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PubMed2006

The chemistry of phospholipid binding by the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p as determined by EPR spectroscopy.

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

Binding Sites↗

PubMed2000

Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry.

A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.

Animals↗

PubMed1995

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa.

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsaturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k1 = 0.003 hr-1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k1 = 0.145 and 0.162 hr-1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37 degrees C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A2 may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation.

Adult↗

PubMed1985

Phospholipid methylation in skeletal muscle membranes.

Phospholipid methylation is thought to modulate such vital cellular processes as calcium transport, receptor function, and membrane microviscosity. As these processes are fundamental to the function of muscle cells and are thought to be altered in disease states, we have characterized several features of phospholipid methylation reactions in skeletal muscle and have defined appropriate assay conditions. In rat leg muscle, methyltransferase activity was assayed radiometrically by measuring the incorporation of methyl groups from S-adenosyl-L-[methyl-3H]methionine into membrane phospholipids, the methylated derivatives of which were separated by thin-layer chromatography. Contrary to previous investigations of whole muscle, phospholipid methyltransferase activity was clearly present in skeletal muscle membranes, being highly localized in sarcoplasmic reticulum and present to a lesser extent in sarcolemma. Both the reaction products and the reaction kinetics were consistent with sequential methylation of phospholipids by two methyltransferase enzymes. S-adenosylhomocysteine and its analogues were potent inhibitors of phospholipid methylation in sarcoplasmic reticulum. The predominant localization of phospholipid methyltransferase activity in sarcoplasmic reticulum suggests that its functional role in skeletal muscle may be in calcium transport.

Animals↗

PubMedDate not supplied

Esophageal cancer phospholipid characterization by 31P NMR.

Phospholipid extracts of surgical tissue specimens from 18 patients, consisting of normal esophagus, distal esophageal tumor and normal stomach, were analyzed using 31P NMR. The prominent phospholipids detected in these tissues included cardiolipin (CL), phosphatidylethanolamine plasmalogen, phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SPH), phosphatidylinositol (PI), phosphatidylcholine plasmalogen and phosphatidylcholine (PC). Very small quantities of the phospholipids lysophosphatidylcholine, phosphatidic acid, phosphatidylglycerol, and an uncharacterized phospholipid at -0.13 delta also were detected in some of the 54 tissue specimens analyzed. The mean relative concentrations of these phospholipids, in mole percentages of total detected phosphorus, were determined from the acquired spectra and used to differentiate among the three tissue groups. The relative concentrations of the following phospholipids differed significantly (p < 0.001) among the respective tissue groups: normal esophagus vs esophageal tumor, PS, SPH, PI, PC; normal esophagus vs normal stomach, CL, PE, PS, SPH; esophageal tumor vs normal stomach, CL, PE. Membrane phospholipids implicated in modulating the growth and metastases of tumors of epithelial origin can be profiled to discriminate among normal esophagus, distal esophageal tumor and normal stomach using 31P NMR.

Cardiolipins↗

PubMed1993

A fast and sensitive method for detection of phospholipid-binding proteins on nitrocellulose membranes.

We describe a sensitive new method for specific detection and quantitation of phospholipid-binding proteins using two purified lipocortins as model proteins. The method consists of blotting proteins to nitrocellulose membranes followed by incubation with radiolabeled phospholipids and autoradiographic detection of bound phospholipids. It allows specific detection of phospholipid binding proteins to a threshold of about 100 ng/spot and their quantitation up to several micrograms as well as rapid analysis of specific phospholipid binding properties of individual proteins. If performed as a dot-blotting technique, the method permits the simultaneous analysis of a large number of samples and the establishment of Ca2+ and phospholipid binding curves. If blotting is preceded by electrophoresis, the method also allows specific detection and quantitative estimation of phospholipid binding proteins in crude biological preparations and may thus be useful for monitoring and analysis of these proteins in various biological samples.

Animals↗

PubMed1998

Carbonothioate phospholipids as substrate for a spectrophotometric assay of phospholipase A2.

