Human Placental Mitochondria Research Articles

Cholesterol side-chain cleavage, cytochrome P450, and iron-sulfur protein in human placental mitochondria

Cytochrome P450 and the associated iron-sulfur protein have been characterized in human placental mitochondria by means of optical absorbance difference spectrophotometry and electron paramagnetic resonance spectrometry. These two proteins occur in a molar ratio of about 1:1 in human placental mitochondria, and the cytochrome P450 appears to be that form involved in cholesterol side-chain cleavage. Pregnenolone formation from endogenous mitochondrial cholesterol, as measured by radioimmunoassay, follows a biphasic time-course similar to the situation in other steroidogenic tissues. The specific activity of cholesterol side-chain cleavage, and the specific contents of cytochrome P450 and the iron-sulfur protein in the mitochondria, are 2- to 3-fold higher at term than in the 1st and 2nd trimesters. When expressed in terms of the cytochrome P450 content, the rate of pregnenolone formation is high, suggesting that cholesterol side-chain cleavage in human placenta is in an activated state.

Regulation of α-glycerophosphate dehydrogenase activity in human term placental mitochondria

1. 1. α-Glycerophosphate dehydrogenase ( sn-glycerol-3-phosphate:(acceptor) oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from human term placenta was found to be inhibited by ethyleneglycolbis(β-aminoethyl ether)-N,N′- tetraacetic acid (EGTA). Addition of an excess of calcium ions to the incubation medium completely restored the original activity. The concentration of free calcium ion required to activate the α-glycerophosphate dehydrogenase was found to vary between 10 and 100 nM. 2. 2. The pH optimum for α-glycerophosphate dehydrogenase activity varied with substrate concentration. The pH optima were 7.4 and 8.0 in the presence of 2 or 8 mM α-glycerophosphate, respectively. The apparent K m for α-glycerophosphate also varied with pH; the values being 0.4 mM at pH 7.05, 1.5 mM at pH 7.8, and 3.5 mM at pH 8.5. 3. 3. α-Glycerophosphate dehydrogenase activity was inhibited by palmitoylCoA in a competitive manner with an apparent K i value of about 10 μM. This inhibition was less pronounced in the presence of calcium or magnesium ions. 4. 4. The activity of α-glycerophosphate dehydrogenase was inhibited by phospho enolpyruvate, d- and dl-glyceraldehyde 3-phosphate and 3-phosphoglyceric acid, in a competitive manner, the apparent K i values being 0.5, 0.95, 0.12 and 1.5 mM, respectively. 5. 5. α-Glycerophosphate dehydrogenase activity in human placental mitochondria was found to be more sensitive to phospho enolpyruvate, than the activity of the same enzyme in rat skeletal muscle mitochondria, α-Glycerophosphate dehydrogenase activity in rat brown adipose tissue mitochondria was only slightly affected by phosph enolpyruvate under the same conditions. 6. 6. The data obtained suggest that the activity of α-glycerophosphate dehydrogenase in human placental mitochondria may be controlled by changes of the cytosolic level of palmitoyl-CoA, some glycolytic intermediates, and pH.

Some properties of external NADH oxidation by human placental mitochondria

Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg++ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg++ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.

Immunochemical studies on adrenal ferredoxin: Involvement of adrenal ferredoxin-like iron-sulfur proteins in the cholesterol side-chain cleavage reaction of mammalian steroidogenic tissues

The possible immunochemical and functional similarities existing among adrenal ferrdoxin-like iron-sulfur proteins present in the mitochondria of mammalian steroidogenic tissues have been examined by employing a goat antibody produced against homogeneous bovine adrenal ferredoxin. This antibody was found to inhibit the NADPH-cytochrome c reductase and cholesterol side-chain cleavage activities catalyzed by mitochondria prepared from rat adrenals, rat ovaries and the testes of rats which had been treated with human chorionic gonadotropin. No inhibition of the NADH-dependent reduction of cytochrome c catalyzed by these mitochondria was observed in the presence of the anti-adrenal ferredoxin. These results demonstrate that adrenal ferredoxin and the comparable iron-sulfur proteins of ovarian and testicular mitochondria are immunochemically similar and are required for the cholesterol side-chain cleavage reaction occurring in these tissues. Although a precipitin reaction was observed upon double diffusion of the anti-adrenal ferredoxin against human term placental mitochondria, no inhibition of either the NADPH-cytochrome c reductase or the cholesterol side-chain cleavage 4 activity catalyzed by preparations of these mitochondria was observed in the presence of the antibody. These results indicate that the iron-sulfur protein present in human placental mitochondria at term differs immunochemically from other mammalian adrenal ferredoxin-like iron-sulfur proteins.

Inhibition of α-glycerophosphate dehydrogenase activity in human placental mitochondria by phosphoenolpyruvate

Inhibition of α-glycerophosphate dehydrogenase activity in human placental mitochondria by phosphoenolpyruvate

High activity of α-glycerophosphate oxidation by human placental mitochondria

Human term placental mitochondria oxidize α-glycerophosphate at an unusually high rate as compared to other substrates. The apparent K m both for oxidation and α-glycerophosphate dehydrogenase (EC 1.1.99.5) activity of dl-α glycerophosphate determined in a medium containing 2 mM EDTA and 5 mM MgSO 4 was approx. 0.7 mM. EDTA inhibited the α-glycerophosphate oxidation if the later was used at low concentrations. A subsequent addition of MgSO 4 or CaCl 2 restored the orginal activity. EDTA had no effect on mitochondrial respiration at high concentration of α-glycerophosphate. Possible physiological role of relatively high activity of human placental mitochondrial α-glycerophosphate dehydrogenase is discussed.

Multiplicity of mitochondrial monoamine oxidases

There have been only two brief reports which presented direct evidence of the existence of more than one monoamine oxidase in mitochondria although this has been suggested by many workers. Gorkin (1963) partially separated p-nitrophenylethylamine oxidase activity from m-nitro-p-hydroxybenzylamine oxidase activity by ion exchange chromatography of mitochondrial extracts in the presence of nonionic detergent (OP-10). Youdim and Sandler (1967) report the appearance of two or three bands of enzyme activity upon polyacrylamide gel electrophoresis of partially purified monoamine oxidase from rat liver or human placental mitochondria. This paper describes the separation of solubilized liver mitochondrial monoamine oxidases from three species by gel filtration in the presence of detergent. Based on substrate specificity there appear to be at least three different monoamine oxidases present in rat and rabbit liver and at least two in beef liver.