Principles of hydroxamate inhibition of metalloproteases: carboxypeptidase A

Hydroxamic acids of structure RCON(OH)CH(2)CH(CH(2)C(6)H(5))CO(2)H induce micromolar competitive inhibition of catalysis for the enzyme carboxypeptidase A. Enzyme affinity depends on the nature of the acyl group, for RCO equaling HCO, CH(3)CO, FCH(2)CO, F(2)CHCO, F(3)CCO, CH(3)OCH(2)CO, or CH(3)OCO. In acid dissociation these residues yield hydroxamic acid pK(a) values that vary from 7.6 to 10.3. Profiles of inhibitory pK(i) plotted versus pH indicate characteristically a maximum effectiveness near neutrality. Weaker binding to enzyme is generally displayed in either acidic or alkaline solution, with the position of the alkaline limb of the profiles depending on the pK(a) of the inhibitor. A reverse-protonation pattern of association with the enzyme is indicated, in which the hydroxamate anion of the inhibitor displaces a relatively acidic H(2)O ligand (pK(a) of 6) from the active-site zinc ion of carboxypeptidase A. The metal-coordinating, N-substituted hydroxamic acid functional groups exist in solution as a mixture of syn and anti rotamers, with relative abundances that depend on their pK(a). A pyrrolidinone analogue having a conformationally syn-fixed cyclohydroxamic acid was not an especially potent inhibitor. Structure-activity relationships suggest design criteria for hydroxamic acid inhibitors in order to provide most effective binding with metalloenzymes.     

Role of protein dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cell.

The pH profile for the uptake of L-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na+-dependent uptake by System A, and an increasing uptake by Na+-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na+-dependent component, (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicious exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na+-dependent component of uptake retained by L-glutamic acid but not by D-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports L-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic aic or of the norbornane amino acid. The dicarboxylic amino acids take the sequence, aspartic acid less than glutamic acid less than alpha-aminoadipic acid less than S-carboxymethylcysteine, in their rate of mediated, Na+-independent uptake at low pH. Diiodotyrosine and two dissimilas isomers of nitrotyrosine also show acceleration of uptake as the phenolate group on the sidechain is protonated, a result indicating that the acidic group need not be a carboxyl group and need not take a specific position in space to be accepted at the receptor site L. The presence of the carboxyl group does not upset the normal stereospecificity of System L until it falls on the beta-carbon in aspartic acid; even then it is the presence of the carbonyl group and not of the intact carboxyl group nor of its hydroxyl group that cancels out the stereospecificity, as was shown by the absence of normal stereospecificity for aspartic acid and asparagine and its presence in glutamic acid, homoserine and glutamine. In agreement, the uptak of aspartic acid is peculiarly sensitive to the presence of an alpha-methyl group or of other structures that modify the orientation of the sidechain.     

Oleic acid: an efficient inhibitor of glucosyltransferase

Among the extracts from 420 kinds of herbs, Prunus salicina, showing the highest glucosyltransferase inhibition activity, was purified and designated GTI-0163. Structural determination of GTI-0163 revealed it to be an oleic acid-based unsaturated fatty acid. GTI-0163 was an uncompetitive inhibitor of GTase. Among the unsaturated fatty acids, oleic acid showed a significantly higher GTase inhibitory activity than the saturated fatty acids or the ester form of oleic acid. These results strongly suggested that both the number of double bonds and the existence of free carboxyl groups of fatty acids play an important role in GTase inhibitory activity.     

Determination of the location of fluorescent probes attached to fatty acids using parallax analysis of fluorescence quenching: effect of carboxyl ionization state and environment on depth.

