggregation and fusion of acidic liposomes at concentrations of about 1 mM, 75 μM and 75 μM, respectively. Aggregation was assayed as increase in turbidity. Fusion was assayed as intermixing of membranes and contents, and was observed in the electron-microscope to form large liposomes. Only at higher concentrations did these amphiphiles induce massive leakage of the liposomes' contents. Similar effects were caused by hexadecylpyridinium bromide (CP) and hexadecyltrimethylammonium bromide (CTAB). The trivalent cation 4-dodecyldiethylenetriamine and the more hydrophobic amphiphile, trioctylmethylammonium chloride, induced fusion at concentrations of about 10–20 μM. Octylamine and heptylamine induced size increase at mM concentrations. They induced membrane intermixing but little or no content intermixing. Thus, these amphiphiles seem to promote size increase either by transfer of lipid or mainly by ‘cracking and annealing’.