Olufunso O. Olorunsogo

Modification of the transport of protons and Ca2+ ions across mitochondrial coupling membrane by N-(phosphonomethyl)glycine

The proton permeability of mitochondrial membranes suspended in 0.15 N NH4Cl was enhanced by N-(phosphonomethyl)glycine (PMG), a broad-spectrum and a non-selective herbicide, in a concentration-dependent manner. Significant decreases in light scattering by these membranes were observed at concentrations greater than or equal to 600 microM PMG. The effect of PMG is therefore several times lower than that of FCCP, a classical uncoupler of oxidative phosphorylation. Using a sensitive pH-glass electrode, PMG significantly enhanced the movement of protons into mitochondrial matrix. Furthermore, the rate of PMG-induced release of Ca2+ ions following its accumulation by energized mitochondria was only slightly over one-half that induced by FCCP (1 microM). Whereas Ca2+ or Mg2+ only marginally reduced the effect induced by PMG, inclusion of glycine into the reaction media did not have any influence whatsoever on the effect induced by PMG. These results indicate that, although PMG increases the permeability of the mitochondrial membrane to protons and to Ca2+, the herbicide does not seem to act like a true protonophore. Its uncoupling effect may, therefore, be due to its ability to act both as a chelator and a mild protonophore.

Hypoglycemic activity of Buchholzia coriacea (Capparaceae) seeds in streptozotocin-induced diabetic rats and mice.

The present study evaluates the possible hypoglycemic activity and ameliorative effects of oral administration of ethanol extracts (EEBC) and butanol fraction (BFBC) of Buchholzia coriacea seeds, a plant in use traditionally for treating diabetes, hypertension, rheumatism, cold, cough and catarrh, in streptozotocin (STZ)-induced diabetic mice and rats. Fasting blood glucose (FBG) levels were evaluated before and after extracts administration. EEBC and BFBC significantly decreased (P<0.05) FBG in hyperglycemic mice and normoglycemic rats within 4 and 12 h, respectively after extract administration. The administration of EEBC, BFBC and glibenclamide (a standard antidiabetic drug) for 10 days significantly lowered (P<0.05) FBG level in STZ-induced diabetic rats by 55%, 64% and 56%, respectively. EEBC and BFBC significantly (P<0.05) decreased hepatic injury induced by STZ as evident in the decreased activity of serum alanine amino transferase and aspartate amino transferase compared to in the STZ-only treated group. Similarly, both extracts significantly decreased (P<0.05) the elevated levels of serum creatinine, urea, total cholesterol, triglyceride and thiobarbituric acid reactive species (TBARS) products in diabetic rats. Serum superoxide dismutase activity was significantly enhanced (P<0.05) by treatments with EEBC, BFBC and glibenclamide. Overall, the results suggest that B. coriacea seeds contain a potent hypoglycemic and antioxidant agent suggested to be a flavone glycoside concentrated in BFBC which may find clinical application in amelioration of diabetes-induced secondary complications.

Comparison of the membrane-bound (Ca2++Mg2+)-ATPase in erythrocyte ghosts from some mammalian species

The properties of the membrane-bound calcium-pumping protein, the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) were compared in erythrocyte ghosts isolated from five mammalian species--human (Homo sapiens), bovine (Bos taurus), porcine (Sus scrofa melitensis), ovine (Ovis aries crassicandus) and caprine (Capra hircus syriaca). The specific activity of the enzyme in porcine erythrocytes is one order of magnitude higher than in the other species. It was also stimulated to various extents by the regulator protein, calmodulin, and by phosphatidylinositol in all the species. Analysis of membrane proteins revealed a number of differences which seem to suggest that the molecular architecture of the red cell membrane influences the activity of the enzyme.

The uncoupling effect of N-(phosphonomethyl)glycine on isolated rat liver mitochondria.

