Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

14-3-3 protein-activated and autoinhibited forms of plasma membrane H(+)-ATPase

Several authors previously showed that the interaction between 14-3-3 proteins and plasma membrane H(+)-ATPase leads to an activated complex in which the enzyme is endowed with more favorable kinetic parameters and a more physiological pH optimum. In this paper we report immunological studies with antibodies covering a different specific region of the protein, including the N- and the C-terminal ends. The results showed that, beside a free and a complexed form, a third form of H(+)-ATPase in the cell must exist with low activity and no more activation due to the loss of a part of the C-terminal regulatory domain. A model in which 14-3-3 proteins activate H(+)-ATPase by protecting it from a specific proteolytic attack is presented and its generalization is discussed.     

Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.     

In vitro comparative metabolism of 6,7-3H estrone and 6,7-3H estrone-3-sulfate in maternal and fetal guinea-pig liver]

The metabolism of [6,7-3H] estrone and of [6,7(3)H] estrone-3-sulfate have been comparatively studied in the maternal and fetal guinea-pig livers. The appearance of estradiol-17 beta resulting from the activity of the 17 beta-hydroxysteroid-dehydrogenase is more important in the fetal than in the maternal hepatic tissue. This suggests the direct transformation of estrone-3-sulfate into estradio-3-sulfate in the fetus. After incubation of the [3H] estrone, there is an abundant hepatic conjugation. The glycuroconjugated components are predominant, as well in the maternal as in the fetal hepatic tissue. For the latter-one the sulfoconjugation is inexistant. The sulfatasic activity shown after the incubation of [3H] estrone-3-sulfate is very low in the fetal hepatic tissue; in contrast, this activity is higher in the maternal tissue.     

Phosphorylation-dependent interaction between plant plasma membrane H(+)-ATPase and 14-3-3 proteins

The H(+)-ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H(+)-ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H(+)-ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H(+)-ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H(+)-ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H(+)-ATPase and 14-3-3 proteins was identified and partially purified from maize root.     

Preparation of high-specific-activity D-[3-3H]pantothenic acid

High-specific-activity D-[3-3H]pantothenic acid (5 Ci/mmol) was prepared from commercially available beta-[3-3H]alanine employing Escherichia coli strain DV1 (panD2 pan F1). This strain is defective in beta-alanine synthesis and pantothenate uptake, and under appropriate growth conditions converted 85 to 90% of the input beta-[3-3H]alanine to extracellular D-[3-3H]pantothenate. The radiolabeled vitamin was purified from the medium by thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The overall yield of D-[3-3H]pantothenic acid was 30% and radiochemical purity was greater than 99%.