The effect of hypolipidemic drugs WY14643 and DH990, and lysophospholipids on the metabolism of oleate in plants

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [¹⁴C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [¹⁴C]oleate or [¹⁴C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [¹⁴C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.     

Studies on lipid metabolism in Tetrahymena pyriformis: properties of acyltransferase systems.

Membrane preparations from Tetrahymena pyriformis catalyzed the acylations of glycerophosphate, isomeric monoacylglycerophosphate, and 1-acylglycerylphosphoryl-choline. Under the optimal conditions, glycerophosphate acyltransferase and 1-acylgly-cerophosphate acyltransferase used saturated and unsaturated acyl-CoA at comparable rates. The specificities of these acyltransferase systems for various acyl-CoAs as compared with the respective maximal velocities do not directly explain the fatty acid distribution in glycerophospholipids. However, the acylation of 2-acylglycerophosphate was highly selective for palmitate when the incubations were carried out in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The 1-acylglycerylphosphorylcholine acyltransferase system showed relatively higher specificity for unsaturated acyl-CoA, which is consistent with the fatty acid pattern of phospholipids. Significant amounts of diglyceride and triglyceride were formed together with phosphatidic acid from acyl-CoA and glycerophosphate, indicating that the enzymes involved in triglyceride synthesis are closely associated with acyltransferase systems involved in phosphatidate synthesis in microsomes. These acyltransferase activities were found mainly in microsomes, and to a lesser extent, in pellicles, too. No significant difference was observed in the properties of acyltransferase systems in microsomes and pellicles.     

The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus

The relationship between the delta 9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied. Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature. Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature. The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids. The spectra of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature. In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S3) vs 1/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature. As with membranes, plots of log S3 vs 1/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature. These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types.     

Effects of pertussis vaccine on the lipid metabolism of hamsters

Administration of pertussis vaccine to hamsters markedly affected their lipid metabolism. Four days after the administration of the vaccine a severe fatty liver was observed. Concomitantly, a rise in the serum levels of free fatty acids, triacylglycerols and ketone bodies was detected. It is suggested that an altered regulation of adipose tissue lipolysis might be at least partially responsible for the observed effects.     

Glutamate metabolism triggered by oxaloacetate in intact plant mitochondria

In Percoll-purified potato tuber mitochondria, glutamate metabolism can be triggered by oxaloacetate, in the presence of ADP and thiamine pyrophosphate. There is a lag phase before O₂ uptake is initiated. During this lag period, oxaloacetate is rapidly converted into α-ketoglutarate and succinate, or into malate at the expense of the NADH generated by α-ketoglutarate dehydrogenase. The ratio of the flux rates of both pathways is strongly dependent on the glutamate concentration in the medium. When all the oxaloacetate is consumed, a rapid O₂ uptake is initiated. The effects of malonate on glutamate metabolism triggered by oxaloacetate and on α-ketoglutarate oxidation are reported. It is concluded that the inhibition of the succinate dehydrogenase by either malonate or oxaloacetate does not affect the rate of α-ketoglutarate dehydrogenase functioning. All the metabolites accumulated are excreted by the mitochondria in the supernatant. Some of them are then reabsorbed. These results emphasize the importance of the anion carriers in the overall process.     

Perturbation of lipid metabolism in L-M cultured cells by elaidic acid supplementation: formation of fatty alcohols

Supplementation of culture medium with elaidic acid (400 μg/flask) in L-M cells results in the formation of an otherwise undetected lipid component. We have identified this lipid component to be a mixture of free fatty alcohols containing primarily elaidyl alcohol with cetyl, stearoyl, and oleoyl alcohols as minor constituents. Formation of fatty alcohols by fatty acid supplementation seems to be specific with $\text{trans}$ fatty acids (i.e., elaidate, $\text{trans}$ vaccenate, and linolelaidate); addition of stearate and oleate to the L-M cells does not produce fatty alcohols. The fatty alcohols accumulated by the $\text{trans}$ fatty acid supplementation are associated with both the particulate and supernatant fractions of the cells.     

The effect of altered lipid composition on the transport of various amino acids in Candida albicans.

Candida albicans cells grown on alkanes of different chain lengths (C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, and C₁₈) exhibited a low growth rate and gradual increase in the total lipid content with the increase in the length of alkanes. There was a significant change in the phospholipids and sterols content of various alkane-grown cells compared to glucose-grown cells. In glucose-grown cells, the transport of various amino acids, e.g., proline, glutamic acid, lysine, glycine, phenylalanine, serine, methionine, and leucine was found to be energy dependent and against a concentration gradient. In alkane-grown cells, the transport of lysine, proline, serine, and methionine was reduced, however, there was no effect on the uptake of glycine, glutamic acid, phenylalanine, and leucine. The results were interpreted as different carrier(s) responsible for amino acid uptake responsed differently to the change of lipid environment.     