Abstract The rates of oxygen consumption by rat liver mitochondria, respiring on either succinate, a two-site substrate, or β-hydroxybutyrate, a three-site substrate, and in the presence of varying concentrations of the isopropylamine salt of N -(phosphonomethyl) glycine (PMG) have been measured polarographically. The respiratory control ratios of these mitochondria were shown to be significantly reduced, by at least 10 per cent by the addition of 3.95 × 10 −5 M PMG. There was a larger decrease in these ratios, up to 50 per cent, as the concentration of the herbicide was raised to 1.25 × 10 −3 M. At concentrations ranging from 3.12 × 10 −4 M to 1.25 × 10 −3 M, PMG restored respiration of mitochondria previously inhibited by oligomycin. Adenosine triphosphatase (ATPase) activity was enhanced by the addition of PMG. In this respect, the maximal increase, 3-fold, was obtained at 6.25 × 10 −4 M PMG. These findings suggest that N -(phosphonomethyl) glycine uncouples oxidative phosphorylation in isolated rat liver mitochondria.

Inhibition of energy-dependent transhydrogenase reaction by N-(phosphonomethyl)glycine in isolated rat liver mitochondria

The pattern of interaction of various concentrations of N-phosphonomethyl derivative of glycine (PMG) with membrane-bound nicotinamide nucleotide transhydrogenase has been investigated in intact mitochondria isolated from rat liver. Lower concentrations of PMG ( less than 1.50 . 10(-4)M) had no significant effect (12% inhibition) on the activity of the enzyme when the reaction was supported by energy generated from succinate oxidation. Inhibition increased as the concentration of the herbicide was raised: at 3.12 . 10(-4) the degree was 28% and at 1.25 . 10(-3)M PMG, 46% (maximal inhibition). Similar results were obtained when ATP was used as the source of energy. These observations indicate that like thyroxine, an uncoupler of oxidative phosphorylation, PMG interacts with both oxidative phosphorylation and energy-dependent transhydrogenase reaction.

Inhibition of succinate-linked reduction of pyridine nucleotide in rat liver mitochondria ‘in vivo’ by N-(phosphonomethyl)glycine

The pattern of the interaction of N-(phosphonomethyl)glycine (PMG), a broad-spectrum and non-selective herbicide with succinate-linked reduction of pyridine nucleotide, was investigated in liver mitochondria isolated 5 h after albino rats were given i.p. injections of PMG. Although there was no appreciable inhibition of the reduction of pyridine nucleotide at dosage levels less than 150 mg PMG/kg, the extent of inhibition increased as the dose was raised to 240 mg PMG/kg. Maximal inhibition of 34.5% and 45.4% were obtained at 240 mg PMG/kg when externally added ATP and high-energy intermediate, respectively, were used as the source of energy. These findings suggest that the inhibitory effect of PMG may be due to its uncoupling effect on oxidative phosphorylation.

Erythrocyte membrane Ca2+-pumping ATPase of hypertensive humans: Reduced stimulation by calmodulin

The Ca2+-pumping ATPase of erythrocyte plasma membranes of hypertensive humans (HTN) show, in the absence ofcalmodulin, a low Vmax comparable to that of the enzyme of the erythrocyte membranes of normotensive humans (NTN). Although the addition of calmodulin (1.5 μgper ml) increased the maximum activity of the calcium pump of membranes of HTN and NTN individuals by at least 2-fold and 4-fold, respectively, the activator protein partially purifed from the erythrocytes of HTN individuals enhanced the activity of the enzyme in a fashion similar to that of the protein obtained from the haemolysate of NTN individuals. A determination of the dependence of the activity of the pump on concentration of ATP revealed that the Km (ATP) of the enzyme of membranes of HTN individuals is 52% higher than that of the enzyme of membranes of NTN individuals, while the Vmax (1.75±0.28 μmol ATP mg protein−1 h−1) of the pump is 46% lower in the membranes of HTN humans than that of the enzyme of membranes of normal individuals (3.25 ±0.42 μmol ATP mg protein−1 h−1) . It seems likely from these results that elevated erythrocyte Ca2+ concentration associated with essential hypertension may be due to a defective interaction between the Ca2+-pumping ATPase and the calmodulin Ca2+ complex,

Distinct metal ion requirements for the phosphomonoesterase and phosphodiesterase activities of calf intestinal alkaline phosphatase.

