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P C Schmid

Publications and source records attributed to P C Schmid.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.

Animals↗
PubMed1991

Generation of the mitochondrial permeability transition does not involve inhibition of lysophospholipid acylation. Acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity is not found in rat liver mitochondria.

The possible involvement of acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles in regulating the mitochondrial permeability transition have been examined by direct methods. 1-Acyllysophospholipid acyltransferase activity found in mitochondrial preparations obtained by differential centrifugation is inhibited by several transition-inducing agents and by glutathione disulfide. However, marker enzyme analysis employing mitochondria prepared by Percoll density gradient centrifugation or fractionated by a shear force-dependent method indicate that this activity is associated with contaminating microsomes and not with mitochondria. The absence of phospholipid acylation-deacylation cycles in isolated mitochondria is demonstrated by the absence of 18O incorporation from H2(18)O into phospholipid acylester carbonyl groups, confirming conclusions arrived at from marker enzyme data by a definitive independent approach. Mitochondria prepared by differential centrifugation and Percoll density gradient centrifugation are shown to be equivalent in requirements for induction of the permeability transition and the apparent rate of this process. It is concluded that 1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles are not factors regulating the transition in isolated mitochondria. However, mitochondrial phospholipase A2 activity remains as a potential regulating factor, whereas the action of transition-inducing agents on microsomal 1-acyllysophospholipid acyltransferase may be important in mechanisms of cell injury.

Acyl Coenzyme A↗
PubMed1991

Remodeling of rat hepatocyte phospholipids by selective acyl turnover.

Acyl turnover of rat hepatocyte phospholipids and triacylglycerols was assessed by incubating the cells in media containing 40% H2(18)O and measuring the time-dependent incorporation of 18O into ester carbonyls by gas chromatography-mass spectrometry of hydrogenated methyl esters. Incorporation of 18O into 22-carbon acyl groups was low in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, whereas in phosphatidylethanolamine, it was about the same as in the other acyl groups. Incorporation of 18O into individual molecular species of phosphatidylcholine and phosphatidylethanolamine was determined after phospholipase C hydrolysis, derivatization to dinitrobenzoates, and separation by high-performance liquid chromatography. In most molecular species, acyl groups at the sn-1 and sn-2 positions became 18O-labeled at drastically different rates, indicating remodeling through deacylation-reacylation. Molecular species expected to arise de novo from acylation of glycerophosphate exhibited similar rates of 18O incorporation at the sn-1 and sn-2 positions. The data suggest that hepatocyte phospholipids are continually synthesized, remodeled by deacylation-reacylation at specific turnover rates up to 10-15%/h, and degraded. This acyl turnover probably does not involve the majority of intracellular unesterified fatty acids whose 18O incorporation was found to be very low. In contrast, the oxygens of extracellular unesterified fatty acids were readily exchanged with the media. This exchange was enzyme-catalyzed, possibly by lipases released into the media from damaged cells. Incorporation of 18O into exogenously added fatty acids was also rapid and resulted in enhanced uptake of 18O-labeled fatty acids into cellular lipids, primarily triacylglycerols and phosphatidylcholine, without drastic change of the intracellular free fatty acid pool.

Animals↗
PubMed1991

Phosphatidyl-Tris rather than N-acylphosphatidylserine is synthesized by Rhodopseudomonas sphaeroides grown in Tris-containing media.

We have synthesized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(N-oleoyl)serine (N-acyl-PS) and 1,2-dioleoyl-sn-glycero-3-phospho-Tris (phosphatidyl-Tris) and have characterized both phospholipids by their chemical and chromatographic properties, as well as by their IR, 13C NMR, and 1H NMR spectra. Comparison of these data with those reported for a phospholipid isolated from Rhodopseudomonas sphaeroides grown in Tris-supplemented media [Donohue et al. (1982) Biochemistry 21, 2765-2773] indicates that R. sphaeroides synthesizes phosphatidyl-Tris rather than N-acyl-PS.

Acylation↗
PubMed1990

Differential turnover of phospholipid acyl groups in mouse peritoneal macrophages.

Phospholipid acyl turnover was assessed in mouse peritoneal exudate cells which consisted primarily of macrophages. The cells were incubated for up to 5 h in media containing 40% H218O, and uptake of 18O into ester carbonyls of phospholipids was determined by gas chromatography-mass spectrometry of hydrogenated methyl esters. The uptake was highest in choline phospholipids and phosphatidylinositol, less in ethanolamine phospholipids, and much less in phosphatidylserine. Acyl groups at the sn-1 and sn-2 positions of diacyl glycerophospholipids, including arachidonic and other long-chain polyunsaturated fatty acids, acquired 18O at about the same rate. Acyl groups of alkylacyl glycerophosphocholine exhibited lower rates of 18O uptake, and acyl groups of ethanolamine plasmalogens (alkenylacyl glycerophosphoethanolamines) acquired only minimal amounts of 18O within 5 h, indicating a low average acyl turnover via free fatty acids. Pulse experiments with exogenous 3H-labeled arachidonic acid supported the concept that acylation of alkenyl glycerophosphoethanolamine occurs by acyl transfer from other phospholipids rather than via free fatty acids and acyl-CoA. The 18O content of intracellular free fatty acids increased gradually over a 5-h period, whereas in extracellular free fatty acids it reached maximal 18O levels within the first hour. Arachidonate and other long-chain polyunsaturated fatty acids were found to participate readily in deacylation-reacylation reactions but were present only in trace amounts in the free fatty acid pools inside and outside the cells. We conclude that acyl turnover of macrophage phospholipids through hydrolysis and reacylation is rapid but tightly controlled so that appreciable concentrations of free arachidonic acid do not occur.

