Biomedical subjects
Publications and source records attributed to Z Aleksandrowicz.
The activity of alpha-glycerophosphate shuttle in homogenate and in a reconstructed system (isolated mitochondria and cytoplasm) from rat skeletal muscle is presented. The influence of some inhibitors of mitochondrial alpha-glycerosphate dehydrogenase on the activity alpha-glycerophosphate shuttle is demonstrated. The possible operativity of alpha-glycerophosphate shuttle in skeletal muscle in vivo is discussed.
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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.
alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.
1. alpha-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 (beta-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 alpha-glycerophosphate dehydrogenase was found to vary between 10 and 100 nM. 2. The pH optimum for alpha-glycerophosphate dehydrogenase activity varied with substrate concentration. The pH optima were 7.4 and 8.0 in the presence of 2 or 8 mM alpha-glycerophosphate, respectively. The apparent Km for alpha-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. alpha-Glycerophosphate dehydrogenase activity was inhibited by palmitoyl-CoA in a competitive manner with an apparent Ki value of about 10 muM. This inhibition was less pronounced in the presence of calcium or magnesium ions. 4. The activity of alpha-glycerophosphate dehydrogenase was inhibited by phosphoenolpyruvate, D- and DL-glyceraldehyde 3-phosphate and 3-phosphoglyceric acid, in a competitive manner, the apparent Ki values being 0.5, 0.95, 0.12 and 1.5 mM, respectively. 5. alpha-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. alpha-Glycerophosphate dehydrogenase activity in rat brown adipose tissue mitochondria was only slightly affected by phosphenolpyruvate under the same conditions. 6. The data obtained suggest that the activity of alpha-glycerophosphate dehydrogenase in human placental mitochondria may be controlled by changes of the cytosolic level of palmitoyl-CoA, some glycolytic intermediates, and pH.
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Human term placental mitochondria oxidize alpha-glycerophosphate at an unusually high rate as compared to other substrates. The apparent Km both for oxidation and alpha-glycerophosphate dehydrogenase (EC 1.1.99.5) activity of DL-alpha glycerophosphate determined in a medium containing 2mM EDTA and 5 mM MgSO4 was approx. 0.7 mM. EDTA inhibited the alpha-glycerophosphate oxidation if the later was used at low concentrations. A subsequent addition of MgSO4 or CaCl2 restored the original activity. EDTA had no effect on mitochondrial respiration at high concentration of alpha-glycerophosphate. Possible physiological role of relatively high activity of human placental mitochondrial alpha-glycerophosphate dehydrogenase is discussed.
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1. Citrate was oxidized by human placental mitochondria at a rate half that for isocitrate, and cis-aconitate at a rate by 20% lower as with isocitrate. The apparent Km values for these substrates were similar. 2. Oxidation of citrate was stimulated by L-malate but the stimulation was abolished by benzene 1,2,3-tricarboxylate and butylmalonate. 3. Citrate uptake by placental mitochondria was inhibited by benzene 1,2,3--tricarboxylate, and raised specifically by L-malate. Stimulation by fumarate and succinate was found to be due to conversion of these substrates to L-malate. 4. It is concluded that human placental mitochondria contain malate-stimulated tricarboxylate carrier system.
1. Phenylpyruvate inhibits pyruvate plus malate oxidation in human and rat skeletal muscle mitochondria in state 3 and in the uncoupled state, it has, however, no effect in state 4. 2. Inhibition by phenylpyruvate of pyruvate oxidation by intact uncoupled rat muscle mitochondria was competitive, with the Ki value about 0.18 mM. 3. It is suggested that the inhibition of pyruvate oxidation is due to the action of phenylpyruvate on muscle pyruvate dehydrogenase, and is the principal cause of the elevated concentration of pyruvate and lactate in blood plasma of phenylketonuric patients.
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.
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