A continuous spectrophotometric assay for phospholipase A2 (PLA2) was developed using novel carbonothioate phospholipids. These phospholipid analogues contain a carbonothioate bond in the place of the sn-2 ester of the natural substrates of phospholipase A2 and were synthesized in a one-pot two-step reaction. Phospholipase A2 from cobra venom (Naja naja atra) hydrolyzes carbonothioate phospholipids and liberates a free thiol, alkylmercaptan, which is reacted with 5,5'-dithiobis(2-nitrobenzoic acid) to yield a product that absorbs at 412 nm. The kinetic studies on PLA2 hydrolysis of carbonothioate phospholipids were carried out in pure phospholipid forms and in Triton X-100 mixed micelles. The hydrolysis of pure carbonothioate phospholipids exhibits an interfacial activation phenomenon. The hydrolysis of phospholipid in mixed Triton X-100 micelles follows classical Michaelis-Menten kinetics. In a mixed micellar system, the catalytic efficiency observed with this series of substrates is two orders of magnitude lower than that of the hydrolysis of the natural substrate dipalmitoyl phosphocholine. However, these substrates bind to the enzyme over 10 times tighter than does the natural substrate. Application of this carbonothioate assay to screen both reversible and irreversible enzyme inhibitors of phospholipase A2 is also demonstrated.

Elapid Venoms↗

PubMed1996

Phospholipids and nuclear RNA.

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.

Animals↗

PubMed1994

Exposure of anionic phospholipids upon platelet activation permits binding of beta 2 glycoprotein I and through it that of IgG antiphospholipid antibodies. Studies in platelets from patients with antiphospholipid syndrome and normal subjects.

Patients with antiphospholipid syndrome, whether primary or secondary to systemic lupus erythematosus, may have thrombocytopenia. Their antibodies to anionic phospholipids might bind to phospholipids on the platelet wall but anionic phospholipids are asymmetrically located in the inner leaflet. In addition, antibodies to anionic phospholipids may require beta 2 glycoprotein I (beta 2GPI) as a cofactor in order to bind to phospholipids. In turn, beta 2GPI has high affinity for anionic phospholipids. Loss of this asymmetry occurs upon platelet activation and could thus permit such antibody-beta 2GPI-platelet interaction. We studied this by flow cytometry using purified beta 2GPI-FITC labelled and similarly labelled affinity-purified polyclonal antibodies to cardiolipin or phosphatidylserine (aPL) obtained from sera of patients with primary antiphospholipid syndrome. Five percent of resting platelets were bound by aPL in the presence of beta 2GPI. Such binding increased when we activated platelets with various agonists, reaching 31% with the concurrent use of thrombin and the calcium ionophore A23187. Platelet activation resulted in the expression of GMP140 but this did not correlate with aPL binding. This probably reflects that the expression of GMP140, which depends on their secretion of alpha granules, has different agonist responses and occurs at different times than do microvesicle formation and expression of prothrombinase activity which coincide with the loss of phospholipid asymmetry on the platelet wall. When we studied the binding of purified beta 2GPI we also found that it binds preferentially to activated platelets and that it seems to be a prerequisite for the binding of aPL onto them. Our findings indicate that aPL from patients with antiphospholipid syndrome may bind to activated platelets through beta 2GPI.

Anions↗

PubMed1994

Microdynamics of the phospholipid bilayer in cardiomyopathic hamster heart cell membrane.

To investigate the microdynamics and the structural architecture of the membrane phospholipid bilayer during the course of cardiomyopathy, membrane fractions were prepared from hearts of cardiomyopathic Syrian hamsters (BIO 14.6) aged 4, 18 and 31 weeks and compared with age-matched control hamsters (F1b). Membrane cholesterol, phospholipids and phospholipid fatty acids were measured by thin-layer chromatography, gas-liquid chromatography and high performance liquid chromatography. Microdynamics of the phospholipid bilayer were determined by a nanosecond fluorometer using pulsed excitation of a fluorescent probe, diphenyl-hexatriene. At the age of 4 weeks, there was no difference in lipid compositions and microdynamics between the BIO 14.6 and F1b. At the age of 18 weeks, saturated fatty acids, 18:0 and 22:0 increased and 20:0, 20:2 and 32:4 decreased in the BIO 14.6. At the age of 31 weeks, adding to the above changes in phospholipid fatty acids, unsaturated fatty acids 20:4 and 22:6 decreased, moreover membrane phospholipids, especially phosphatidylinositol and phosphatidylethanolamine significantly decreased. The viscosity and the wobbling angle of phospholipid molecules were decreased significantly. We have previously demonstrated that intracellular Ca2+ accumulation might be responsible for the pathogenesis of cardiomyopathy. Thus, we conclude that cardiomyopathic membrane may alter its structure and function with age. These alterations in cell membranes might be involved in the cardiac hypertrophy and dysfunction through impaired Ca2+ handling in cardiomyopathic hamsters.

