Zenon Aleksandrowicz

High activity of α-glycerophosphate oxidation by human placental mitochondria

Abstract Human term placental mitochondria oxidize α-glycerophosphate at an unusually high rate as compared to other substrates. The apparent Km 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 MgSO4 was approx. 0.7 mM. EDTA inhibited the α-glycerophosphate oxidation if the later was used at low concentrations. A subsequent addition of MgSO4 or CaCl2 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.

Isolation and regulatory properties of mitochondrial malic enzyme from rat skeletal muscle

Mitochondrial malic enzyme (L-malate:NADP+ oxidoreductase (oxalo-acetate-decarboxylating), EC 1.1.1.40) has been isolated from rat skeletal muscle by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA 34. Specific activity of the purified enzyme was 25 micromol/min per mg of protein which corresponds to about 840-folf purification. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of KHCO3 and pyruvate at about 15% of the rate of the forward reaction. The Km values determined at pH 7.2 for malate, NADP and Mn2+ were 0.33 mM, 6.8 microM and 7.1 microM, respectively. The Km values for pyruvate, NADPH and KHCO3 were 8.3 mM, 19.6 microM, and 24.4 mM, respectively. Purified enzyme showed allosteric properties at low concentration of malate and this characteristic can be modified by succinate and fumarate which do not affect the maximum velocity of the reaction. The pH optimum for decarboxylation reaction was between 7.2 and 8.4. Possible metabolic role of mitochondrial malic enzyme in skeletal muscle is discussed.

Effect of some steroids and α-tocopherol on cytochrome c induced extramitochondrial NADH oxidation by human and rat skeletal muscle mitochondria

Abstract 1. 1. Progesterone at concentrations 50 μ M was found to exert an inhibitory effect on cytochrome c induced NADH oxidation by skeletal muscle mitochondria. This inhibition was only partial in the case of human and almost complete in the case of rat skeletal muscle mitochondria. The inhibitory effect of progesterone may be reversed by α-tocopherol. 2. 2. In human skeletal muscle mitochondria other steroids tested were without effect on NADH oxidation in the presence of cytochrome c. In the case of rat skeletal muscle mitochondria the relative order of the strength of the inhibitory action of steroids tested was:progesterone⪢deoxycorticosterone = oestradiol>corticosterone. 3. 3. Oxidation of NAD-linked substrates ( pyruvate + L -malate ) in both human and rat skeletal muscle mitochondria was inhibited at the same extent. 4. 4. It is concluded that in rat skeletal muscle mitochondria the pathways of exogenous NADH oxidation in the presence of added cytochrome c and of intramitochondrial NADH oxidation are similar in their sensitivity to progesterone. In human skeletal muscle mitochondria these pathways differ in their sensitivity to the progesterone action.

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.

Inhibition of anion transport across the mitochondrial membrane by amytal

Abstract It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p -trifluoromethoxyphenylhydrazone. Using [ 14 C] succinate and [ 14 C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P i , succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [ 14 C]succinate/anion (P i , succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P i translocation across the mitochondrial membrane.

Comparative studies on NADP-linked dehydrogenases in some tissues of fish and crustaceans

Abstract 1. 1. The activities of isocitrate dehydrogenase (EC 1.1.1.42), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were measured in extracts of various tissues obtained from three species of fish and three crustaceans. 2. 2. The activity of isocitrate dehydrogenase greatly exceeded that of malic enzyme in fish muscle and was only slightly higher in crustaceans muscle. 3. 3. Very low activity of glucose-6-phosphate dehydrogenase was detected in all somatic muscle investigated. 4. 4. In the heart muscle of fish the activities of the three enzymes studied were greater than those in somatic muscle. 5. 5. However the isocitrate dehydrogenase/malic enzyme and isocitrate dehydrogenase/glucose-6-phosphate dehydrogenase activities ratios were generally similar in somatic and heart muscle of fishes. 6. 6. Insignificant differences were observed between the livers and gills of fish with respect to the three enzyme activities tested. 7. 7. Fish livers and gills displayed significantly higher glucose-6-phosphate dehydrogenase activity levels than somatic muscle of fish. 8. 8. Slight differences were observed in isocitrate dehydrogenase and matic enzyme activities between somatic muscle and livers or gills.

Intracellular distribution of fumarase in rat skeletal muscle

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11-19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.

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

Abstract 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 phosphoenolpyruvate, 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 phosphoenolpyruvate, 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 phosphenolpyruvate 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.

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.

The inhibition by bromothymol blue of anion translocation across the mithochondrial membrane

1. In rat liver mitochondria bromothymol blue inhibited the exchange of [14C]succinate for succinate, malonate, L-malate and inorganic phosphate; the [14C]citrate/citrate and [14C]citrate/malate exchange reactions and the phosphate/hydroxyl exchange were also inhibited by this dye. The inhibition of the rate of succinate, citrate and phosphate uptake by bromothymol blue is found to be competitive. 2. The degree of inhibition by bromothymol blue of the ]14C]succinate/malonate exchange reaction was pH dependent. It has been shown that the inhibition increased linearly while the pH was increased from 6.0 to 8.2. However, the binding rate of bromothymol blue to the mitochondria decreased with the rising pH of the medium. It is concluded that the binding of acidic bromothymol blue was not essential for the inhibitory effect. 3. Other sulfonephthalein derivatives also inhibited [14C]succinate/malonate exchange reaction. At pH 7.2 the relative order of the strength of the inhibitory action of the sulfonephthalein compounds tested was: thymol blue greater than bronocresol green greater than bromothymol blue greater than phenol red greater than bromocresol purple. The results do not indicate any correlation between the pK values of pH values of pH indicators and their extents of inhibition. 4. It is suggested that the negatively charged bromothymol blue interacts with the positively charged centers of the anion carrier systems causing inhibition of membrane permeability for anions.