Characterization of an acyl-coenzyme a thioesterase associated with the envelope of spinach chloroplasts

The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell.     

Purification and properties of acyl coenzyme A thioesterase II from Rhodopseudomonas sphaeroides

A high molecular weight acyl coenzyme A (acyl-CoA) thioesterase, designated thioesterase II, has been purified 5300-fold from photoheterotrophically grown cells of Rhodopseudomonas sphaeroides. In contrast to R. sphaeroides acyl-CoA thioesterase I [Boyce, S.G., & Lueking, D.R. (1984) Biochemistry 23, 141-147], thioesterase II has a native molecular mass (Mr) of 120,000, is capable of hydrolyzing saturated and unsaturated acyl-CoA substrates with acyl chain lengths ranging from C4 to C18, and is completely insensitive to the serine esterase inhibitor diisopropyl fluorophosphate. Palmitoyl-CoA and stearoyl-CoA are the preferred (lowest Km) saturated acyl-CoA substrates and vaccenoyl-CoA is the preferred unsaturated substrate. However, comparable Vmax values were obtained with a variety of acyl-CoA substrates. Unlike a similar thioesterase present in cells of Escherichia coli [Bonner, W.M., & Bloch, K. (1972) J. Biol. Chem. 247, 3123-3133], R. sphaeroides thioesterase II displays a high ratio of decanoyl-CoA to palmitoyl-CoA activities and exhibits little ability to hydrolyze 3-hydroxyacyl-CoA substrates. Only 3-hydroxydodecanoyl-CoA supported a measurable rate of enzyme activity. With the purification of thioesterase II, the enzymes responsible for greater than 90% of the acyl-CoA thioesterase activity present in cell-free extracts of R. sphaeroides have now been identified.     

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.     

Purification and characterization of the 2-methyl branched-chain Acyl-CoA dehydrogenase, an enzyme involved in NADH-dependent enoyl-CoA reduction in anaerobic mitochondria of the nematode, Ascaris suum

The 2-methyl branched-chain acyl-CoA dehydrogenase was purified to homogeneity from mitochondria of the parasitic nematode, Ascaris suum. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. The enzyme migrated as a single protein band with Mr = 42,500 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the enzyme is a tetramer composed of identical subunits. The enzyme exhibited absorbance maxima at 272, 375, and 452 with a ratio 7.9:0.8:1.0, respectively. FAD content was estimated to be 0.9 mol/mol of subunit and the absorption coefficient of FAD at 452 nm was 14.1 mM-1 cm-1. The purified enzyme dehydrogenated both 2-methylbutyryl-CoA and 2-methylvaleryl-CoA with apparent Km and Vmax values of 18 microM and 1.62 mumol/min/mg and 21 microM and 1.58 mumol/min/mg, respectively. This enzyme also appeared to dehydrogenate butyryl-CoA, valeryl-CoA, and octanoyl-CoA but at a much lower rate. The enzyme did not dehydrogenate propionyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and palmitoyl-CoA. Tiglyl-CoA and 2-methyl-2-pentenoyl-CoA were identified as reaction products from 2-methylbutyryl- and 2-methylvaleryl-CoA, respectively. Dehydrogenating activity with both substrates was inhibited by tiglyl-CoA, acetoacetyl-CoA, and straight chain acyl CoAs of increasing chain length. N-Ethylmaleimide and p-hydroxymercuribenzoate had little effect on dehydrogenating activity but the heavy metals Hg2+ and Ag2+ were potent inhibitors. Physiologically, the dehydrogenase functions as a branched-chain enoyl-CoA reductase. Incubations of A. suum submitochondrial particles, NADH, tiglyl-CoA, purified A. suum electron-transfer flavoprotein, and the 2-methyl branched-chain acyl-CoA dehydrogenase resulted in the rotenone-sensitive, dehydrogenase-dependent formation of 2-methylbutyryl-CoA.     

Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.     

