Biomedical subjects

P M Stevenson

Publications and source records attributed to P M Stevenson.

At least 19 recordsLinked to original sources

PubMed1994

Cytosolic NADP(+)-dependent isocitrate dehydrogenase. Isolation of rat cDNA and study of tissue-specific and developmental expression of mRNA.

Immunoscreening and DNA hybridization were used to isolate a 1.72-kilobase pair cDNA encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase from a rat liver, lambda gt11 cDNA library. The identity of the cDNA was confirmed by comparison of the deduced amino acid sequence with sequences of peptides obtained from purified ovarian cytosolic isocitrate dehydrogenase. The 1.72-kilobase pair cDNA sequence translated into a protein of 414 amino acid residues with a molecular mass of 46,681 Da. The amino acid sequence contains a tripeptide (AKL) at the COOH terminus which represents a possible peroxisomal targeting sequence. The deduced amino acid sequence of the rat liver cytosolic isocitrate dehydrogenase showed 70 and 59% identity with sequences reported for NADP(+)-dependent isocitrate dehydrogenases from porcine mitochondria and yeast cytosol respectively. Northern blot analysis demonstrated a 13-fold increase in expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase mRNA during the gonadotropin-induced development of the immature rat ovary. In comparative studies, the cytosolic and mitochondrial isocitrate dehydrogenase mRNAs were found to differ in size (2.2 and 1.8 kilobases, respectively) and to be differentially expressed in various tissues of the rat. Distinct digestion patterns were also obtained in Southern blot analysis of rat genomic DNA.

Amino Acid Sequence↗

PubMed1992

The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones.

Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone-induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membrane/mass protein and a change in the intrinsic permeability of the phospholipid bilayer.

Animals↗

PubMed1992

Changes in catalase activity and concentration during ovarian development and differentiation.

The ovaries of immature rats were used to prepare a peroxisome-enriched fraction by differential centrifugation. Following gonadotropin stimulation, which caused large numbers of follicles to develop into corpora lutea, the specific activity of catalase in the peroxisome-enriched fraction increased 5-fold, while catalase recovered in the post-30,000 x g supernatant did not increase in activity. The increase in catalase specific activity in the peroxisome enriched fraction was shown to be due to an increased concentration of the enzyme as determined by Western blotting. Catalase in pig granulosa cells also increased in specific activity as the follicles aged and luteinized. This increase appeared to parallel increases in the concentration of cytochrome P-450scc. We conclude there is a differential regulation of the peroxisomal and cytosolic pools of rat ovarian catalase.

Animals↗

PubMed1991

A study of the control of NADP(+)-dependent isocitrate dehydrogenase activity during gonadotropin-induced development of the rat ovary.

The concentration of cytoplasmic NADP(+)-dependent isocitrate dehydrogenase increased 20.2-fold during gonadotropin-induced development of the immature rat ovary. Measurement was by protein (Western) blotting using polyclonal antibodies raised against purified enzyme from the porcine corpus luteum. The increase in enzyme concentration during development correlated well with the 18.5-fold increase observed for the specific activity of the enzyme in the cytosolic fraction. An immunochemical similarity was demonstrated between the cytoplasmic enzyme from the ovary, testes, placenta, skeletal muscle, brain, liver, kidney, mammary and adrenal gland. However the mitochondrial NADP(+)-dependent isocitrate dehydrogenase from these tissues was found to be immunochemically distinct from the cytoplasmic enzyme. The concentration of the substrate D(+/-)-threo-isocitrate in the ovaries was measured by fluorometry and found to increase 3.1-fold during hormone-induced development. The intracellular concentration of substrate was estimated to be of the same order of magnitude as the enzyme concentration. We conclude that the increase in cytoplasmic NADP(+)-dependent isocitrate dehydrogenase activity observed during the gonadotropin-stimulated development of the rat ovary is due to increased concentration of enzyme rather than to an activation of the enzyme. The activity of the enzyme in vivo appears to be regulated by the availability of the substrate D(+/-)-threo-isocitrate.

Animals↗

PubMed1990

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum.

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.

Animals↗

PubMed1990

Composition of the inner mitochondrial membrane of porcine corpus luteum.

