Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Further enzymatic characteristics of a thylakoid protein kinase

The enzymatic characteristics of a protein kinase purified from thylakoids are further described. ATP (KM approximately 30 microM) and Mg2+ ion (greater than 1.0 mM) were required for activity, while ADP was a competitive inhibitor (Ki = 100 microM). Activity was 55% inhibited by the sulfhydryl inhibitor p-chloromercuribenzoate (1 mM) and was less sensitive to substituted maleimides. Lysine-rich histones (H1) were utilized as an exogenous phosphorylation substrate both by thylakoid-bound kinase and by isolated enzyme; threonine was predominantly phosphorylated by the in situ enzyme, whereas the isolated enzyme phosphorylated closely related serine residues as determined by peptide mapping. Detergents that proved useful in extracting the kinase from thylakoids markedly inhibited activity of the isolated enzyme, whereas Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid had little effect. The enzyme could be freed from detergent and behaved as an active monomer on size-exclusion chromatography. The phosphate contents of the light-harvesting chlorophyll a/b protein complex of photosystem II isolated from maximally phosphorylated thylakoid membranes of spinach and pea were equivalent to approximately 6% and approximately 19% phosphorylation, respectively. Corresponding values for nonphosphorylated membranes were approximately 3% and approximately 14.5%.     

A novel nonhemorragic protease from the African puff adder (Bitis arietans) venom.

A nonhemorrhagic proteinase B-20 from the venom of Bitis arietans has been purified to apparent electrophoretic homogeneity by chromatography on Sephadex G-100, Q-Sepharose, and CM-cellulose. It has a molecular weight of 20 k Da as determined by size exclusion chromatography on Sephadex G-100 and migrated as a single 20-k Da band on SDS polyacrylamide. It has an optimum pH of 6-8 and is inactive at pH 4.0. EDTA and 1,10-phenanthroline strongly inhibited the enzyme suggesting it is a metalloenzyme. Also it is inhibited by antipain but is unaffected by trasylol, antitrypsin, and pepsptatin. Colombin, an identified active component of Aristolochia albida used in the treatment of snake poisoning, did not inhibit the protease activity. It lost over 90% of its activity in the presence of 0.5 microM Hg(2+) but the inhibition was completely blocked in the presence of 10 microM mercaptoethanol implicating sulfhydryl groups in the catalytic entity of the protein. The activity was also inhibited competitively by glutathione and cysteine with inhibition binding constants K(i) of 240 and 40 microM, respectively. The enzyme is unaffected by several divalent cations but activated by 1 mM Fe(3+). It had a prolyl endopeptidase and thermolysin-like activity. The enzyme displayed a fast acting alpha-fibrinolytic and delayed gamma-fibrinolytic activity when tested on human fibrinogen. The relevance of these findings is discussed.     

Effects of ganglioside GM1 on DNA synthesis in isolated nuclei and on the activity of DNA polymerase alpha derived from S-phase HeLa cells

Ganglioside GM1 inhibited either DNA synthesis in isolated nuclei or the activity of DNA polymerase alpha fractionated from S-phase HeLa cells. The concentrations of GM1 necessary for 50% inhibition were about 5 microM and 10 microM for nuclei and DNA polymerase alpha, respectively. The GM1 inhibition of the enzyme activity was suppressed by the addition of 0.05% Triton X-100. Neither gangliotetraosylceramide (asialo-GM1) nor free N-acetylneuraminic acid inhibited the enzyme activity. These facts suggest that GM1, probably in the form of micelles, could influence the enzyme activity by behaving as a polyanionic macromolecule. The kinetic studies indicate that the GM1 inhibition of the enzyme activity was not competitive with the substrate, deoxythymidine triphosphate, but rather with the template DNA. Binding of GM1 and DNA polymerase alpha was suggested by the cocentrifugation of GM1 and the enzyme fraction after their preincubation. It was also observed that other acidic glycolipids, i.e., brain sulphatide and seminolipid, also inhibited the enzyme activity, whilst neutral galactosylceramide did not. The inhibitory influences of these sulphate esters of glycolipids were, similarly to GM1, suppressed by the addition of 0.05% Triton X-100.     

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.     

Total CA activity in isolated perfused guinea pig lung by 18O-exchange method

The rate of exchange of 18O between alveolar CO2 and lung water was measured in isolated perfused guinea pig lungs to quantify carbonic anhydrase (CA) activity. The average lung CA activity, with a reaction velocity constant of 5.32 +/- 2.2 s-1, is sufficient to accelerate CO2 reactions in lung water by two orders of magnitude over the uncatalyzed rate at 22 degrees C and a PCO2 of 40 Torr. Three sulfonamide inhibitors of CA with different human erythrocyte membrane permeabilities were used to determine the availability of the enzyme to the perfusate. Ethoxzolamide, the most permeable at 0.1 microM (100 times its inhibition constant, of Ki) inhibited 85% of enzyme activity after exposure of the lung for 3 min and 94% of enzyme activity after 30 min, whereas 1.25 microM (320 times its Ki) acetazolamide (1/165 as permeable) only inhibited CA 28% at 3 min and 75% at 30 min. Benzolamide (less than 1/1,000 as permeable) at 4 microM (1,000 times its Ki) inhibited only approximately 17% of control CA activity by 5 min and 48% by 30 min after the start of perfusion. These data indicate the CA available to pulmonary capillary plasma is approximately 10% of the total lung CA activity, in agreement with published measurements on the homogenized lung.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

