Inhibition of leukotriene function can modulate particulate-induced changes in bone cell differentiation and activity.

Aseptic loosening remains the major problem facing arthroplasty longevity with particulates from component materials touted as the cause of periprosthetic osteolysis. Proposed mechanisms in aseptic bone loss include: increased resorption, increased differentiation of osteoclasts (and/or macrophages locally), and decreased osteoblastic bone formation. Leukotrienes participate in osteoclastic bone resorption. We investigated inhibiting leukotrienes synthesis, using ICI 230487, to ameliorate the effects of particulates on osteoclast pit formation and also assessed the effects of alendronate, a bisphosphonate, on pit formation. Three particulates were used: ultra high molecular weight polyethylene (UHMWPE), polymethylmethacrylate (PMMA) and hydroxyapatite (HA). Osteoclast resorption was increased with UHMWPE, PMMA, and HA particles. Interventions with alendronate and ICI 230487 reduced particulate-induced osteoclast resorption. Both ICI 230487 and alendronate reduced osteoclast numbers at higher doses. To assess the effect of particulates on osteoclast and macrophage differentiation, mouse bone marrow was cultured and stained for tartrate resistant acid phosphatase colonies (TRAP+, osteoclasts) and nonspecific esterase positive colonies (NSE+, macrophage precursors). Particulates increased both TRAP+ and NSE+ colony formation. These increases were inhibited by ICI 230487. Particulates also inhibited osteoblast function assessed by the development of mineralized nodules and alkaline phosphatase positive (AP+) colony area. ICI 230487 partly protected osteoblast function from this particulate effect. Blockade of leukotriene production may prove a useful therapeutic intervention for particulate-induced aseptic loosening by inhibiting resorptive activity, reducing the pro-inflammatory cell populations induced and recruited by these particulates, as well as ameliorating the negative effects of inflammatory mediators on osteoblast function.

Ion chromatography on a new moderate capacity anion exchange column.

The mechanisms of retention of anions on a new solvent-compatible, moderate capacity (170 microequivalent per column) anion-exchange column are described. Retention based on anion exchange is shown using both sodium hydroxide and 4-cyanophenolate eluants. Plots of the log k' vs. log C, (where k' is the capacity factor and C is the eluant concentration) are shown to be linear with slopes approximating the charge of the eluant as predicted by the standard anion-exchange equation. Sodium hydroxide is shown to be a relatively weak eluant, capable of separating monovalent anions that are difficult to separate on other columns. Sodium 4-cyanophenolate is shown to be a strong eluant, capable of eluting polyvalent anions. The retention of nonionic alkylphenones is also described. In plots of log k' vs. percentage acetonitrile in the eluant, these compounds were found not to follow the solvophobic mechanism typical of reverse-phase HPLC columns. Results are compared with other anion exchange columns. Applications include the determination of lysophosphatidyl inositol in brain microsomes and methacrylic acid in esterase-treated samples.

Acylation of lysophosphatidylcholine by brain membranes.

Brain microsomes catalyze the acylation of lysophosphatidylcholine (lysoPtdCho) in the presence and absence of added CoA derivatives. The catalytic activity is distributed widely in various subcellular fractions from rat or bovine cerebral cortex as measured by the conversion of 1-[14C]palmitoyl-sn-glycero-3-phosphocholine to [14C]PtdCho. Analysis of this latter compound revealed that the dipalmitoyl derivative is the predominant molecular species, which is formed in this reaction by transacylation between two [14C]lysoPtdCho molecules. This lysoPtdCho: lysoPtdCho transacylation reaction was enhanced several-fold by the addition of oleoyl-CoA, which also is an effective donor of acyl groups in the acyl-CoA: lysoPtdCho acyltransferase-catalyzed reaction. Measurements of the initial velocity of the transacylation reaction were used to determine kinetic constants. Apparent Km values for lysoPtdCho in the presence and absence of oleoyl-CoA were 29 microM and 104 microM, respectively, and the corresponding maximal velocities were 0.11 and 1.06 nmol.min-1.mg-1, respectively. Oleoyl-CoA at 4 microM produced half-maximal stimulation of the transacylation reaction. CoA also stimulated the rate of conversion of [14C]lysoPtdCho to [14C]PtdCho, either in the presence or absence of oleoyl-CoA, with a half-maximal effect of CoA at 80 microM. These results may be important in understanding the regulation of PtdCho synthesis and the mechanism by which acyl group composition of this compound is controlled.

