Conformations of carnitine and acetylcarnitine and the relationship to mitochondrial transport of fatty acids

The conformations of carnitine and acetylcarnitine by EHT and CNDO/2 molecular orbital calculations show that carnitine has two low energy conformers. One of these is an extended conformer corresponding to a charge separated species, while the other is a folded conformer having a charged onium head and carboxylate anion in close proximity forming an internal ionic bond. These results suggest that the folded conformation is responsible for the active transport of acetyl- and acyl-carnitine by enzymes which transfer acyl groups into the mitochondria for subsequent fatty acid oxidation.     

Oxidation of acetate,acetyl CoA and acetylcarnitine by pea mitochondria

Acetylcarnitine was rapidly oxidised by pea mitochondria. (-)-carnitine was an essential addition for the oxidation of acetate or acetyl CoA. When acetate was sole substrate, ATP and Mg²⁺ were also essential additives for maximum oxidation. CoASH additions inhibited the oxidation of acetate, acetyl CoA and acetylcarnitine. It was shown that CoASH was acting as a competitive inhibitor of the carnitine stimulated O₂ uptake. It is suggested that acetylcarnitine and carnitine passed through the mitochondrial membrane barrier with ease but acetyl CoA and CoA did not. Carnitine may also buffer the extra- and intra-mitochondrial pools of CoA. The presence of carnitine acetyltransferase (EC 2.3.1.7) on the pea mitochondria is inferred.     

Stimulation of mitochondrial reactive oxygen species production by unesterified,unsaturated fatty acids in defective human spermatozoa

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P < 0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.     

Stimulation of potassium cycling in mitochondria by long-chain fatty acids

Nonesterified long-chain fatty acids (myristic, palmitic, oleic and arachidonic), added at low amounts (around 20 nmol/mg protein) to rat liver mitochondria, energized by respiratory substrates and suspended in isotonic solutions of KCl, NaCl, RbCl or CsCl, adjusted to pH 8.0, induce a large-scale swelling followed by a spontaneous contraction. Such swelling does not occur in alkaline solutions of choline chloride or potassium gluconate or sucrose. These changes in the matrix volume reflect a net uptake, followed by net extrusion, of KCl (or another alkali metal chloride) and are characterized by the following features: (1) Lowering of medium pH from 8.0 to 7.2 results in a disappearance of the swelling-contraction reaction. (2) The contraction phase disappears when the respiration is blocked by antimycin A. (3) Quinine, an inhibitor of the K⁺/H⁺ antiporter, does not affect swelling but suppresses the contraction phase. (4) The swelling phase is accompanied by a decrease of the transmembrane potential and an increase of respiration, whereas the contraction is followed by an increase of the membrane potential and a decrease of oxygen uptake. (5) Nigericin, a catalyst of the K⁺/H⁺ exchange, prevents or partly reverses the swelling and partly restores the depressed membrane potential. These results indicate that long-chain fatty acids activate in liver mitochondria suspended in alkaline saline media the uniporter of monovalent alkali metal cations, the K⁺/H⁺ antiporter and the inner membrane anion channel. These effects are presumably related to depletion of mitochondrial Mg²⁺, as reported previously [Arch. Biochem. Biophys. 403 (2002) 16], and are responsible for the energy-dissipating K⁺ cycling. The uniporter and the K⁺/H⁺ antiporter are in different ways activated by membrane stretching and/or unfolding, resulting in swelling followed by contraction.     

Stimulation of potassium cycling in mitochondria by long-chain fatty acids

Nonesterified long-chain fatty acids (myristic, palmitic, oleic and arachidonic), added at low amounts (around 20 nmol/mg protein) to rat liver mitochondria, energized by respiratory substrates and suspended in isotonic solutions of KCl, NaCl, RbCl or CsCl, adjusted to pH 8.0, induce a large-scale swelling followed by a spontaneous contraction. Such swelling does not occur in alkaline solutions of choline chloride or potassium gluconate or sucrose. These changes in the matrix volume reflect a net uptake, followed by net extrusion, of KCl (or another alkali metal chloride) and are characterized by the following features: (1) Lowering of medium pH from 8.0 to 7.2 results in a disappearance of the swelling-contraction reaction. (2) The contraction phase disappears when the respiration is blocked by antimycin A. (3) Quinine, an inhibitor of the K(+)/H(+) antiporter, does not affect swelling but suppresses the contraction phase. (4) The swelling phase is accompanied by a decrease of the transmembrane potential and an increase of respiration, whereas the contraction is followed by an increase of the membrane potential and a decrease of oxygen uptake. (5) Nigericin, a catalyst of the K(+)/H(+) exchange, prevents or partly reverses the swelling and partly restores the depressed membrane potential. These results indicate that long-chain fatty acids activate in liver mitochondria suspended in alkaline saline media the uniporter of monovalent alkali metal cations, the K(+)/H(+) antiporter and the inner membrane anion channel. These effects are presumably related to depletion of mitochondrial Mg(2+), as reported previously [Arch. Biochem. Biophys. 403 (2002) 16], and are responsible for the energy-dissipating K(+) cycling. The uniporter and the K(+)/H(+) antiporter are in different ways activated by membrane stretching and/or unfolding, resulting in swelling followed by contraction.     

