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 properties of NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the green alga Chlamydomonas reinhardtii

NADP-dependent non-phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9), previously described in higher plants, has been now found to be present in eukaryotic green algae, but in neither cyanobacteria nor non-photosynthetic microorganisms. The enzyme from the unicellular green alga Chlamydomonas reinhardtii, strain 6145c, has been purified to apparent electrophoretic homogeneity. The non-phosphorylating enzyme was effectively separated from the NADP-dependent phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) dye-ligand chromatography on Reactive Red-120 agarose. The purified enzyme exhibited an optimum pH in the 8.5–9.0 range and a specific activity of approx. 8 μmol·(mg protein)⁻¹·min⁻¹. The native protein was characterized as a homotetramer with a molecular weight of 190 000, a Stokes radius of 5.2 mn, and an isoelectric point of 6.9. From kinetic studies, K_m-values of 9.8 and 51 μM were calculated for NADP and D-glyceraldehyde 3-phosphate, respectively, an absolute specificity for both substrates being observed. L-Glyceraldehyde 3-phosphate was a potent non-competitive inhibior (K_i, 48 μM). The reaction products NADPH and D-3-phosphoglycerate inhibited enzyme activity in a competitive manner with respect to NADP (K_i, 78 μM) and D-glyceraldehyde 3-phosphate (K_i, 1.2 mM), respectively. Thermal inactivation occurred above 45°C and was effectively prevented by either substrate. The presence of essential vicinal thiol groups is suggested by the inactivation produced by diamide, with D-glyceraldehyde 3-phosphate, but not NADP, behaving as a protective agent. The enzyme's possible physiological role in photosynthetic metabolism is discussed briefly.     

Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39

l-allo-Threonine aldolase (l-allo-threonine acetaldehyde-lyase), which exhibited specificity for l-allo-threonine but not for l-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of l-allo-threonine (K_m=1.45 mM, V_max=45.2 μmol min⁻¹ mg⁻¹). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5′-phosphate participates in the enzymatic reaction. The enzyme does not act on either l-serine or l-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (l-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or l-threonine aldolase (EC 4.1.2.5).     

Isolation of 3-methylcrotonyl-coenzyme A carboxylase from bovine kidney

3-Methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidation pathway, was highly purified from bovine kidney. The native enzyme has an approximate molecular weight of 835,000 as measured from exclusion limits by polyacrylamide gel electrophoresis at pH 7.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated two subunits, identified as a biotin-free subunit (A subunit; M_r = 61,000) and a biotin-containing subunit (B subunit; M_r = 73,500). The biotin content of the enzyme was 1 mol/ 157,000 g protein, consistent with an AB protomeric structure for the enzyme. The isoelectric point of the enzyme was found to be 5.4. Maximal MCase activity was found at pH 8 and 38 °C in the presence of Mg²⁺ and an activating monovalent cation such as K⁺. Kinetic constants (K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 75 μm; ATP, 82 μm; HCO₃^?, 1.8 mm. Certain acyl-CoA derivatives, including crotonyl-CoA, (2Z)-3-ethylcrotonyl-CoA, and acetoacetyl-CoA, were also substrates for the enzyme. Some data on inhibition of the enzyme by acyl-CoA derivatives, and sulfhydryl- and arginyl-reagents, are presented.     

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.     

Enzymatic production of α-dehydrobiotin from biotin

The enzymatic production of α-dehydrobiotin (α-DHB), an antibiotic, from biotinyl-CoA using acyl-CoA oxidase and from biotin using a coupling system of biotinyl-CoA synthetase and acyl-CoA oxidase was developed. Acyl-CoA oxidase was found to show activity for biotinyl-CoA. K_m and V_max values of acyl-CoA oxidase for biotinyl-CoA were 75 μM and 3.92 μmol min⁻¹ mg⁻¹, respectively. Optimum reaction conditions for the α-DHB production from biotin were examined. The maximum production of α-DHB (4.29 μmol ml⁻¹) was obtained, when the reaction was carried out at 30°C for 36 h in a mixture consisting of 100 mM potassium phosphate buffer (pH 8.0), 20 mM biotin, 20 mM ATP, 60 mM CoA, 20 mM MgCl₂, 2 units of biotinyl-CoA synthetase, 90 units of acyl-CoA oxidase and 25 units of catalase in a total volume of 0.6 ml under aerobic conditions. The product was purified from 14 ml of the reaction mixture and 10 mg of crystals with white needle form were obtained. From NMR, mass spectra and other physical analyses, this compound was identified as (+)-trans-α-DHB.     

