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Mitochondrial fraction prepared from human term placentae were incubated with [14C]-cholesterol in the presence of citrate, cis-aconitate or isocitrate. A stimulatory effect of these metabolites on cholesterol to progesterone conversion was found. In contrast to the other metabolites tested, citrate supported progesterone biosynthesis was found to be inhibited by manganese ions. This effect occurred in intact mitochondria only and was accompanied by a decreased citrate consumption.

Summary It has been shown that mitochondria from human placenta incubated in the presence of 2-oxoglutarate or its precursors utilize acetoacetate at the rate about 1 nmol/min/mg protein. Utilization of acetoacetate is completely inhibited by arsenite. Mitochondria from human placental tissue show activity of 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. It is proposed that acetoacetate utilization by placental mitochondria proceeds via the conversion to acetoacetyl-CoA catalysed by 3-oxoacid-CoA transferase, and then to acetyl-CoA, catalysed by acetoacetyl-CoA thiolase.

It has been shown that ADP, ATP, NAD(P), and NAD(P)H significantly stimulate pyruvate production from malate by intact uncoupled human term placental mitochondria. No stimulation by ADP was observed when mitochondria were incubated in the presence of NAD(P) or NAD(P)H or when mitochondrial membrane had been disrupted. Atractyloside and oligomycin were without effect on ADP- and ATP-stimulated pyruvate production. Other dinucleotides tested such as GDP, UDP, and CDP, stimulated pyruvate production only slightly when mitochondria were incubated in the absence of phosphate. The rate of pyruvate production by intact mitochondria is commensurate with partly purified NAD(P)-linked malic enzyme activity as measured by NAD(P) reduction as far as the effects of pH of hydroxymalonate on these both processes is concerned. It is concluded that pyruvate production by intact human placental mitochondria is catalyzed by NAD(P)-linked malic enzyme and that this process is stimulated by ADP and ATP.

1. 1. α-Glycerophosphate dehydrogenase activity in sonic particles and in intact mitochondria isolated from human term placenta was studied. 2. 2. The V max of this enzyme activity in mitochondria was much higher than in sonic particles; the K max for α-glycerophosphate was about 1 mM in both preparations. 3. α-Glycerophosphate dehydrogenase activity was inhibited by ethyleneglycol-bis(β-aminoethyletherl- N , N '-tetraacetic acid (EGTA) or EDTA, the inhibition being more pronounced in the case of intact mitochondria. 3. 4. In the presence of these inhibitors, the mitochondrial enzyme exhibited sigmoid-shaped kinetics, whereas in sonic particles competitive inhibition was observed. 4. 5. Phosphoenolpyruvate and palmitoyl-CoA exerted a stronger inhibitory effect on the enzyme activity in mitochondria than in sonic particles. 5. 6. It is concluded that catalytic properties of α-glycerophosphate dehydrogenase change during sonication of mitochondria.

Summary It has been shown that mitochondria isolated from human placenta contain β -hydroxybutyrate dehydrogenase activity. The properties of the β -hydroxybutyrate dehydrogenase from human early placenta are very similar to those of the enzyme isolated from other mammalian tissues.

The inhibition of [14C]acetate incorporation in the presence of amytal, rotenone and cyanide in squalene and cholesterol fractions was observed m placenta and liver tissue.

1. A procedure for the isolation of tightly coupled mitochondria from human early placenta is described. 2. Mitochondria obtained by this method were able to oxidize Krebs cycle intermediates, pyruvate, glutamate, glutamine, palmitoyl-carnitine, alpha-glycerophosphate and beta-hydroxybutyrate. 3. These mitochondria incubated in the medium containing ethylene diamine tetraacetic acid and bovine serum albumin and no added Mg2+ ions exhibited a high respiratory control and adenosine diphosphate:oxygen (ADP:O) ratios corresponding to the theoretical values for all substrates tested. Addition of Mg2+ ions markedly reduced the respiratory control index and ADP:O ratio. 4. Adenosine triphosphatase (ATPase) activity in the obtained mitochondrial preparation was stimulated about tenfold by Mg2+. Oligomycin inhibited Mg2+-stimulated ATPase activity by about 25 per cent, but completely inhibited this activity in the absence of Mg2+ ions. 5. It is concluded that the effect of Mg2+ ions on the respiratory control and ADP:O ratio reported in this paper is exerted mainly through the Mg/+-stimulated oligomycin-insensitive ATPase activity.

In human term placental mitochondria the equilibrium rate of the reaction catalyzed by 20α-hydroxysteroid dehydrogenase was shown to be shifted to progesterone formation. The enzyme activity was more specific for NAD+ than NADP+. NADH strongly inhibited the progesterone biosynthesis from 20α-dihydroprogesterone. This inhibition could be reversed by the conditions with favor the NADH oxidation. Regulation of progesterone biosynthesis by the redox state of phosphopyridine nucleotides in human placental mitochondria is discussed.

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 μmol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn 2+ and Co 2+ were by far more effective activators than Mg 2+ and Ni 2+ , whereas the reaction did not proceed in the presence of Ca 2+ . The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for l -malate and NAD were 2.82 and 0.33 m m ; and in the presence of fumarate 1.18 and 0.22 m m , respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.