Inhibition by hydroxymalonate of malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

It has been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate or fumarate was inhibited by hydroxymalonate—an inhibitor of malic enzyme. No inhibition was observed when mitochondria were incubated in the presence of citrate or isocitrate. The degree of inhibition by hydroxymalonate of partly purified NAD(P)-linked malic enzyme activity was identical to that of both malate dependent pyruvate and progesterone formation by intact mitochondria. These data strongly support a previous suggestion that malic enzyme plays an important role in the malate dependent progesterone biosynthesis by human placental mitochondria.     

Correlation between the malate dependent progesterone and citrate biosynthesis in the mitochondrial fraction of human term placenta. The stimulatory effect of ADP and ATP

The relationship between malate dependent conversion of cholesterol to progesterone and citrate biosynthesis in human term placental mitochondria has been investigated. It has been shown that ADP and ATP (but not AMP) stimulate, significantly, both progesterone and citrate formation. The stimulatory effect of these adenine nucleotides was dependent on the presence of Mn2+ in the incubation medium. When Mn2+ was omitted or replaced by Mg2+ only negligible stimulatory effect of ADP and ATP was observed. Atractyloside and oligomycin were without effect on ADP and ATP stimulated progesterone and citrate production. Other dinucleotides tested as: GDP, UDP and CDP stimulated both progesterone and citrate formation only slightly. In all the experiments presented the rate of progesterone biosynthesis was found to be significantly correlated with the rate of citrate production. The experimental results presented in this paper suggest that the stimulatory effect of ADP and ATP on malate dependent progesterone biosynthesis is a consequence of an increased conversion of malate to tricarboxylic Krebs cycle intermediates. The possible mechanism by which ATP and ADP stimulate the citrate formation in human placental mitochondria is discussed.     

Inhibition by fatty acids of the cholesterol side-chain cleavage in the course of progesterone biosynthesis in the mitochondrial fraction of human term placenta

1. Saturated and unsaturated fatty acids inhibit the progesterone biosynthesis from cholesterol in the human placenta mitochondrial fraction. 2. Inhibition of the progesterone biosynthesis by fatty acids could be relieved by ATP, CoA and carnitine. 3. The inhibitory effect of fatty acids is due to their action on the cholesterol side-chain mixed-function oxidase system but not on the NADPH-regenerating system.     

Dual functional roles of ATP in the human mitochondrial malic enzyme

Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.     

Evidence for the role of malic enzyme in the rapid oxidation of malate by cod heart mitochondria

Mitochondria isolated from the heart of cod (Gadus morrhua callarias) oxidized malate as the only exogenous substrate very rapidly. Pyruvate only slightly increased malate oxidation by these mitochondria. This is in contrast with the mitochondria isolated from rat and rabbit heart which oxidized malate very slowly unless pyruvate was added. Arsenite and hydroxymalonate (an inhibitor of malic enzyme) inhibited the respiration rate of mitochondria isolated from cod heart, when malate was the only exogenous substrate. Inhibition caused by hydroxymalonate was reversed by the addition of pyruvate. In the presence of arsenite, malate was converted to pyruvate by cod heart mitochondria. Cod heart mitochondria incubated in the medium containing Triton X-100 catalyzed the reduction of NADP+ in the presence of L-malate and Mn2+ at relatively high rate (about 160 nmoles NADPH formed/min/mg mitochondrial protein). The oxidative decarboxylation of malate was also taking place when NADP+ was replaced by NAD+ (about 25 nmol NADH formed per min per mg mitochondrial protein). These results suggest that the mitochondria contain both NAD+- and NADP+-linked malic enzymes. These two activities were eluted from DEAE-Sephacel as two independent peaks. It is concluded that malic enzyme activity (presumably both NAD+- and NADP+-linked) is responsible for the rapid oxidation of malate (as the only external substrate) by cod heart mitochondria.     

NADP-linked malic enzyme and malate metabolism in ageing tomato fruit

Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP⁺) EC 1.1.1.40, malic enzyme, has been purified 40-fold to a homogeneous state using affinity chromatography and gel permeation chromatography. The M_r is 260–265 K with four subunits each of 64–65 K. The enzyme has some competitive or non-competitive inhibitors, particularly some of the Krebs cycle acids and exhibits a rapid rise in activity at the same time as activity of the enzymes of the Krebs cycle are decreasing in the tomato mitochrondrion. The malic enzyme is restricted to the cytosol. The relevance of this information to malate metabolism in plants is discussed.     

The physiological role of malic enzyme in grape ripening

The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring l-enantiomer of malic acid, its very selective C₄-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO₂/HCO ₃ ^- and NADPH/NADP ratios. However, a very limited incorporation of ¹⁴C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-¹⁴C] alanine to grape berries before and after the onset of ripening, indicate that the reverse reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.Abbreviation ME Malic enzyme (l-malate: NADP oxidoreductase)     

Properties of the aromatase system associated with the mitochondrial fraction of human placenta

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35–50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16α-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction     

The cytoplasmic 60 kDa progesterone receptor isoform predominates in the human amniochorion and placenta at term

Background  

The mechanism that initiates human parturition has been proposed to be 'functional progesterone withdrawal' whereby the 116 kDa B-isoform of the progesterone receptor (PR-B) switches in favour of the 94 kDa A-isoform (PR-A) in reproductive tissues. Recently, other PR isoforms, PR-S, PR-C and PR-M generated from the same gene have been identified and partially characterised.     

The role of malate in hormone-induced enhancement of mitochondrial respiration

Shortly after the injection of glucagon, epinephrine, norepinephrine, vasopressin, or angiotensin II into fasted rats, mitochondria isolated from their livers contained elevated concentrations of malate and oxidized citrate, alpha-ketoglutarate, and, in some cases, succinate more rapidly than mitochondria from fasted, control rats. The administration of tryptophan, lactate, or ethanol and refeeding of rats fasted 24 h result in similar elevations of mitochondrial malate concentration and oxidation of added substrates. Treatments that resulted in elevated mitochondrial malate resulted also in increased uptake of added citrate, alpha-ketoglutarate, pyruvate, and, in some cases, succinate. It is postulated that the well-documented effect of gluconeogenic hormones on mitochondrial oxidation of carboxylic substrates may be mediated by malate which not only yields oxalacetate to support the tricarboxylic acid cycle but also facilitates the transport of added substrates, and which is regenerated in the tricarboxylic acid cycle.