The oxidation of dicarboxylic acid CoA esters via peroxisomal fatty acyl-CoA oxidase

Evidence supporting a common peroxisomal beta-oxidation pathway for the coenzyme A thioesters of medium-chain-length dicarboxylic acids (DCn-CoA) and monocarboxylic acids (MCn-CoA) has been obtained. Using the mono-CoA esters of dodecanedioic acid (DC12-CoA) and lauroyl-CoA (MC12-CoA) as substrates, parallel inductions of activities and parallel increases in specific activities during purification of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) from rat liver after di(2-ethylhexyl)phthalate treatment were seen. The purified enzyme was used for antiserum production in rabbits; antiserum specificity was verified by immunoblot analysis. Coincident losses of oxidase activities with MC12-CoA and DC12-CoA were found in immunotitration experiments with rat liver homogenates, supporting the hypothesis that peroxisomal fatty acyl-CoA oxidase is solely responsible for the oxidation of medium-chain length dicarboxylic acid substrates. Kinetic studies with purified enzyme using the mono-CoA esters of sebacic (DC10-CoA), suberic (DC8-CoA), and adipic (DC6-CoA) acids along with DC12-CoA revealed substrate inhibition. Although these substrates exhibited similar calculated Vmax values, with decreasing chain length, the combination of increasing Km values and decreasing substrate inhibition constant (Ki) caused the maximum obtainable velocity to decrease. These studies offer an explanation for the previously observed limit of the ability of peroxisomes to chain-shorten dicarboxylates and increased urinary excretion of adipic acid when peroxisomal oxidation of dicarboxylic acids is enhanced.     

Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.

The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes.     

Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis

A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.     

Purification and characterization of a long-chain acyl-CoA hydrolase from rat liver microsomes.

A long-chain acyl-CoA hydrolase from rat liver microsomes has been purified by solvent extraction and gel chromatography to homogeneity as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme was a monomer of molecular weight 59 000. In a sucrose gradient it sedimented at 4.3 S. The isoelectric point, pI was 6.9, and the Stokes radius was approx. 31 A. The enzyme hydrolyzed long-chain fatty acyl-CoA (C7--C18) with maximum activity for palmitoyl-CoA. Bovine serum albumin activation of the enzyme was related to the ratio acyl-CoA/bovine serum albumin, and at high ratios, acyl-CoA inhibited the enzyme activity. Disregarding the substrate inhibition, an apparent Km of 65 nmol/mg protein or 1-10(-7) M and a V of 750 nmol/mg protein per min were calculated. The enzyme was inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide. Reactivation by means of dithiothreitol was not complete.     

Purification and characterization of a fatty acyl-CoA hydrolase from the uropygial glands of Peking ducks (Anas domesticus)

In a previous communication the occurrence of a medium-chain acyl-CoA hydrolase designated thioesterase-B in the uropygial gland of mallard ducks was reported [L. Rogers, P. E. Kolattukudy, and M. J. de Renobales (1982) J. Biol. Chem. 257, 880-886]. In the present study, thioesterase-B was purified from the postmicrosomal supernatant of homogenized uropygial glands from Peking ducks (Anas domesticus). Most of the contaminating thioesterase activities were removed by ammonium sulfate fractionation. The 55% ammonium sulfate supernatant, containing thioesterase-B, was chromatographed on hydroxylapatite followed by gel filtration on Sephadex G-100. The remaining contaminants were removed by chromatofocusing followed by desalting on Sephadex G-75. This procedure gave a 26% yield with a nearly 200-fold purification. Gel filtration of the purified enzyme showed that the molecular weight of the native enzyme was 56,300, whereas sodium dodecyl sulfate-gel electrophoresis of components separated by chromatofocusing showed that the purified enzyme contained enzymatically active proteins of molecular weights 59,400, 58,300, 56,000, and 55,800. The four species differed slightly in pI (4.9, 4.7, 4.45, and 4.40) but they were kinetically and immunologically indistinguishable. All four had the same N-terminal sequence. The purified thioesterase preparation showed a pH optimum of 9.3 with C12-CoA but the pH optimum was dependent on the chain length of the acyl group. At pH 8.0, C10 was the preferred substrate with less activity on C12, C8, and C14. The enzymatic activity was stimulated by bovine serum albumin and was inhibited by p-hydroxymercuribenzoate. Involvement of active serine in catalysis was suggested by inhibition of the enzyme by diethylpyrocarbonate, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Degradation of chloroaromatics: purification and characterization of maleylacetate reductase from Pseudomonas sp. strain B13.