In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issue) was used to determine the location (depth) of anthroyloxy and carbazole probes attached to model membrane inserted fatty acids. A monotonic increase in depth was found as the number of carbon atoms between the attachment site of the probe and the fatty acyl carboxyl group is increased. It was also found that depth is sensitive to pH, with an increase in probe depth upon protonation of the fatty acid carboxyl group of around 0.5-2.5 A, depending on probe location and identity. This result shows that carboxyl protonation causes an increase in depth all along a fatty acid chain. In addition, it indicates that parallax analysis is very sensitive to small changes in depth. At a given pH, no significant change in probe depth was observed in vesicles containing anionic phospholipid or at various ionic strengths, suggesting these parameters do not strongly regulate fatty acyl chain location. It was also found that there is a decrease of the apparent depth of each of the fatty acyl attached probes both at longer excitation wavelengths and at longer emission wavelengths. This is consistent with there being a distribution of depth for each fluorophore, with shallower fluorophore dominating the fluorescence at red-shifted wavelengths. Solvent relaxation effects also appear to contribute to this wavelength dependence.     

Inhibition of yeast phosphatidic-acid synthesis by free fatty acids

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl-CoA thioesters. When palmitoyl-CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1-Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl-CoA thioesters as acyl donor. These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1-Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.     

Identification of the acyl transfer site of fatty acyl-protein synthetase from bioluminescent bacteria

Fatty acid activation, transfer, and reduction by the fatty acid reductase multienzyme complex from Photobacterium phosphoreum to generate fatty aldehydes for the luminescence reaction is regulated by the interaction of the synthetase and reductase subunits of this complex. Identification of the specific site involved in covalent transfer of the fatty acyl group between the sites of activation and reduction on the synthetase and reductase subunits, respectively, is a critical step in understanding how subunit interactions modulate the flow of fatty acyl groups through the fatty acid reductase complex. To accomplish this goal, the nucleotide sequence of the luxE gene coding for the acyl-protein synthetase subunit (373 amino acid residues) was determined and the conserved cysteinyl residues implicated in fatty acyl transfer identified. Using site-specific mutagenesis, each of the five conserved cysteine residues was converted to a serine residue, the mutated synthetases expressed in Escherichia coli, and the properties of the mutant proteins examined. On complementation of four of the mutants with the reductase subunit, the synthetase subunit was acylated and the acyl group could be reversibly transferred between the reductase and synthetase subunits, and fatty acid reductase activity was fully regenerated. As well, sensitivity of the acylated synthetases to hydroxylamine cleavage (under denaturation conditions to remove any conformational effects on reactivity) was retained, showing that a cysteine and not a serine residue was still acylated. However, substitution of a cysteine residue only ten amino acid residues from the carboxyl terminal (C364S) prevented acylation of the synthetase and regeneration of fatty acid reductase activity. Moreover, this mutant protein preserved its ability to activate fatty acid to fatty acyl-AMP but could not accept the acyl group from the reductase subunit, demonstrating that the C364S synthetase had retained its conformation and specifically lost the fatty acylation site. These results provide evidence that the flow of fatty acyl groups in the fatty acid reductase complex is modulated by interaction of the reductase subunit with a cysteine residue very close to the carboxyl terminal of the synthetase, which in turn acts as a flexible arm to transfer acyl groups between the sites of activation and reduction.     

Role of proton dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cells

The pH profile for the uptake of l-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na⁺-dependent uptake by System A, and an increasing uptake by Na⁺-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na⁺-dependent component. (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicuous exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another.A small Na⁺-dependent component of uptake retained by l-glutamic acid but not by d-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports l-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic acid or of the norbornane amino acid.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).     

Synthesis,Biological Evaluation and Molecular Docking of Deferasirox and Substituted 1,2,4‐Triazole Derivatives as Novel Potent Urease Inhibitors: Proposing Repositioning Candidate

A series of new deferasirox derivatives were synthesized through the reaction of monosubstituted hydrazides with 2‐(2‐hydroxyphenyl)‐4H‐benzo[e][1,3]oxazin‐4‐one. For the first time, deferasirox and some of its derivatives were evaluated for their in vitro inhibitory activity against Jack bean urease. The potencies of the members of this class of compounds are higher than that of acetohydroxamic acid. Two compounds, bearing tetrazole and hydrazine derivatives (bioisoester of carboxylate group), represented the most potent urease inhibitory activity with IC₅₀ values of 1.268 and 3.254 μm , respectively. In silico docking studies were performed to delineate possible binding modes of the compounds with the enzyme, urease. Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its crucial amino acid residues, CME592 and His593. The overall results of urease inhibition have shown that these target compounds can be further optimized and developed as a lead skeleton for the discovery of novel urease inhibitors     