Acylation↗
PubMed1988

Determination of ester carbonyl 18O/16O ratios in phospholipids by gas chromatography-mass spectrometry.

A method is described for the quantitation of 18O enrichment in phospholipid acyl groups. Methyl esters are prepared by transesterification with sodium hydroxide in methanol and are hydrogenated prior to gas chromatography-mass spectrometry (GC-MS). Estimation of 18O content of the resulting esters is made using the m/z 74 and m/z 76 fragments, which contain both the ester oxygens. The accuracy of the method is demonstrated with synthetic methyl esters and phosphatidylcholines containing a known amount of 18O.

Carbon Isotopes↗
PubMed1987

N-Acylation of ethanolamine phospholipids by acyl transfer does not involve hydrolysis.

N-Acylethanolamine phospholipids occur in infarcted but not in normal canine myocardium. Their synthesis is catalyzed by a membrane-bound, Ca2+-requiring N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of various phospholipids including phosphatidylethanolamine to the amino group of ethanolamine phospholipids. When dog heart mitochondria are incubated in media containing Ca2+ and H2(18)O, the resulting N-acylethanolamine phospholipids do not accumulate 18O in either the amide or 1-O-acyl groups. The results indicate that acyl transfer occurs without hydrolysis, most likely through an acyl-enzyme complex which may be covalently linked.

Acylation↗
PubMed1987

Assessment of phospholipid deacylation-reacylation cycles by a stable isotope technique.

Incorporation of 18O into glycerophospholipids was determined after incubating mouse peritoneal exudate cells for 1 or 2 h in media containing 40% H(2)18O. Gas chromatography-mass spectrometry of hydrogenated fatty acid methyl esters showed highest amounts of 18O in choline phospholipids and phosphatidylinositol. Acyl groups generally present at the sn-1 position contained at least as much carbonyl 18O as those at the sn-2 position. Considering the route of 18O incorporation via free fatty acid derived through ester hydrolysis in H(2)18O, acyl turnover in certain peritoneal exudate cell phospholipids may equal or exceed 20% per h.

Acylation↗
PubMed1986

Separation of phosphatidylethanolamine from its oxidation and hydrolysis products by high-performance liquid chromatography.

1-Palmitoyl-2-linoleoyl-phosphatidylethanolamine degrades relatively quickly when subjected to common storage and handling procedures. The degradation products consist of compounds in which double bonds in the sn-2 position acyl chain are partially oxidized and of products arising from the hydrolysis of the acyl ester bonds. Thin-layer chromatography (TLC), which is widely utilized to isolate and to ascertain the purity of phospholipids, does not readily separate the oxidation products from the parent lipid class. High-performance liquid chromatography (HPLC), however, employing a normal phase column and an isocratic, UV-transparent solvent system, can be employed to produce a rapid analytical or preparative of phosphatidylethanolamine (PE) from these degradative impurities.

Chromatography, High Pressure Liquid↗
PubMed1986

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain.

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.

Acylation↗
PubMed1985

Properties of rat liver N-acylethanolamine amidohydrolase.

Rat liver microsomes and mitochondria contain an amidohydrolase which catalyzes the hydrolysis of N-acylethanolamine to ethanolamine and fatty acid. The enzyme is active over a wide range of pH, does not require divalent cations, and is inhibited by sulfhydryl-reactive agents. The detergents Triton X-100, sodium cholate, and sodium dodecyl sulfate are also inhibitory, but sodium taurodeoxycholate has little effect and was therefore used to solubilize the enzyme. The solubilized enzyme exhibits high substrate specificity for long-chain amides of ethanolamine. Amides of propanolamine or higher homologs are hydrolyzed at a drastically slower rate, and isomers prepared from long-chain amine and short-chain hydroxy acid are neither substrates nor inhibitors of the enzyme. Neither ceramide (N-acylsphingosine) nor N,O-diacylethanolamine is hydrolyzed. Both particulate and soluble enzyme preparations also catalyze the synthesis of N-acylethanolamine from ethanolamine and fatty acid, probably by the amidohydrolase acting in reverse.

Amidohydrolases↗
PubMed1985

Occurrence of N-acylethanolamine phospholipids in fish brain and spinal cord.