1,25-Dihydroxyvitamin D3 metabolism. The effect of dietary calcium and phosphorus

Rats maintained on tritiated 1,25-dihydroxyvitamin D₃ as their sole source of vitamin D and placed on diets differing in calcium content had similar intestinal levels of tritiated 1,25-dihydroxyvitamin D₃. Since 1,25-dihydroxyvitamin D₃ administration eliminated adaptation of intestinal calcium transport, it appears that increased production of 1,25-dihydroxyritamin D₃ is responsible for the stimulation of calcium transport by low dietary calcium. When maintained on tritiated 1,25-dihydroxyvitamin D₃, rats fed a low-phosphorus diet had somewhat higher levels of tritiated 1,25-dihydroxyvitamin D₃ in the duodenum and plasma than rats on a normal-phosphorus diet. In addition to stimulating 1,25-dihydroxyvitamin D₃ synthesis, low dietary phosphorus may increase the accumulation of 1,25-dihydroxyvitamin D₃ in both intestine and plasma.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

The multiple effects of ethylenediaminetetraacetate in several model lipid peroxidation systems

Experiments were performed which illustrate the various ways EDTA can influence lipid peroxidation. Either detergent-dispersed linoleate, or liposomes made from extracted microsomal phospholipids were utilized as substrates for peroxidation. Peroxidation was accomplished using Fe²⁺ or Fe³⁺. In systems utilizing Fe²⁺, EDTA chelation facilitated Fe²⁺ autoxidation which in turn caused peroxidation of detergent-dispersed linoleate. Peroxidation was not initiated during EDTA-Fe²⁺ autoxidation when the substrate lipids were in a liposomal configuration. Systems utilizing Fe³⁺ required an enzyme (either xanthine oxidase or NADPH-cytochrome P₄₅₀ reductase) to reduce the iron for peroxidative activity. EDTA chelation of Fe³⁺ enhanced the xanthine oxidase and NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of detergent-dispersed linoleate, presumably by facilitating the reduction of Fe³⁺. Catalase and mannitol inhibited both EDTA-Fe²⁺- and EDTA-Fe³⁺-dependent lipid peroxidation. EDTA-Fe³⁺ was not capable of initiating peroxidation of phospholipid liposomes following enzymatic reduction by either enzyme, but ADP-chelated iron effectively initiated liposomal peroxidation in similar systems. With xanthine oxidase-catalyzed peroxidation of liposomes with ADP-Fe³⁺, the inclusion of EDTA-Fe³⁺ caused a modest enhancement of activity. EDTA-Fe³⁺ greatly stimulated NADPH-cytochrome P₄₅₀ reductase-catalyzed peroxidation of liposomes with ADP-Fe³⁺. In contrast, the addition of EDTA, rather than EDTA-Fe³⁺ inhibited the liposomal peroxidation catalyzed by either enzyme with ADP-Fe³⁺ when the EDTA concentration exceeded the concentration of Fe³⁺.     

Hepatic heme metabolism in selenium-deficient rats: effect of phenobarbital.

Hepatic heme metabolism was examined in selenium (Se)-deficient and Se-adequate (control) rats. Administration of phenobarbital stimulated heme synthesis in the liver in both Se-deficient and Se-adequate rats. In contrast to these results, phenobarbital increased microsomal heme oxygenase (MHO) activity six- to eightfold in Se-deficient but not control rats. These data suggest that the previously reported abnormalities of cytochrome P-450 induction in Se-deficient rats are related to increased degradation of hepatic heme.     

The preparation, properties, and metabolism of 14C-bicarbonate-labelled transferrin

¹⁴C-bicarbonate-labelled transferrin was prepared in order to study the role of bicarbonate in the cell-mediated release of iron from transferrin. ¹⁴C-bicarbonate bound to transferrin only in the presence of iron and with a ratio of bound bicarbonate to bound iron of one. The transferrin-¹⁴C-bicarbonate complex was very stable in Tris-HCl buffered at pH 7.5–9.0 even in the presence of excess non-radioactive bicarbonate. However, oxalate, citrate, and phosphate promoted a rapid exchange of transferrin-bound ¹⁴C-bicarbonate with bicarbonate present in the medium.Rabbit reticulocytes effected a temperature-dependent release of ¹⁴C-bicarbonate from transferrin at the same rate at which they incorporated ⁵⁹Fe from transferrin — suggesting the existence of a coordinated mechanism in the cells for the release of both iron and bicarbonate from transferrin.     

The effect of spermidine on endonuclease inhibition by agarose contaminants

A simple method for the determination of molecular weight and effective size of proteins is proposed. The procedure consists in comparison of sedimentation coefficients of reversed micelles of aerosol OT in octane in the presence and in the absence of solubilized protein.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

The lipid integrity of membranes of guinea pig alveolar macrophages studied by nanosecond fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene: The effect of stimulation by concanavalin A and formyl peptides1

Time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene was used to monitor physical changes in the membranes of guinea pig alveolar macrophages following stimulation by N-formyl peptides (either N-formylmethionylphenylalanine (FMP) or N-formyl methionyl leucylphenylalanine (FMLP)) and concanavalin A. The anisotropy of diphenylhexatriene in macrophages showed a dependence on stimulation both in the rate of decay and in the value of anisotropy at infinite time. Subtle differences were observed between the effect of concanavalin A and FMLP on the membrane lipid fluidity as detected by fluorescence anisotropy. Concanavalin A stimulation of macrophages decreased the value of the anisotropy at infinite times in the range of 0–20 °C and increased the value at 25–40 °C; and at all temperatures it decreased the rate of decay of anisotropy. At temperatures below 25 °C, the response to FMLP was similar to concanavalin A, but above 25 °C, FMLP only slightly modified the anisotropy decay profile. Another physical parameter, calcium permeability, was examined because Ca⁺² fluxes are dependent upon membrane properties. The temperature-dependent profiles of concanavalin A and FMP-stimulated ⁴⁵Ca⁺² efflux from alveolar macrophages were similar. The rate and extent of ⁴⁵Ca⁺² efflux increased from 4 to 22 °C, with no further increases observed up to 37 °C. This pattern correlated well with observed changes in membrane fluidity.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.