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of M_r 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.     

Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis

The study of acyl transfer activity of a wide spectrum amidase from Rhodococcus sp. R312, overproduced in an Escherichia coli strain, revealed that the ‘bi-bi-ping-pong’ type reaction was efficient with only four very-short chain (C₂–C₃) aliphatic amides as substrates. The optimum working pH was 7.0 for all neutral amides. Very short-chain aliphatic carboxylic acids were 10–1000-fold less efficient and the corresponding optimum working pH values depended on the acid used. Very polar molecules, such as water, hydroxylamine and hydrazine, were good acyl acceptors. An [acyl donor]/[acyl acceptor] ratio lower than 0.3-0.5 had to be maintained to avoid enzyme inhibition by excess acyl donor. The different acyl-enzyme complexes generally exhibited high affinity for hydroxylamine or hydrazine (except the propionyl-enzyme complex), so that the residual hydrolysis activities were almost totally inhibited at appropriate acyl acceptor concentrations. Molar conversion yields were higher with hydrazine as acyl acceptor (e.g., 97% with acetamide as acyl donor) because of the higher V_max values, but in all cases, interesting quantities of short-chain hydroxamic acids (2.9-6.5 g l⁻¹) and acid hydrazides (6.4–7.8 g l⁻¹) could be quickly obtained (10–60 min) with small amounts of enzyme (0.04-0.20 g l⁻¹).     

Characterization of an intradiol dioxygenase involved in the biodegradation of the chlorophenoxy herbicides 2,4-D and 2,4,5-T

Hydroxyquinol 1,2-dioxygenase, an intradiol dioxygenase, which catalyzes the cleaving of the aromatic ring of hydroxyquinol, a key intermediate of 2,4-D and 2,4,5-T degradation, was purified from Nocardioides simplex 3E cells grown on 2,4-D as the sole carbon source. This enzyme exhibits a highly restricted substrate specificity and is able to cleave hydroxyquinol (K_m for hydroxyquinol as a substrate was 1.2 μM, V_max 55 U/mg, K_cat 57 s⁻¹ and K_cat/K_m 47.5 μM s⁻¹), 6-chloro- and 5-chlorohydroxyquinol. Different substituted catechols and hydroquinones are not substrates for this enzyme. This enzyme appears to be a dimer with two identical 37-kDa subunits. Protein and iron analyses indicate an iron stoichiometry of 1 iron/65 kDa homodimer, α₂ Fe. Both the electronic absorption spectrum which shows a broad absorption band with a maximum at 450 nm and the electron paramagnetic resonance spectra are consistent with a high-spin iron(III) ion in a rhombic environment typical of the active site of intradiol cleaving enzymes.     

Two flavonoid-specific malonyltransferases from cell suspension cultures of Petroselinum hortense: Partial purification and some properties of malonyl-coenzyme A: Flavone/flavonol-7-O-glycoside malonyltransferase and malonyl-coenzyme A: Flavonol-3-O-glucoside malonyltransferase

Two malonyltransferases were isolated from irradiated cell suspension cultures of parsley (Petroselinum hortense) and extensively purified. One enzyme was most active with flavone and flavonol 7-O-glycosides as substrates; the other enzyme preferentially malonylated flavonol 3-O-glucosides. The substrate specificity of the enzymes in vitro was in good agreement with the pattern of malonylated flavonoid glycosides occurring in the cell cultures in vivo. The apparent K_m values for the most efficient substrates, including the donor of the acyl residue, malonyl-CoA, were about 4–20 μm. Both malonyltransferases had an apparent molecular weight of approximately 50,000.     

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.     