The roles of Mg2+ and Zn2+ ions in promoting phosphoryl transfer catalysed by alkaline phosphatase are yet to be fully characterised. We investigated the divalent metal ion requirements for the monoesterase and diesterase activities of calf intestinal alkaline phosphatase. The synergistic effect of Mg2+ and Zn2+ in promoting the hydrolysis of para-nitrophenyl phosphate (monoesterase reaction) by alkaline phosphatase is not observed in the hydrolysis of the diesterase substrate, bis-para-nitrophenyl phosphate. Indeed, the diesterase reaction is inhibited by concentrations of Mg2+ that were optimal for the monoesterase reaction. This study reveals that the substrate specificities of alkaline phosphatases and related bimetalloenzymes are subject to regulation by changes in the nature and availability of cofactors, and the different cofactor requirements of the monoesterase and diesterase reactions of mammalian alkaline phosphatases could have significance for the biological functions of the enzymes.

EFFECT OF REACTIVE OXYGEN SPECIES ON THE ERYTHROCYTE CALCIUM-PUMP FUNCTION IN PROTEIN-ENERGY MALNUTRITION

The presence of detectagle amounts of non-heme iron in erythrocyte ghost membranes have been postulated to lead to the initiation of membrane lipid peroxidation and the attendant perturbation of membrane functions. We have investigated the presence of non-heme iron and endogenous products of lipid peroxidation in erythrocyte membranes of normal and kwashiorkor (KWA) subjects and assessed the susceptibility of the membranes to exogenously generated reactive oxygen species. The modulation of the basal and calmodulin-stimulated calcium-pumping activity of these membranes by reactive oxygen species was also assessed. The results show the presence of significant amounts of non-heme iron and endogenous free radical reaction products in the red cell membranes of KWA subjects compared with that of normal children. Estimation of the extent of lipid peroxidation in the presence of exogenously generated reactive oxygen species further revealed that erythrocyte ghost membranes of KWA subjects are more susceptible to oxidative stress than those of normal individuals. Although both the basal and calmodulin-stimulated activities of the membrane-bound Ca2+-pump enzyme in normal and KWA subjects were inhibited by oxygen-free radicals, the erythrocyte enzyme in KWA subjects showed higher susceptibility to inhibition by oxygen free radicals than that of normal individuals. We propose that the reduced erythrocyte calcium-pump function in KWA is not unconnected with excessive generation of reactive oxygen species.

Erythrocyte membrane (Ca2++Mg2+)-ATPase in human protein-energy malnutrition

Calmodulin-free ghost membranes were prepared from erythrocytes of kwashiorkor children and from healthy children in the same age bracket. In the absence of calmodulin, the specific activity of Mg2+-dependent Ca2+-pumping ATPase (Ca2++Mg2+-ATPase) of kwashiorkor membranes was more than 40 percent lower than the specific activity of the normal enzymes, whose maximum velocity was increased by at least four-fold by the modulator protein. In constrast, the maximum velocity of the enzymes of kwashiorkor membranes was enhanced by calmodulin by about 11/2 times the basal activity of the normal enzymes and by 2 times the basal activity of the kwashiorkor enzymes. The affinity of the pump for ATP was lower in the membranes of kwashiorkor children (Km for ATP=30.6±2.8 μM ATP) in comparison to normal membranes (Km for ATP=21.7±2.0 μM ATP). Similarly, calmodulin-affinity of the enzymes, was lower in kwashiorkor membranes than in the normal membranes irrespective of source of calmodulin. Calmodulin from haemolysates of kwashiorkor red cells activated the enzymes of normal and kwashiorkor membranes to the same degree as calmodulin partially purified from the haemolysate of healthy children. A determination of the dependence of the activity of the pump on calcium in the absence and presence of calmodulin reveals that the affinity of the kwashiorkor enzymes for Ca2+ is at least 70 percent lower than that of enzymes of normal membranes. Altogether, these findings suggest that the Ca2+-pumping ATPase of kwashiorkor membranes is less functional than the enzymes of healthy erythrocytes.