Kinetic behaviour of acetylcholinesterase from muscle microsomal membranes

In order to determine whether catalytic hydrolysis of acetylcholine, observed in muscle microsomes enriched in sarcoplasmic reticulum membranes, was carried out by true acetylcholinesterase we studied the substrate specificity of this enzyme, its kinetic behaviour and its sensitivity against several reversible inhibitors. The results showed that the enzyme from muscle microsomes had acetylcholine (or acetylthiocholine) as the preferent substrate and was also able to hydrolyze acetyl-beta-methylcholine. The enzyme had a Km of 100-120 microM, being inhibited by a high substrate concentration. Acetylcholinesterase in this source was competitively inhibited by BW-284-c-51, eserine and decamethonium with ki values of 0.025 microM, 0.021 microM and 65 microM, respectively. The enzyme was poorly inhibited by the pseudocholinesterase inhibitor ethopropazine. The results show that the hydrolytic enzyme is indeed acetylcholinesterase.     

Purification and characterization of malate synthase from the glucose-grown wood-rotting basidiomycete Fomitopsis palustris

Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 microM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K1, 8.5 microM) and glycolate (Ki, 17 microM), and uncompetitively by coenzyme A (Ki, 100 microM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2+ (Km, 0.4 microM), which also served to significantly prevent enzyme inactivation during storage.     

Effect of bile acid analogs on 7 alpha-dehydroxylase activity in Eubacterium sp. V.P.I. 12708

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM.     

Mechanism of 4-HNE mediated inhibition of hDDAH-1: implications in no regulation

Nitric oxide synthase is inhibited by NG-methylated derivatives of arginine whose cellular levels are controlled by dimethylarginine dimethylamino-hydrolase (DDAH). DDAH-1 is a Zn(II)-containing enzyme that through hydrolysis of methylated l-arginines regulates the activity of NOS. Herein, we report the kinetic properties of hDDAH-1 and its redox-dependent regulation. Kinetic studies using recombinant enzyme demonstrated Km values of 68.7 and 53.6 microM and Vmax values of 356 and 154 nmols/mg/min for ADMA and L-NMMA, respectively. This enzymatic activity was selective for free ADMA and L-NMMA and was incapable of hydrolyzing peptide incorporated methylarginines. Subsequent studies performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching >or=100 microM were needed to confer significant inhibition of DDAH activity. However, exposure of DDAH to the lipid oxidation product, 4-HNE, dose-dependently inhibited DDAH activity with 15% inhibition observed at 10 microM, 50% inhibition at 50 microM, and complete inhibition at 500 microM. Mass spectrometry analysis demonstrated that the mechanism of inhibition resulted from the formation of Michael adducts on His 173, which lies within the active site catalytic triad of hDDAH-1. These studies were performed with pathophysiologicaly relevant concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Because DDAH is the primary enzyme involved in methylarginine metabolism, the loss of activity of this enzyme would result in impaired NOS activity and reduced NO bioavailability.     

The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive

The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA.     

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.     

Biochemical studies in marine species--I. NADP(+)-dependent isocitrate dehydrogenase from turbot liver (Scophthalmus maximus L.)

1. An NADP+-dependent isocitrate dehydrogenase was extracted from turbot liver. The enzyme required divalent cations (Mg2+ or Mn2+) for its activity but was inhibited by high salt concentrations. 2. The enzyme had an optimum activity in the pH range between 7.5 and 9.0. The enzymic activity increased with temperature (in the range of 5 to 68 degrees C) with an Ea of 23.5 kJ/mol and a Q10 of 1.34. 3. The apparent Km values for the substrates were 6.5 microM for NADP+, 56 microM for Mg2+, 87 microM for Mn2+ and 4.2 and 73.5 microM for the complexes Mg-isocitrate and Mn-isocitrate, respectively. The physiological significance of these results is discussed. 4. The enzyme was inhibited by citrate and adenine nucleotides. The degree of inhibition depended on the relation between the concentrations and that of magnesium. A possible regulating mechanism is proposed.     

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C

A nonradioactive spectrometric assay for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC) is described. l-alpha-Phosphatidylinositol from bovine liver was used as substrate in the presence of the micelle-forming detergent deoxycholic acid. PI-PLC isolated from Bacillus cereus and crude cytosol fractions from porcine brain were used as enzyme sources. PI-PLC activity was determined by measuring the release of 1-stearoyl-2-arachidonoyl-sn-glycerol with reversed-phase HPLC and UV detection at 200 nm. PI-PLC from B. cereus was not inhibited by the putative PI-PLC inhibitors U-73122 and ET-18-OCH(3) at 100 microM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC(50) of 75 microM. PI-PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100 microM to approximately 30 to 50%.     

Antifungal antibiotics and Siba inhibit 1-aminocyclopropane-1-carboxylic acid synthase activity

The antifungal antibiotics Sinefungin and A9145C isolated from Streptomyces griseolus and the synthetic nucleoside Siba, which are analogs of S-adenosylmethionine, inhibit the activity of 1-aminocyclopropane 1-carboxylic acid synthase from tomato fruits. Sinefungin and Siba were shown to be more potent inhibitors than A9145C. In extracts of green fruits, the enzyme activity was inhibited by Sinefungin with an I50 of 1 microM, which was similar to that caused by aminoethoxyvinylglycine, and by Siba with an I50 of 100 microM; in extracts from red tomatoes, the I50's were 25 microM and 100 microM, respectively. The inhibition of ACC synthase by these analogs could be reversed by gel filtration chromatography.     

Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine.

The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO2; 30 microM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 microM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 microM) and N-methyl-N'-nitro-N-nitrosoguanidine (200 microM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.