Animals↗

PubMed1993

Exchange of phospholipids between Escherichia coli cells and environment.

We studied the exchange of phospholipids between Escherichia coli K-12 cells and the suspension medium containing inactivated guinea-pig serum. In this medium, the release of 3H-labelled phospholipids was proportional both to the quantity of serum and to the temperature of incubation. No phospholipids were released when no guinea-pig serum was added to the medium, or the incubation temperature was 4 degrees C. The release of phospholipids into the medium was accompanied by an uptake of serum phospholipids by the cells, as demonstrated by incorporation of labelled phospholipids from the suspension medium. We conclude that an exchange occurs between the cellular phospholipids and those of the medium. Control tests with 3H-thymidine showed that cellular lysis was not involved.

Animals↗

PubMed1986

High energy phosphates, phospholipids, and calcium in ischemic renal tubular cell injury.

Substantial data is available supporting critical roles for high energy phosphates, calcium, and phospholipids in ischemic cell injury. Although it has been proposed that one of these compounds may be most critical and defines the "final common pathway" for lethal cell injury, it is more accurate to define the pathogenesis of ischemic cell injury as the simultaneous derangement of several critical metabolic process which act in concert to produce a cascade of events that finally lead to plasma and subcellular membrane dysfunction incompatible with the maintenance of cell viability and integrity. As detailed in Figure 1, a tentative scheme is proposed interrelating high energy phosphate depletion, cellular calcium derangements, and membrane phospholipid degradation and loss. Ischemia directly leads to declines in the rate of oxidative phosphorylation due to a lack of oxygen availability. A fall in cellular ATP levels develops. Ischemia also promotes a redistribution of intracellular calcium pools and results in phospholipase activation and phospholipid degradation. When phospholipid degradation occurs concomitantly with a decline in high energy phosphate levels, phospholipid synthesis cannot keep pace with phospholipid degradation and net membrane phospholipid loss occurs. An increase in plasma membrane calcium permeability develops and the influx of calcium down its electrochemical gradient from extracellular to intracellular spaces occurs. This calcium is taken up and sequestered in mitochondria and causes further alterations in mitochondrial structure and function, leading to further declines in cellular ATP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Kidney Injury↗

PubMed1990

Lateral diffusion of erythrocyte phospholipids in model membranes comparison between inner and outer leaflet components.

The physical properties of lipid bilayers with a similar composition to the outer and inner leaflets of the human erythrocyte membrane have been examined in protein-free model systems. The outer leaflet (OL) was represented by a phospholipid mixture containing phosphatidylcholine and sphingomyelin extracted from human erythrocytes, while a mixture of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine represented the inner leaflet (IL). The ratio of cholesterol to phospholipid was varied in both mixtures. The lateral diffusion coefficient of fluorescent phospholipids diluted in such lipid mixtures was determined by the modulated fringe pattern photobleaching technique. Contrast curves with a single exponential decay, indicative of homogeneous samples, were obtained only for temperatures above 15 degrees C and for a cholesterol to phospholipid molar ratio below 0.8. The rate of lateral diffusion was approximately five times faster in IL than in OL multilayers, in agreement with former results obtained in human erythrocytes (Morrot et al. 1986). Varying the cholesterol to phospholipid ratio from 0 to 0.8 (mol/mol) enabled us to decrease the diffusion constant by only a factor of approximately 2 for both IL and OL mixtures. The order parameter of a spin-labeled phospholipid was determined in the different systems and found to be systematically smaller in IL mixtures than in OL mixtures. The present study indicates that the difference in lipid diffusivity of the two erythrocyte leaflets may be accounted for solely by a difference in phospholipid composition, and may be independent of cholesterol and protein asymmetry.