Effects of lysophospholipids and diacylglycerols on the transfer of arachidonic acid to phospholipids and triacylglycerols in rat brain membranes.

Brain membranes catalyze the acylation of lysophospholipids and diacylglycerols (DAG) to form the respective phospholipids and triacylglycerols (TAG). These acylation reactions were examined using brain plasma membrane-enriched fractions by measuring the incorporation of [14C]arachidonic acid into TAG and individual phospholipids under a variety of conditions. In the absence of added lipid substrates, the amount of [14C]arachidonic acid incorporated into TAG in the presence of ATP, Mg2+, and CoA was approx twice the amount incorporated into phosphatidylositol (PtdIns), and more than 10 times the amount incorporated into phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer). These results suggest the presence of endogenous DAG, lysoPtdIns, and the required enzymes in the membrane preparations for acylation reactions. The addition of DAG, lysoPtdCho or lysoPtdIns to the incubation system resulted in a 2-20-fold increase in the rate of incorporation of labeled arachidonic acid into TAG, PtdCho or PtdIns, respectively. LysoPtdEtn and lysoPtdSer were poor substrates for the synthesis of PtdEtn and PtdSer. On the other hand, the addition of lysoPtdSer stimulated the incorporation of [14C]arachidonic acid into TAG and into most phospholipids, especially phosphatidic acid, the synthesis of which was enhanced more than 10-fold. Exogenous lysoPtdCho and lysoPtdIns inhibited the incorporation of [14C]arachidonate into TAG in the presence of DAG, and DAG inhibited the incorporation of [14C]arachidonic acid into phospholipids in the presence of lysophospholipids. In general, [14C]palmitic acid was less effectively incorporated into lipids than arachidonic acid. These results suggest reciprocal regulatory effects of DAG and lysophospholipids on acyltransfer to phospholipids and triacylglycerol in brain membranes.

Lithium effects on inositol phospholipids and inositol phosphates: evaluation of an in vivo model for assessing polyphosphoinositide turnover in brain.

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.

Ion chromatographic determination of inositol tris- and tetrakisphosphates in rat brain.

Ion chromatography has been applied to the determination of inositol 2,4,5-trisphosphate (Ins 2,4,5-P3), inositol 1,3,4-trisphosphate (Ins 1,3,4-P3), inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). Other common polyanionic metabolites, including ATP and GTP, do not interfere with the determinations even at concentrations exceeding those normally found in tissue extracts. Assay of rat brain for Ins 1,4,5-P3 and Ins 1,3,4,5-P4 by the method of standard addition gives values which are within 10% of the amount calculated by external calibration. When rat brain homogenates are spiked with Ins 1,4,5-P3, the recoveries are greater than 90%. Treatment of animals with lithium chloride and pilocarpine produces increases in total inositol trisphosphates from 14 to 64 nMol/g wet weight, and increases in Ins 1,3,4,5-P4 from 0.9 to 18 nMol/g wet weight in tissue extracts obtained from the cerebral cortex. Using chemically suppressed conductivity, detection limits in brain are estimated to be 50 pMol/g wet weight for inositol trisphosphates and 22 pMol/g wet weight for inositol tetrakisphosphate.

Metabolism of phosphatidylinositol in plasma membranes and synaptosomes of rat cerebral cortex: a comparison between endogenous vs exogenous substrate pools.

The metabolism of phosphatidylinositols (PI) labeled with [14C]arachidonic acid within plasma membranes or synaptosomes was compared to the metabolism of PI prelabeled with [14C]arachidonic acid and added exogenously to the same membranes. Incubation of membranes containing the endogenously-labeled PI pool in the presence of Ca2+ resulted in the release of labeled arachidonic acid, as well as a small amount of labeled diacylglycerol. Labeled arachidonic acid was effectively reutilized and returned to the membrane phospholipids in the presence of adenosine triphosphate (ATP), CoA, and lysoPI. Although Ca2+ promoted the release of labeled diacylglycerol from prelabeled plasma membranes, this amount was only 17% of the maximal release, i.e., release in the presence of deoxycholate and Ca2+. This latter condition is known to fully activate the PI-phospholipase C, and incubation of prelabeled plasma membranes resulted in a six-fold increase in labeled diacylglycerols. On the other hand, when exogenously labeled PI were incubated with plasma membranes in the presence of Ca2+, the labeled diacylglycerols released were 59% of that compared to the fully activated condition. The phospholipase C action was calcium-dependent, regardless of whether exogenous or endogenous substrates were used in the incubation. In contrast to plasma membranes, intact synaptosomes had limited ability to metabolize exogenous PI even in the presence of Ca2+, although the activity of phospholipase C was similar to that in the plasma membranes when assayed in the presence of deoxycholate and Ca2+. These results suggest that discrete pools of PI are present in plasma membranes, and that the pool associated with the acyltransferase is apparently not readily accessible to hydrolysis by phospholipase C.