Stimulation of guanylate cyclase activity by several fatty acids.

Guanylate cyclase of plasma membrane of isolated rat fat cells was activated 7 to 11 fold by oleic acid, linoleic acid, linolenic acid or arachidonic acid. The activation of the enzyme by linoleic acid or oleic acid was influenced by the concentration of enzyme protein and that of the fatty acid. At 158 μg/ml of enzyme protein, 0.6 mM linoleic acid produced maximal activation of 12 fold which was partially reversed by washing. Particulate guanylate cyclase of cerebral cortex and liver was also activated by linoleic acid.     

Stimulation of human platelet guanylate cyclase by fatty acids.

Guanylate cyclase from human platelets was over 90% soluble, even when assayed in the presence of Triton X-100. A time-dependent increase in activity occurred when the enzyme was incubated at 37 degrees and this spontaneous activation was prevented by dithiothreitol. Arachidonic acid stimulated the soluble enzyme activity approximately 2- to 3-fold. Linear double reciprocal plots of guanylate cyclase activation as a function of arachidonic acid concentration were obtained with a Ka value of 2.1 muM. A Hill coefficient of 0.98 was obtained indicating that one fatty acid binding site is present for each catalytic site. Concentrations of arachidonic acid in excess of 10 muM caused less than maximal stimulation. Dihomo-gamma-linolenic acid and two polyunsaturated 22 carbon fatty acids stimulated the activity of guanylate cyclase to the same degree as did arachidonic acid. The methyl ester of arachidonic acid was much less effective. Diene, monoene, and saturated fatty acids of various carbon chain lengths as well as prostaglandins E1, E2, and F2alpha, had little or no effect. These data indicate that the structural determined required for stimulation by fatty acids of soluble platelet guanylate cyclase is a 1,4,7-octatriene group with its first double bond in the omega6 position. This structural group is similar to the substrate specificity determinants of fatty acid cyclooxygenase, the first enzyme of the prostaglandin synthetase complex. However, conversion of arachidonic acid to a metabolite of the cyclooxygenase pathway did not appear to be required for activation of the cyclase since activation occurred in the 105,000 X g supernatant fraction and pretreatment of this fraction with aspirin did not alter the ability of arachidonic acid to activate guanylate cyclase. Kinetic studies showed that the stimulation of guanylate cyclase by arachidonic acid is primarily an effect on maximal velocity. Arachidonic acid did not alter the concentration of free Mn2+ required for optimal activity. It is concluded that the activity of the soluble form of guanylate cyclase in cell-free preparations of human platelets can be increased by a lipid-protein interaction involving specific polyunsaturated fatty acids.     

Stimulation of pyruvate oxidation in asparagus mitochondria by asparagusic acids

Effects of asparagusic acids on pyruvate oxidation in asparagusmitochondria were studied. We found that asparagusic acids stronglystimulated pyruvate oxidation, whereas -lipoic acid only slightaffected it. These results suggest that asparagusic acids mayplay essential roles in the pyruvate oxidation of asparagus. ¹ To whom all correspondences should be addressed. (Received June 25, 1973; )     

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.     

Stimulation of guanylate cyclase of fibroblasts by free fatty acids.

The membranous guanylate cyclase of Balb 3T3 fibroblasts was stimulated by a fraction of calf serum extracted by ether. Stimulation was observed with Mg2+ as the only bivalent cation in the presence of Lubrol PX. The activator co-chromatographed with free fatty acids, and several of these were found to stimulate guanylate cyclase. Among the saturated fatty acids, myristic acid had the highest activity. Stimulating activity diminished as the hydrocarbon chain of the fatty acid was lengthened or shortened. Introduction of an unsaturated bond enhanced the activation by the longer fatty acids. This pattern of specificity is similar to that observed for the effect of fatty acids on many other membranous functions. Under appropriate conditions fatty acids were found to stimulate guanylate cyclase activity in the absence of Lubrol PX. The relationship among the effects of Mg2+, Mn2+, Lubrol PX, and fatty acids on enzyme activity was examined. On the basis of these studies, it appears that fatty acids stimulate the enzyme by a mechanism different from nonionic detergents or Mn2+.     

Esterification of fatty acids at room temperature by chloroform-methanolic HCl-cupric acetate

A new procedure for the preparation of methyl esters from free fatty acids under mild conditions was investigated. Free fatty acids are dissolved in a mixture of chloroform-methanolic HCl-cupric acetate and kept at room temperature for 30 min for complete esterification. The method is suitable for esterification of long-chain acids, such as 18:0, and for very long chain acids, such as 24:0. Fatty acids from brain glycerophosphatides, which included a high concentration of polyenes such as 20:4 (n - 6), 22:4 (n - 6), and 22:6 (n - 3), were also esterified by the same procedure, and neither artifact formation nor loss of unsaturated acids was observed.