N-Acylethanolamine phospholipids were identified in the central nervous system of the fresh water fish, pike (Esox lucius) and carp (Cyprinus carpio), at levels ranging from 0.1 to 0.9% of total phospholipid. The N-acylethanolamine phospholipids of carp brain were isolated and characterized by chemical, biochemical and spectroscopic methods. Two major species, 1,2-diacyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 30%) and 1-O-(1'-alkenyl)-2-acyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 70%) were identified. The N-acyl groups of each species consisted primarily of 16:0 (approx. 60%) but also contained 16:1, 18:0 and 18:1 (approx. 10% each) and a number of trace constituents. The N-acylethanolamine phospholipids had O-acyl and O-alkenyl group compositions similar but not identical to those of the ethanolamine phospholipids of the same tissue. N-Acylethanolamine phospholipids were present in all subcellular fractions of carp brain, except mitochondria.

Animals↗
PubMed1985

Effects of phospholipase A2 inhibitors on ruthenium red-induced Ca2+ release from mitochondria.

The pharmacologic agents verapamil, nifedipine, diltiazem, prenylamine, N-oleoylethanolamine, R 24571, trifluoperazine, dibucaine, and quinacrine are examined as potential inhibitors of rat liver mitochondrial phospholipase A2 acting on endogenous phospholipid. Their potency as inhibitors of the enzyme is compared to their activities as inhibitors of phospholipase A2-dependent swelling and ruthenium red-induced Ca2+ release in intact mitochondria. For verapamil, diltiazem, trifluoperazine, dibucaine, and quinacrine, there is complete agreement between the relative potencies as inhibitors of phospholipase A2 and the two other processes. Nifedipine and prenylamine, which are weak inhibitors of phospholipase A2, produce a permeable inner membrane, provided that the mitochondrial have accumulated Ca2+. R 24571, which strongly inhibits the enzyme, disrupts mitochondria by a Ca2+-independent mechanism. N-Oleoylethanolamine, which is an effective inhibitor of swelling, does not inhibit phospholipase A2 or ruthenium red-induced Ca2+ release. The results support a proposed scheme wherein ruthenium red-induced Ca2+ release is viewed as reverse activity of the Ca2+-uptake uniporter occurring subsequent to decline in the proton motive force. The latter effect is proposed to arise from a specific phospholipase A2-dependent increase in inner-membrane H+ conductance of mitochondrial subpopulations. It is further shown that mitochondrial membranes display cyclic oscillations in free fatty acid content which are not dependent on the presence of Ca2+ or on the capacity to generate acylcoenzyme A.

Animals↗
PubMed1979

Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function.

Liver mitochondria treated with N-ethylmaleimide can accumulate Ca2+ but cannot retain it. Ca2+ loss following uptake occurs in parallel with a proton uptake and collapse of the membrane potential. Respiration is not activated during Ca2+ release and cannot be stimulated by uncoupler. After Ca2+ release and accompanying phenomena are nearly complete, the mitochondria undergo a large amplitude swelling. Nupercaine inhibits the premature release of Ca2+, proton uptake, decline in membrane potential, inhibition of uncoupler-stimulated respiration, and large amplitude swelling. Ruthenium red also prevents these effects. Neither Sr2+ or Mn2+ will substitute for Ca2+ to induce these effects in N-ethylmaleimide-treated mitochondria. The effects of N-ethylmaleimide plus Ca2+ on mitochondria are not accompanied by a significant alteration in the content or composition of phospholipids but are accompanied by small increases in the mitochondrial content of free fatty acids. Free fatty acids accumulate more rapidly in response to limited Ca2+ loading in the absence of N-ethylmaleimide than they do in its presence. In the absence of N-ethylmaleimide, polyunsaturated fatty acids and saturated plus monounsaturated fatty acids accumulate at nearly equal rates. In the presence of N-ethylmaleimide, polyunsaturated fatty acids accumulate more rapidly than saturated plus monounsaturated fatty acids. Any condition or agent tested which inhibited swelling and the other effects produced by Ca2+ plus N-ethylmaleimide also prevented the more rapid accumulation of polyunsaturated, compared to saturated plus monounsaturated, fatty acids. In the light of a positional analysis of phospholipid acyl moieties, these data suggest that 1-acyllysophospholipids accumulate in swelling mitochondria but not in response to noraml Ca2+ loading or when swelling is blocked by other agents. The free fatty acid accumulation, per se, is not responsible for swelling, but levels of exogenous palmitic acid as low as 1 nmol/mg of protein dramatically alter the dependence of swelling velocity on Ca2+ concentration, producing a shift from a sigmoidal- to a hyperbolic-like relationship. This same alteration is brought about by aging the mitochondrial preparation at 0 degrees C. Either pyruvate or DL-carnitine prevents the effect of exogenous palmitate and restores the Aa2+ swelling dependence of aged N-ethylmaleimide-treated mitochondria to that of fresh N-ethylmaleimide-treated mitochondria. Intramitochondrial acylcoenzyme A or acylcarnitine, or both, therefore, to be the modulator of Ca2+ sensitivity rather than free fatty acid. The findings are discussed in terms of the role of intramitochondrial phospholipase and other phospholipid metabolizing enzymes in the mechanisms of N-ethylmaleimide plus Ca2+ effects on mitochondria.

Animals↗