Decapitation-induced changes in inositol phosphates in rat brain.

Decapitation resulted in a time-dependent production of inositol phosphates in rat brain. This production was analyzed by measuring both the radioactivity and the concentrations of inositol phosphates generated from [3H]inositol-labeled phospholipids. Both measurements produced the same time-dependent changes, including a rapid decrease in inositol 1,4,5-trisphosphate within 1.5 min, a 6-fold increase in inositol 1,4-bisphosphate to a maximum at 1.5 min, a 5-fold rise in inositol 4-monophosphate to a maximum at 2.5 min, and little change in inositol 1-monophosphate. The temporal changes in the mass and radioactivity of these compounds, together with the decrease in labeling of phosphatidylinositol 4,5-bisphosphates, support the idea that the inositol phosphates originate from the hydrolysis of phosphatidylinositol 4,5-bisphosphates and not from either the direct hydrolysis of phosphatidylinositol 4-phosphates or phosphatidylinositols.

Purification of lysophosphatidylcholine transacylase from bovine heart muscle microsomes and regulation of activity by lipids and coenzyme A.

Heart muscle microsomes catalyze the transacylation of lysophosphatidylcholine (lyso PC) to produce phosphatidylcholine (PC). The enzyme which catalyzes this reaction, lyso PC:lyso PC transacylase, has been isolated and characterized from bovine heart muscle microsomes. The purification of the enzyme was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The purified enzyme was nearly homogeneous and consisted of a single molecular species of 128 kDa as determined by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The catalytic activity of the enzyme was dependent on the presence of either CoA or acyl-CoA, both of which maximally stimulated at concentrations of approx. 10 microM. Analysis of the PC produced in the reaction showed that the enzyme catalyzed a transacylation in which both acyl groups arose from lyso PC. Furthermore, the enzyme did not possess acyl-CoA:lyso PC acyltransferase activity, lysophospholipase or acyl-CoA hydrolase activity, nor did it catalyze transacylation from lyso PC to lysophosphatidylethanolamine, lysophosphatidylinositol or lysophosphatidylserine. Although transacylation was highly specific for lyso PC as the substrate, various unsaturated fatty acyl-CoA derivatives served as activators. Palmitoyl-CoA and stearoyl-CoA did not significantly activate, although acetyl-CoA was an effective activator. Further modulation of activity was produced by palmitic acid and PC, both of which further activated the enzyme in the presence of oleoyl-CoA, whereas arachidonic acid, oleic acid, phosphatidylethanolamine and phosphatidylserine had no effect on activity. The high activity of this transacylase and its regulation by lipids suggests an important role for disaturated PC species in membranes and a mechanism for controlling the metabolism of lyso PC.

Determination of inositol phosphates and other anions in rat brain.

Anion chromatography methods have been developed to determine the concentrations of inositol phosphates and other water-soluble metabolites in rat brain. The cerebral cortex, hippocampus, and striatum were analyzed by chemically suppressed conductivity detection. Animals were sacrificed by directed microwave irradiation for more accurate estimations of in vivo concentrations. The effects of different sample preparation methods are compared. Trichloroacetic acid, formic acid, and perchloric acid extraction result in lower recoveries of phosphocreatine and other anions than does homogenization with water followed by chloroform-methanol extraction. The effects of postdecapitative ischemia on metabolite concentrations are determined. These methods will be used to study the effects of pharmacological agents on inositol phosphates and should be broadly applicable to the analysis of a variety of biological systems.

Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase.

The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors.

Arachidonic acid uptake by phospholipids and triacylglycerols of rat brain subcellular membranes.