Diffusion↗

PubMed1989

Interactions among phospholipids of guinea pig rough microsomes, effect of fat deficiency.

The fatty acid composition and the steady-state fluorescence anisotropy (rs) of 1,6-diphenyl-1,3,5-hexatriene (DPH) were determined for each of the five major phospholipid (PL) classes present in the liver rough microsomes (RM) of guinea pigs fed with control and fat-deprived diets. In order to obtain information about PL-PL interactions and their contribution to the overall membrane fluidity the experimental rs of phospholipid mixtures were compared to the molar weighed average values of the individual phospholipid rs values. The PL ratios in the mixtures were the same to those found in the RM membranes. Binary mixtures of phosphatidylcholine (PC) with phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) show higher values of rs than those estimated from the individual component parameters indicating a 'rigidizing' interaction. The rigidizing effect of PE was also observed when this phospholipid was sonicated with mixtures of PC with PS and PI. However, no rigidizing effect of PE was observed in the PC bilayers when SM was included in the composition suggesting that PE-SM interactions prevent 'rigidizing' effects of PE. Besides, in spite that PC-PI and PC-PS mixtures have 'rigidizing' interactions, the incorporation of PI and PS to PC-PS and PC-PI mixtures, respectively, have a 'fluidizing' effect. In consequence, phospholipid polar head groups interact in RM membranes modifying the molecular packing and/or the rotational diffusion of acyl chains. The complexity and variety of constituent phospholipids could prevent major changes in the fluidity. The comparison of results obtained with PL mixtures of control and fat-deficient animals indicate that a change in the number of double bonds does not evoke a significant difference between either the 'rigidizing' of 'fluidizing' effects. However, there is a general tendency indicating that phospholipids with higher double bond index evoke lower 'rigidizing' and 'fluidizing' interactions. Moreover, PL of animals fed a fat-deficient diet have less fluidity than those of control animals.

Animals↗

PubMed1976

Palmitic acid-1-14C incorporation and turnover in lung phospholipids of rats treated with chlorphentermine, RMI 10.393 and Ro 4-4318.

The effects of 3 lipidosis-inducing drugs on the incorporation and turnover of palmitic acid-1-14C in lung phospholipids was studied. In rats treated with 1 dose of chlorphentermine or RMI 10.393, the incorporation of palmitate-1-14C into most lung phospholipid fractions was moderately decreased, but markedly lowered after 1 dose of Ro 4-4318. Eight doses of chlorphentermine and RMI 10.393 strongly inhibited the incorporation of palmitate-1-14C into lung phospholipids, whereas with 8 doses of Ro 4-4318 the incorporation was highly increased. Thirty hours after the last of 3 injections of the labeled palmitic acid the turnover of most lung phospholipids was considerably lower in chlorphentermine- and RMI 10.393-treated rats than in controls. Ro 4-4318, however, induced a highly increased turnover of most phospholipids. After 54 h, this effect had practically disappeared. Our studies showed that phospholipid storage after treatment with chlorphentermine and RMI 10.393 is mainly due to decreased degradation of phospholipids, whereas increased synthesis accounts for the effect of Ro 4-4318.

Amitriptyline↗

PubMed1991

Phospholipid profiles of human colon cancer using 31P magnetic resonance spectroscopy.