An inner mitochondrial membrane fraction was prepared from porcine corpus luteum. The concentrations of the respiratory cytochromes, cytochrome P-450scc, cholesterol, ubiquinone, cardiolipin and the total phospholipids were measured. The fatty acid compositions of cardiolipin and the total phospholipid fraction were determined. Comparative data from porcine heart and liver were obtained using the same methods. Differences in both the concentration and the fatty acid composition of the phospholipids were observed between the tissues. It appeared that the phospholipid bilayer was expanded relative to haem a in luteal mitochondria. It is proposed that in the ovary this expansion may be necessary to accommodate cytochrome P-450scc and its substrate, cholesterol.

Animals↗

PubMed1988

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization.

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.

Animals↗

PubMed1986

Ferredoxin reductase levels in the ovaries of pigs and superovulated rats during follicular cell growth and luteinization.

The concentration of ferredoxin reductase, a component of the mitochondrial steroidogenic electron transport chain, was measured in the ovaries of pigs and superovulated rats by a protein blotting procedure using polyclonal antibodies to the purified protein. The concentration of ferredoxin reductase in porcine granulosa cells doubled during growth of follicles from small (1-2 mm diameter) to large (6-12 mm diameter). The concentration doubled again during the period of luteinization. This is in contrast to the rate of cholesterol side-chain cleavage, which showed little change during follicular growth but increased by more than tenfold during luteinization. A similar large increase in cholesterol side-chain cleavage occurs during the period of luteinization in the ovaries of superovulated rats, but as for the pig, only a small increase in ferredoxin reductase was observed. A threefold increase in the yield of mitochondrial protein from tissue homogenates was found between the granulosa cells of small-medium follicles and the cells of the corpora luteum. The increase in ferredoxin reductase during follicular development and luteinization, therefore, correlates well with the increase in mitochondria in the cells, but does not correlate with the dramatic increase in cholesterol side-chain cleavage activity which occurs during luteinization. Based on these results, it is unlikely that the level of ferredoxin reductase limits the expression of the full steroidogenic activity of the granulosa cells of the ovary.

Animals↗

PubMed1985

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P-450scc incorporated into vesicles prepared from these phospholipids.

Cytochrome P-450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P-450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450scc.

Animals↗

PubMed1985

Interactions between ATP and cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries.

ATP stimulated the rate of [4-14C]cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries. The effect of ATP was apparently similar to the stimulatory effect of choriogonadotropin on mitochondrial [4-14C]cholesterol utilization. Enhancement of the rate of steroidogenesis by ATP and choriogonadotropin were not additive. ATP seemed to promote both cholesterol uptake into the inner mitochondrial membrane and the supply of electrons for [4-14C]cholesterol utilization from both endogenous substrate and succinate.

2,4-Dinitrophenol↗

PubMed1985

Creatine kinase, steroidogenesis and the developing ovarian follicle.

Creatine kinase activity was present throughout follicular development and luteinization in rat ovary. The activity of creatine kinase in ovary was about 1/4 that of brain and 1/15th that of heart. The ovary contained the same creatine kinase isoenzyme as brain, and the type did not change during follicular development. Creatine kinase was found to be concentrated in the steroidogenic thecal and luteal cells of the ovary. The activity of creatine kinase was stimulated 23% by choriogonadotropin in luteal tissue.

Animals↗

PubMed1985

A comparison of the lipid classes and essential fatty acid content of rat plasma lipoproteins and ovary.

Plasma lipoproteins, prepared from the blood of superovulated rats by precipitation with phosphotungstate/Mg2+, were similar to HDL and LDL prepared by 24 hr centrifugation. Analyses of the fatty acid compositions of the major lipid classes in HDL, LDL and ovary revealed that the esterified fatty acids of plasma were markedly shorter and more saturated than those of the ovary. Elongation of essential fatty acids by ovary may be important in disposing of carbon fragments generated by the incomplete oxidation of fatty acids during steroidogenesis. The function of HDL and/or LDL in supplying polyunsaturated fatty acids to ovary may be more important than their role in delivering cholesterol, which the ovary can make.

Animals↗

PubMed1985

The effect of calcium on cholesterol side-chain-cleavage enzyme in superovulated rat ovaries.