Fatty acyl coupled catalase

Bovine liver catalase (hydrogen-peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was derivatized by 9″(10″)-[4′-{2-(4,6-dichloro-1,3,5-triazinyl)oxy}butoxy]stearic acid and the fatty acyl-coated enzyme was separated from native catalase and excess reagent by hydroxyapatite chromatography. The derivatization of catalase resulted in coupling the long-chain fatty acyl residues to lysine, histidine and arginine, while other amino acids remained essentially unaffected. The fatty acyl-coated enzyme was water soluble at pH > 7.0 but became octanol and ether soluble at pH < 6.5. The derivatized enzyme retained 50–80% of the catalatic- and peroxidative-specific activities. The free carboxyl function of the coupled long-chain fattyl acyl residues could serve as substrate for ATP-dependent CoA-thioesterification catalyzed by the rat liver microsomal long-chain fatty acyl-CoA synthase.     

Fatty acyl coupled catalase

Bovine liver catalase (hydrogen-peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was derivatized by 9"(10")-[4'-(2-(4,6-dichloro-1,3,5-triazinyl) oxy)butoxy] stearic acid and the fatty acyl-coated enzyme was separated from native catalase and excess reagent by hydroxyapatite chromatography. The derivatization of catalase resulted in coupling the long-chain fatty acyl residues to lysine, histidine and arginine, while other amino acids remained essentially unaffected. The fatty acyl-coated enzyme was water soluble at pH greater than 7.0 but became octanol and ether soluble at pH less than 6.5. The derivatized enzyme retained 50-80% of the catalatic- and peroxidative-specific activities. The free carboxyl function of the coupled long-chain fattyl acyl residues could serve as substrate for ATP-dependent CoA-thioesterification catalyzed by the rat liver microsomal long-chain fatty acyl-CoA synthase.     

Design, synthesis and biological evaluation of tyrosine-based hydroxamic acid analogs as novel histone deacetylases (HDACs) inhibitors

Histone deacetylases (HDACs) are a promising target for treating cancer and some other disorders. Herein, based on the structure of our previously reported tetrahydroisoquinoline-based hydroxamic acids, a novel series of tyrosine-based hydroxamic acid derivatives was designed and synthesized as HDACs inhibitors. Compared with tetrahydroisoquinoline-based hydroxamic acids, tyrosine-based hydroxamic acid derivatives exhibited more potent HDAC8 inhibitory activity. However, the antiproliferative activities and HeLa cell nuclear extract inhibition of several selected tyrosine-based hydroxamic acids were moderate.     

Dual effects of caffeoyl-amino acidyl-hydroxamic acid as an antioxidant and depigmenting agent

Anti-aging and depigmentation have both been an important subject of study for skin disease and the cosmetic industry. Caffeic acid (CA) has shown synergistically enhanced antioxidant activity when conjugated with amino acids. Hydroxamic acid (NHOH) is a well-known metal chelator, potentially having both tyrosinase inhibitory activity and free radical scavenging activity. We prepared caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) and found that caffeoyl-prolyl-hydroxamic acid (CA-Pro-NHOH) contained excellent antioxidant activity and tyrosinase inhibitory activity by various bioassay systems. Also, CA-Pro-NHOH showed mild melanogenesis inhibitory activity in Mel-Ab cells.     

Inhibition of adenylyl cyclase activity in brain membrane fractions by arachidonic acid and related unsaturated fatty acids

Pretreatment of mouse brain membranes with arachidonic acid (AA) and related unsaturated fatty acids at 30 degrees C for 10 min decreased basal activity and isoproterenol/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and forskolin-stimulated activities of adenylyl cyclase to a level less than 5% of control. The presence of the carboxyl group on the fatty acids was essential for the inhibition, because no such inhibition was found with ethyl arachidonate or AA attached to diacylglycerols and phospholipids. The AA-mediated inhibition was observed when the activity was measured in the presence of Mn2+ or forskolin and was insensitive to pertussis toxin or guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), indicating a mechanism independent of GTP-binding proteins. In addition, the fact that stimulators of the adenylyl cyclase catalytic unit, ATP, GTP gamma S and forskolin, when present during pretreatment, attenuate the inhibitory effect of AA may suggest that the catalytic unit is a target of AA. Bovine serum albumin suppressed the inhibition when present in the mixtures for pretreatment, but could not restore the adenylyl cyclase activity that had been reduced by AA, indicating an irreversible inhibition by AA. The effect of AA was found to be additive to P-site-mediated inhibition. The present study suggests the existence of another mechanism of regulation of adenylyl cyclase by unsaturated fatty acids.     