Polyamine oxidase of millet shoots

Polyamine oxidase was purified ca 168-fold from the acetone powder extract of millet shoots. The light yellow enzyme had maximum absorption at 278,380 and 460 nm. The absorption at 380 and 460 nm was decreased by the addition of spermidine. The enzyme (M, ca 80 000) showed a high specificity for spermine and spermidine (K_ms 6 × 10⁻⁵ M and 5 × 10⁻⁷ M respectively). The enzyme was inhibited by quinacrine and acriflavine.     

Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

The Streptomyces glaucescens fabH gene, encoding β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an M_r of 37,000, while gel filtration analysis determined a native M_r of 72,000 ± 3,000 (mean ± standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a K_m value of 3.66 μM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with K_m values of 2.4 μM for acetyl-CoA, 0.71 μM for butyryl-CoA, and 0.41 μM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.     

Chloroplastic Glyceride Isoform of Dihydroxyacetone Phosphate Reductase from Dunaliella tertiolecta: Purification and Characterization

The chloroplastic glyceride isoform of dihydroxyacetone phosphate reductase (Gly-DHAPR) in the photosynthetic unicellular green algae, Dunaliella, plays key role in the synthesis of glycerol-P and glycerides. A four-step procedure has been developed to purify the Gly-DHAPR from the chloroplasts of Dunaliella tertiolecta. The enzyme was purified 462-fold to apparent electrophoretic homogeneity by precipitation of Rubisco by polyethylene glycol-4000, and successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. The overall yield of the purified enzyme was 5.1% with a specific activity of 425 μmol. min^?1. mg^?1 protein, and a subunit molecular mass of 37 kD. The Gly-DHAPR had little preference for NADH or NADPH, but was highly specific for DHAP. The purified enzyme was slightly stimulated by 50 mM NaCl, KCl or by 25 mM MgCl₂. Detergents, lipids, fatty acids, or long-chain acyl-CoA derivatives inhibited the Gly-DHAPR. The Gly-DHAPR differs in properties from the other chloroplastic osmoregulatory isoform of DHAP reductase from Dunaliella, but has significant similarities with the glyceride isoforms from higher plants for glycerol-P and triglyceride synthesis.     

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K_m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min ⁻¹nmol⁻¹ P-450 protein and with a purified rabbit P-450_2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min⁻¹ lnmo⁻¹ P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.     

Probing reactivity and substrate specificity of both subunits of the dimeric Mycobacterium tuberculosis FabH using alkyl-CoA disulfide inhibitors and acyl-CoA substrates

The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C₆-C₂₀ acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C₁₂ acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C₁₂ acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C₁₈-C₂₀ acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

Some properties of nitrite and hydroxylamine reductases from Derxia gummosa

NADH-nitrite and -hydroxylamine reductases were co-purified from Derxia gummosa. The stoichiometries for the reduction of nitrite and hydroxylamine to ammonia were 3 NADH:1 NO⁻₂:1 NH₃ and 1 NADH:1 NO⁻₂:1 NH₃. The K_m values for nitrite and hydroxylamine were 4.8 μM and 5.3 mM, respectively, and for NADH they were 6.3 μM for nitrite reductase and 150 μM for hydroxylamine reductase. The optimal pH value for both enzyme activities was 8.5. Both activities were inhibited by NADH in the absence of the appropriate substrate, namely nitrite or hydroxylamine. Studies with amino acid modifiers indicate that histidine, glutamate/aspartate, sulphydryl and tyrosine are essential components of the enzyme protein. Kinetic studies show that nitrite and hydroxylamine were competitive for the same binding site on the enzyme. The results indicate that although nitrite and hydroxylamine reductases are associated with the same enzyme, its main function is the reduction of nitrite to ammonia. Azaserine inhibited the induction of the enzyme.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

Purification and properties of an ld-dipeptidase FROM Bacillus sphaericus

• 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min⁻¹·mg⁻¹) and l-Lys-d-Ala (sp. act. 11 μmol · min⁻¹·mg⁻¹) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
• 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
• 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (M_r 38,000).
• 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
• 5.5. This enzyme may have a function in peptidoglycan metabolism.