Cholesterol side-chain-cleavage enzyme activity in mitochondria isolated from heavily luteinized rat ovaries was determined using isocitrate as an electron donor and [4-14C]cholesterol to start the reaction. The activity of the enzyme was reduced when more than 10 microM calcium was added to the mitochondrial preparations. When 100 microM EGTA or 2 mM ATP was added to the reaction an increase in enzyme activity was observed and ATP was able to partially overcome the calcium-induced inhibition.

Adenosine Triphosphate↗

PubMed1984

Changes in coenzyme A and carnitine concentrations in superovulated rats.

Changes in the concentrations of total coenzyme A, acetyl CoA, free carnitine and acetylcarnitine were measured in ovaries from immature rats before and after superovulation with 50 I.U. pregnant mare's serum gonadotropin. In addition, the concentrations of total CoA and total acid-soluble carnitine were measured in liver, adrenal glands and skeletal muscle from the same rats. Ovarian concentrations of total CoA, free carnitine and acetylcarnitine increased 3-fold on gonadotropin stimulation, whereas there was no marked change in total CoA and acid-soluble carnitine concentrations in the other organs. In ovary, the ratio of free CoA to acetyl CoA was about 2:1 during the growth period of follicular development and during active steroidogenesis in the luteal phase, but less than 1 when replication stopped and ovulation occurred. These results show that during periods of high energy demand the ovary has a good capacity to accommodate fatty acid oxidation, and supports the evidence that fatty acids are the major source of reducing equivalents for steroidogenesis at these times.

Acetyl Coenzyme A↗

PubMed1984

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum.

Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins. The luteal and adrenal proteins had similar absorbance spectra and molecular weights. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450scc. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450scc.

Adrenal Glands↗

PubMed1984

Properties of bovine luteal cytochrome P-450scc incorporated into artificial phospholipid vesicles.

Bovine luteal cytochrome P-450scc was purified and incorporated into artificial phosphatidylcholine vesicles. The vesicle reconstituted cytochrome used membrane bound cholesterol as substrate and cholesterol binding varied with the phosphatidylcholine fatty acyl composition and was stimulated by the presence of cardiolipin in the vesicle. 22R -hydroxycholesterol and 20 alpha, 22R - dihydroxycholesterol bound to the cytochrome up to 300 times more tightly than cholesterol and decreased the affinity of the cytochrome for CO by 100-200-fold. The properties of the cytochrome closely paralleled those reported for cytochrome P-450scc purified from the bovine adrenal gland.

Adrenal Glands↗

PubMed1984

Cyclic AMP and steroidogenesis in superovulated rat ovaries.

During the gonadotropin stimulated differentiation of ovarian follicles into corpora lutea, the concentrations of total cyclic AMP and protein bound cyclic AMP increased only marginally. In contrast, there was a 10 fold increase in progesterone production. After acute stimulation of luteinized ovaries with choriogonadotropin total cyclic AMP levels increase more than 6 times while bound cyclic AMP and plasma progesterone rose by 80 and 60% respectively. Our results question the role of cyclic AMP in the basal production of progesterone by the ovary, and suggest a relationship between bound cyclic AMP and progesterone synthesis only exists after acute gonadotropin stimulation.

Animals↗

PubMed1984

The composition and distribution of lipid granules in the rat ovary.

Lipid granules in ovaries of immature rats were confined to the interstitial tissue, and comprised 70% cholesteryl esters and 20% triacylglycerols, the balance being phospholipid and free cholesterol. Following treatment with gonadotropin the interstitial granules disappeared as cholesteryl esters were hydrolysed, but reformed in the follicle as it developed, first in the theca, then in the outer granulosa and finally in the inner cells. The cholesteryl ester: triacylglycerol ratio fell during follicular growth, but in the corpus luteum the ratio in the widely distributed granules was 1:1. Esterified fatty acids in both cholesteryl esters and triacylglycerols became longer and more unsaturated as development progressed. The same progression of granules across the follicles was evident in ovaries of normal adult rats. We concluded that lipid granules in interstitial tissue supplied the substrates for synthesis of new cells in adjacent developing follicles, and those in corpora lutea were a prerequisite for steroidogenic competence.

Animals↗