Urease inhibition by polymer analogs of substrate and thiophosphamides

Inhibition of soybean urease by polymeric substrate analogues, urea and thiourea polydisulfides (PDSU and PDSTU, respectively), or three thiophosphoric acid amides (TPAA), tri-(N-3-hydroxyphenyl)thiophosphamide (1), tri-(N-4,4'-aminodiphenyl)thiophosphamide, and di-oxy-(N-alpha-piridyl)thiophosphamide (3) was studied in aqueous solutions at various pH values. The inhibitory effects of all these substances were reversible and competitive with the lowest inhibition constant Ki 2.8 microM for TPAA-1 at pH 3.85. Above and below this pH value, Ki increased reaching 24 [mu]M at pH 7.2. All test substances inhibited urease comparably with known inhibitors such as thiols (cysteamine, etc.) and hydroxamic acid derivatives, but were less efficient than phosphorodiamidates. Structural features of possible urease inhibitors of higher efficiency were proposed.     

Inhibition of in vitro cholesterol synthesis by fatty acids.

Inhibitory effect of 44 species of fatty acids on cholesterol synthesis has been examined with a rat liver enzyme system. In the case of saturated fatty acids, the inhibitory activity increased with chain length to a maximum at 11 to 14 carbons, after which activity decreased rapidly. The inhibition increased with the degree of unsaturation of fatty acids. Introduction of a hydroxy group at the alpha-position of fatty acids abolished the inhibition, while the inhibition was enhanced by the presence of a hydroxy group located in an intermediate position of the chain. Branched chain fatty acids having a methyl group at the terminal showed much higher activity than the corresponding saturated straight chain fatty acids with the same number of carbons. With respect to the mechanism for inhibition, tridecanoate was found to inhibit acetoacetyl-CoA thiolase specifically without affecting the other reaction steps in the cholesterol synthetic pathway. The highly unsaturated fatty acids, arachidonate and linoleate, were specific inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA synthase. On the other hand, ricinoleate (hydroxy acid) and phytanate (branched-chain acid) diminished the conversion of mevalonate to sterols by inhibiting a step or steps between squalene and lanosterol.     

Synthesis and biological evaluation of O-methyl and O-ethyl NSAID hydroxamic acids

This paper reports the synthesis of O-methyl and O-ethyl NSAID hydroxamic acids, their antimicrobial activities, and their ability to inhibit urease and soybean lipoxygenase activities. Ibuprofen and fenoprofen hydroxamic acids with free hydroxy groups present the highest antimicrobial activity, while indomethacin and diclofenac analogs show significantly lower antimicrobial activity. Diclofenac hydroxamic acid 4e exerts the highest anti-urease activity. Indomethacin O-ethyl hydroxamic acid 3h and ibuprofen O-benzyl hydroxamic acid 4b exert significant inhibitory activities on soybean lipoxygenase. Fenoprofen and indomethacin O-ethyl hydroxamic acids 3b and 3h and diclofenac and indomethacin O-benzyl analogs 4g and 4i highly inhibit lipid peroxidation. The highest antioxidant activity was shown by fenoprofen derivative 3b.     