Maleylacetate reductase of Pseudomonas sp. strain B13 was purified to homogeneity by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100. The final preparation gave a single band by polyacrylamide gel electrophoresis under denaturing conditions and a single symmetrical peak by gel filtration under nondenaturing conditions. The subunit M(r) value was 37,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Estimation of the native M(r) value by gel filtration gave a value of 74,000 with a Superose 6 column, indicating that the enzyme is dimeric. The pH and temperature optima were 5.4 and 50 degrees C, respectively. The pI of the enzyme was estimated to be 7.0. The apparent Km values for maleylacetate and NADH were 58 and 30 microM, respectively, and the maximum velocity was 832 U/mg of protein for maleylacetate. Maleylacetate and various substituted maleylacetates, such as 2-chloro- and 2-methyl-maleylacetate, were reduced at significant rates. NADPH was also used as a cofactor instead of NADH with nearly the same Vmax value, but its Km value was estimated to be 77 microM. Reductase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group in the enzyme.     

Characterization of an acyl-coA thioesterase that functions as a major regulator of peroxisomal lipid metabolism.

Peroxisomes function in beta-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor alpha. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.     

Purification and characterization of an oat fructan exohydrolase that preferentially hydrolyzes beta-2,6-fructans.

Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammonium sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogeneity as determined by the presence of a single band (43 kD) on a silver-stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of beta-2,6-linked fructan (neokestin) isolated from oat was used as the substrate to purify fructan hydrolase. Neokestin and small degree of polymerization fructan isomers were used to characterize the substrate specificity of the purified enzyme. The purified fructan hydrolase catalyzed hydrolysis of the terminal beta-2,6 linkage of 6G,6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal beta-2,6 linkage of 6G-kestotriose and approximately 10 times faster than it hydrolyzed the terminal beta-2,1 linkage of chicory inulin. Sucrose and 1-kestose were not substrates. The Km for neokestin (beta-2,6-linked fructans with a degree of polymerization of 7-14) hydrolysis was 2.8% (w/v), and the Vmax was 0.041 mumol min-1 mL-1. The Km for hydrolysis of 6G,6-kestotetraose was 5.6% (w/v), and the Vmax was 0.138 mumol min-1 mL-1. Catalysis was exolytic and by multiple chain attack. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

Purification and characterization of hydroxycinnamoyl D-glucose. Quinate hydroxycinnamoyl transferase in the root of sweet potato, Ipomoea batatas Lam

We have previously proposed a chlorogenic acid biosynthetic pathway which involves a transesterification reaction between hydroxycinnamoyl D-glucose and D-quinic acid. The proposed pathway was based on tracer experimental results (Kojima, M., and Uritani, I. (1972) Plant Cell Physiol. 13, 311-319). The enzyme that catalyzes the above reaction has been purified 160-fold from sweet potato root (Ipomoea batatas Lam.) and characterized. The purified enzyme yielded one band of 26,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000 by gel filtration chromatography. Therefore, the enzyme seems to consist of a single polypeptide of 25,000-26,000 daltons. The isoelectric point of the enzyme was 8.6. The optimum pH of the enzyme reaction was 6.0. The enzyme did not require any metal for activity and showed a broad substrate specificity toward hydroxycinnamoyl D-glucose as donors. The Km and Vmax values were 3.7 mM and 8.5 units/mg of protein for t-cinnamoyl D-glucose, 3.9 mM and 15.1 units/mg of protein for p-coumaroyl D-glucose, and 14.3 mM and 38.1 units/mg of protein for caffeoyl D-glucose. The enzyme showed a strict substrate specificity toward D-quinic acid-related compounds as acceptors; the Km and Vmax values were 16.7 mM and 15.1 units/mg of protein for D-quinic acid, 250 mM and 19.0 units/mg of protein for shikimic acid, and there was no activity with either L-malic acid or meso-tartaric acid. The enzyme activity changed in a manner suggesting its involvement in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.     

Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.

We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.     

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.