Phospholipids of 16 malignant and 11 non-malignant human colon specimens were analyzed using a chloroform-methanol analytical reagent in conjunction with 31P magnetic resonance spectroscopy (MRS) at 202.4 MHz. Sixteen individual generic phospholipids were identified and quantified for statistical intergroup comparisons. Statistically significant elevations in the relative concentrations of lysophosphatidylcholine and phosphatidylcholine plasmalogen were seen in malignant tissues along with significantly depressed levels of sphingomyelin and phosphatidylethanolamine plasmalogen. The malignant and non-malignant tissue groups were further differentiated by the detection of the minor phospholipids, lysophosphatidylcholine plasmalogen, lysophosphatidylethanolamine plasmalogen, lysophosphatidic acid and phosphatidylglycerol exclusively present in the malignant tissues and by significant changes in computed phospholipid metabolic indices that were dominated by choline containing lipids. The 31P MRS methods used represent an advancement over previous protocols for identifying and quantifying major and minor tissue phospholipids making this the first direct study of membrane phospholipids in human colon tissues using 31P MRS. The phospholipid profiles obtained may provide important information regarding the nature of the malignant cell's membrane system and identify markers which may be used to estimate malignant propensity, aggressiveness of disease and provide prognostic information.

Adenocarcinoma↗

PubMedDate not supplied

Metabolic fate of [14C]-ethanol into endothelial cell phospholipids including platelet-activating factor, sphingomyelin and phosphatidylethanol.

The metabolic fate of ethanol into the phospholipid pool of calf pulmonary artery endothelial cells was studied. [14C]-ethanol was incorporated into various endothelial cell phospholipids including phosphatidylethanol (PEth), which may represent a substantial fraction in microdomains of membrane phospholipids. The incorporation into phospholipids was reduced in the presence of pyrazole and cyanamide, inhibitors of ethanol metabolism. Wortmannin, the phosphatidylinositol 3-kinase inhibitor, increased [14C]-PEth formation. [3H]-acetate was also incorporated into endothelial cell phospholipids but in a different pattern. Distribution of [3H]-acetate and [14C]-ethanol into the fatty acyl moiety versus the glycerophosphoryl backbone of the phospholipids was also different. Stimulation of the endothelial cells with ATP increased [3H]-acetate incorporation into platelet-activating factor (PAF) and ethanol decreased it. Ethanol exposure increased ATP-stimulated [3H]-acetate incorporation into sphingomyelin. However, ATP had no effect on the incorporation of [14C]-ethanol into any phospholipids. The results suggest that the two precursors contribute to a separate acetate pool and that the sphingomyelin cycle may be sensitized in ethanol-treated cells. Thus, metabolic conversions of ethanol into lipids and the effect of ethanol on specific lipid mediators, e.g PAF, PEth and sphingomyelin, may be critical determinants in the altered responses of the endothelium in alcoholism.

Acetic Acid↗

PubMed1996

The effects of lidocaine and hypoxia on phospholipid biosynthesis in the isolated hamster heart.

In this study, the effects of lidocaine and hypoxia on the biosynthesis of phospholipids in the hamster heart were examined. Hamster hearts were perfused with [1,3-3H]glycerol under normal and hypoxic conditions, and in the absence or presence of 0.5 mg/mL lidocaine. After perfusion, the radioactivity incorporated into the various phospholipid fractions was determined. With the exception of phosphatidylcholine, the synthesis of phospholipids was generally stimulated by lidocaine perfusion. In contrast, hypoxia caused a general decrease in phospholipid biosynthesis which was partially restored by lidocaine. ATP and CTP levels were severely reduced under hypoxic conditions, but their levels were not restored by lidocaine treatment. The activities of enzymes for phospholipid synthesis were determined under the various perfusion conditions. The activity of phosphatidic acid phosphatase was elevated by lidocaine and decreased by hypoxic treatment. The activity of CTP:phosphatidic acid cytidylyltransferase was increased under hypoxia, with or without lidocaine. Despite the reduction in phosphatidylcholine biosynthesis, no change in the activity of cytidine diphosphocholine (CDPcholine):diacylglycerol cholinephosphotransferase was detected following lidocaine or hypoxic perfusion. However, enzyme activity was inhibited by the presence of lidocaine in the assay mixture. Our results indicate that the reduction in phospholipid biosynthesis under hypoxic conditions was caused mainly by diminishing high-energy nucleotide levels. The enhancement of phospholipid biosynthesis by lidocaine appeared to be mediated in part by modulation of enzyme activities.

Animals↗