Formation of acylphosphatidylglycerol by a lysosomal phosphatidylcholine:bis(monoacylglycero)phosphate acyl transferase

A delipidated soluble fraction prepared from a mitochondrial-lysosomal fraction of rabbit alveolar macrophages that catalyzes transacylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate was also found to transfer oleic acid from [14C]dioleoyl phosphatidylcholine to form acylphosphatidylglycerol. The reaction was dependent on the presence of bis(monoacylglycero)phosphate and was maximal at a concentration of 44 microM when the ratio of fatty acid transferred to fatty acid released was 0.28. Addition of phosphatidylglycerol had only a small effect. Homogenates of rat liver also catalyzed the reaction and after subcellular fractionation the activity was localized to lysosomes. The lysosomal activity was solubilized by delipidation with butanol to give a preparation with a specific activity 2462 times that of the homogenate. Optimal activity of soluble preparations from both macrophages and liver was at pH 4.5, with little activity above 6.0. Release of free fatty acid was also stimulated under conditions of optimal acyl transfer. Both acyl transfer and release of fatty acid were inhibited by Ca2+, detergents, chlorpromazine, lysophosphatidylcholine, and oleic acid. When there was disproportional inhibition, acyl transfer was always more affected. These results suggest that sequential acylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate and then acylphosphatidylglycerol constitute a mechanism in the lysosome for the transport and partition of fatty acids released by the lysosomal phospholipases.     

Nonesterified fatty acids inhibit iron-dependent lipid peroxidation

The effect of various fatty acids on lipid peroxidation of liver microsomes induced by different methods in vitro was studied using oxygen uptake and malonaldehyde (MDA) production. It was observed that fatty acids with a single double bond are effective inhibitors of peroxidation. Stereo and positional isomers of oleic acid were equally effective as oleic acid. There was an absolute requirement for a free carboxyl group, since methyl esters of fatty acids and long-chain saturated and unsaturated hydrocarbons could not inhibit peroxidation. Saturated fatty acids with a chain length of 12-16 carbon atoms showed inhibition, whereas more than 18 carbon atoms reduced the inhibitory capacity. Fatty acids of lower chain length such as capric and caprylic acids did not show inhibition. Fatty acid inhibition was partially reversed by increasing the concentration of iron in the system. Peroxidation induced by methods which were independent of iron was not inhibited by fatty acids. It was observed that intestinal microsomes which were resistant to peroxidation due to the presence of nonesterified fatty acids in their membrane lipids were able to peroxidise by methods which do not require iron. These results suggest that certain fatty acids inhibit peroxidation by chelating available free iron. In addition, they may also be involved in competing with the esterified fatty acids in the membrane lipids which are the substrates for peroxidation.     

MgATP may depress de novo neuronal nuclear PAF generation by promoting the formation of alkylacylglycerophosphate,an inhibitor of alkylglycerophosphate acetyltransferase

MgATP substantially inhibited 1-alkyl-sn-glycero-3-phosphate (AGP) acetyltransferase found in neuronal nuclei. Other nucleotides and the ATP analogue AMP-PNP did not show a comparable inhibition. MgATP inhibition decreased in the presence of bovine serum albumin or the fatty acyl CoA synthetase inhibitor, Triacsin C. MgATP inhibition increased when nuclei were preincubated in 50 mM Tris-HCl (pH 7.4)/1 mM MgCl(2) at 37 degrees C, and preincubations elevated levels of nuclear free fatty acid. Exogenous free fatty acid, added to the acetylation incubations, increased the inhibition seen in the presence of MgATP. Oleoyl CoA, in the absence of MgATP, also inhibited AGP acetylation. These results suggested that MgATP supported the conversion of nuclear free fatty acids to fatty acyl CoA. Fatty acyl CoA may directly inhibit nuclear AGP acetyltransferase, but inhibition brought about by MgATP was competitive for the AGP substrate, suggesting an inhibitor close in structure to AGP. 1-Hexadecyl-2-arachidonoyl-sn-glycero-3-phosphate was identified as a competitive inhibitor for AGP in the acetylation reaction. Neuronal nuclei can convert AGP to 1-alkyl-2-acyl-sn-glycero-3-phosphate (AAcylGP), a reaction dependent upon MgATP and the presence of acetyl CoA or free CoA. This nuclear acylation was increased by free fatty acid addition and was seen using oleoyl CoA in the absence of MgATP. Nuclear AAcylGP formation was inhibited by bovine serum albumin and by Triacsin C. Thus, nuclear AGP acetyltransferase may be regulated by AGP acyltransferase activity and the availability of MgATP, a nucleotide that is rapidly lost during brain ischemia.