In the presence of ATP, MgCl2 and CoASH, different subcellular membrane fractions isolated from rat cerebral cortex exhibited characteristic profiles for the incorporation of [1-14C]arachidonic acid into phospholipids and triacylglycerols. In general, uptake of label by phosphatidylcholines was higher in the synaptic membranes, and that by phosphatidylinositols was higher in the microsomes and somal plasma membranes. A substantial amount of the labeled arachidonate was also incorporated into triacylglycerols, especially in the somal plasma membranes and microsomes. Enzymes mediating the transfer of arachidonic acid to phospholipids were unstable with respect to sample storage and exposure to elevated temperatures. In contrast, the acyltransferase for triacylglycerols was more stable to these factors. Washing the membranes with bovine serum albumin resulted in an enhancement of the incorporation of label into phosphatidylinositols without affecting that of phosphatidylcholines, but the incorporation into triacylglycerols was inhibited. Treatment of synaptosomes and plasma membranes with saponin resulted in an enhancement in the labeling of phospholipids, but the labeling of triacylglycerols was inhibited. Thus, although labeled arachidonic acid was incorporated into phospholipids and triacylglycerols in brain subcellular membranes, these two types of acyltransferases exhibited different properties and responded differently to membrane perturbing agents.

Acylation of lysophosphatidylcholine in bovine heart muscle microsomes: purification and kinetic properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase.

Bovine heart muscle microsomes rapidly convert lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) in the presence of oleoyl-CoA. Both substrates are incorporated into the product, although the rate of incorporation of radiolabel into PC from 1-[14C]palmitoyl-LPC was approximately threefold higher than the rate of incorporation from [14C]oleoyl-CoA. Furthermore, the rate of incorporation of radiolabel from [14C]LPC was stimulated fivefold by the presence of oleoyl-CoA. These results demonstrate the presence of both acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) and an LPC:LPC transacylase (EC 3.1.1.5) in microsomes. Separation of the two enzymatic activities and purification of the acyltransferase was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The isolated acyltransferase was a single species of 64,000 Da as judged by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The substrate specificity of the enzyme was studied by using a series of lysophospholipids as acyl acceptors and acyl-CoA derivatives as acyl donors. The enzyme was catalytically active with LPC as acyl acceptor but displayed little or no activity with lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine. Of the LPC derivatives tested, the highest activity was obtained with 1-palmitoyl-LPC. Wider specificity was exhibited for the nature of the acyl donor, for which arachidonoyl-CoA, linoleoyl-CoA, and oleoyl-CoA were highly active substrates. These properties of the acyltransferase are in accord with a role of the enzyme in determining the composition of PC in myocardium.

Determination of inositol phosphates and other biologically important anions by ion chromatography.

Ion chromatography has been applied to the simultaneous, multi-component determination of biologically important anions. More than 20 different biologically important anions were separated on high performance ion-exchange columns and detected using chemically suppressed conductivity. Application of the technique to the separation of inositol mono-, bis-, and trisphosphates shows that these compounds can be separated from the other ions tested and can be detected at concentrations that may be found in vivo. For inositol monophosphate, the conductivity was proportional to the amount of compound from less than 20 pmol to more than 400 nmol. Although alternative methods are available for assaying each of these anions individually, the advantages of ion chromatography lie in the sensitivity of detection, the speed of separation, and the ability to simultaneously determine numerous ions. This method should be broadly applicable to studies of second messengers, measurements of reaction rates, and various metabolic studies.

Ion chromatographic determination of sugar phosphates in physiological samples.

Ion chromatography is shown to be capable of simultaneous determination of biologically important anions. Application of this technique is illustrated for the separation and quantification of the major anions present in rat brain and liver tissues. Sugar phosphates and carboxylic acids are separated on high-performance anion-exchange columns and are detected using chemically suppressed conductivity. Detection limits range from 20 to 100 pmol for the anions tested, including inositol phosphates, lactate, pyruvate, glucuronic acid-1-phosphate, fructose-6-phosphate and glucose-6-phosphate. The coefficient of variation for the determination of most anions was in the range 5-10%. Many of these anions are either difficult to separate with other methods, or require expensive radiochemical techniques for detection. This method should be applicable to other biological studies, from the flow of carbons in photosynthesis to the study of synaptic transmission.

Purification of long-chain acyl-CoA hydrolase from bovine heart microsomes and regulation of activity by lysophospholipids.

Long-chain acyl-CoA hydrolase (EC 3.1.2.2) has been purified 12,000-fold from bovine heart muscle microsomes by extraction with Miranol detergent, followed by column chromatography on Reactive Blue agarose and DEAE-cellulose. The purified enzyme was nearly homogeneous on polyacrylamide gel electrophoresis and had a molecular weight of 41,000 in the presence of dodecyl sulfate. The specificity and kinetic properties of the enzyme were studied using several acyl-CoA derivatives as potential substrates. The enzyme showed a wide degree of specificity with little dependence on either the fatty acyl chain length or the degree of unsaturation of the acyl group. The kinetic properties were in accord with the Michaelis-Menten equation under most conditions, although high concentrations of substrates generally inhibited the enzyme. Arachidonoyl-CoA, which was the most effective substrate, had a Km value of 0.4 microM and a Vmax value of 6.0 mumol min-1 mg-1. The enzyme was strongly and specifically inhibited by constants of 16 and 30 nM, respectively. Other lysolipids and detergents such as deoxycholate and Triton X-100 were weak inhibitors. These properties and others distinguish this enzyme from other acyl-CoA hydrolases and support the idea that lysophospholipids may be important in vivo in the regulation of lipid metabolism.

Lysophospholipase activity in rat brain subcellular fractions.

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [32P]glycerophosphocholine from [32P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg2+, Cu2+, and Zn2+ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25-200 microM), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 micrograms/mg protein and inhibition at higher concentrations. Addition of cholesterol (50-250 micrograms/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because of their detergent properties.

Purification and properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes.

Acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase has been purified approximately 3000-fold from bovine brain microsomes by detergent solubilization followed by ion-exchange and affinity chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single protein of molecular weight 43,000. The specificity of the purified enzyme was studied by measuring the catalytic activity with various lysophospholipids and acyl-CoA derivatives. Of the lysophospholipids tested, only lysophosphatidylcholine was a substrate. Less specificity was exhibited toward the acyl-CoA derivatives, although the enzyme showed a clear preference for arachidonoyl-CoA and little or no activity with palmitoyl-CoA or stearoyl-CoA. High concentrations of arachidonoyl-CoA inhibited the enzyme. The velocity was a sigmoidal function of the concentration of lysophosphatidylcholine (LPC) with little activity obtained below 20 microM LPC. The specificity and kinetic properties of the enzyme were altered, however, by incorporation of the enzyme into liposomes composed of a mixture of phospholipids. Decanoyl-CoA and myristoyl-CoA, which were effective substrates for the soluble enzyme, did not serve as acyl donors for the liposome-bound acyltransferase. Furthermore, the liposome-bound enzyme, in contrast to the soluble form of the enzyme, was active at concentrations of LPC below the critical micelle concentration. The liposome-bound enzyme was also substantially less susceptible to thermal denaturation and proteolytic digestion. This modulation of the acyltransferase activity by interaction with phospholipids may relate to the kinetic properties and the regulation of the enzyme in vivo.

Partial purification and properties of long-chain acyl-CoA hydrolase from rat brain cytosol.

Long-chain acyl-CoA hydrolase (EC 3.1.2.2) has been partially purified from the 100,000 X g supernatant fraction of rat brain tissue. The purification procedure included chromatography on gel filtration media, DEAE-cellulose, CM-cellulose, and hydroxyapatite. The partially purified enzyme had a specific activity of 7.1 mumol/min-mg, and when analyzed by polyacrylamide gel electrophoresis, revealed one major and three minor bands of protein in the presence of dodecyl sulfate and two major bands of protein in the absence of dodecyl sulfate. The enzyme had a molecular weight of 65,000 and showed no evidence of aggregated or dissociated forms. The highest catalytic activity was exhibited with palmitoyl-CoA and oleoyl-CoA as substrates. Lower activity was found with decanoyl-CoA as the substrate and little or no activity was found with acetyl-CoA, malonyl-CoA, butyryl-CoA, or acetoacetyl-CoA. The enzyme was inhibited by CoA, various metal ions, including Mn2+, Mg2+ and Ca2+, and by bovine serum albumin. Heating the enzyme produced a loss of activity which corresponded to a first-order kinetic process, the rate of which was independent of the choice of substrate used to measure enzyme activity. This finding supports the idea that the purification procedure yields a single species of long-chain acyl-CoA hydrolase.