Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.     

The effects of L-2,4-diaminobutyric acid on the uptake of gamma-aminobutyric acid by a synaptosomal fraction from rat brain

l-2,4-diaminobutyric acid was studied as an inhibitor of gamma-aminobutyric acid uptake by a synaptosomal fraction isolated from rat brain. Competitive inhibition was observed during short-term exposure of the synaptosomal fraction to the inhibitor but noncompetitive inhibition was observed following prolonged exposure. Studies on the mode of action of l-2,4-diaminobutyric acid showed that the synaptosomal fraction was capable of accumulating this compound and that both the uptake and the effectiveness of the inhibitor were sodium-dependent and temperature-sensitive. In addition, the degree of inhibition of gamma-aminobutyric acid uptake was related to the amount of l-2,4-diaminobutyric acid accumulated. It is suggested that the observed noncompetitive inhibition of gamma-aminobutyric acid uptake by l-2,4-diaminobutyric acid is a result of the accumulation of the inhibitor which exerts its effect from within the synaptosomes. Raising the external concentration of gamma-aminobutyric acid to saturating levels did not completely inhibit the accumulation of l-2,4-diaminobutyric acid. Thus, the transport of l-2,4-diaminobutyric acid appears to be mediated, at least in part, by a carrier which is not involved in the transport of gamma-amiuobutyric acid.     

Partial purification and some properties of an L-{alpha}-amino-S-caprolactam-hydrolyzing enzyme from Cryptococcus laurentii

A new L-amlnolactam-hydrolyzing enzyme was partially purifiedfrom cells of Cryptococcus laurentii which can grow on L-aminolactamas a carbon and nitrogen source. The enzyme required a bivalentmetal ion, such as Mn²⁺ or Mg²⁺, and its molecular weight wasroughly estimated to be 1.5?10⁵ Some other properties were alsostudied. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University Sumiyoshi-ku, Osaka 558, Japan. (Received June 20, 1977; )     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

Partial purification and properties of a protein kinase C type enzyme from plants

Partial purification of a protein kinase with a dependence on micromolar concentrations of free calcium has been achieved from seedlings of Amaranthus tricolor. The enzyme has a M_r of 77 600 as determined by gel filtration and 84 500 by SDS-PAGE analysis. Interaction of the enzyme with membranes (inside-out erythrocyte vesicles) is regulated by calcium, a characteristic of animal protein kinase C. Phospholipid and diolein activation of the enzyme is markedly dependent on the phospholipid used and on both calcium and phospholipid concentration. K_m values for Ca²⁺ in the absence of phospholipid was 20–40 μM and in the presence of phosphatidylserine 5–10 μM. Diolein plus phosphatidylserine lowered the K_m to < 1.5 μM. The best activation was achieved at 1OOμM calcium with 40μg/ml phosphatidylserine and 8μg/ml diolein. These properties indicate a protein kinase C type enzyme. The plant enzyme reacted with antiserum directed against the regulatory domain of bovine brain protein kinase C in an immunoblot experiment.     

Partial purification of a lipolytic enzyme from Escherichia coli.

An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.     

Biosynthesis of polymyxin E III. Total synthesis of polymyxin E by a cell-free enzyme system

Polymyxin E, an antimicrobial branched cyclic decapeptide, was synthesized by an enzyme fraction partially purified from crude extracts of the producing organism, $\text{Aerobacillus}\text{polyaerogenes}$. For the synthesis, three constituent amino acids (L-2,4-diaminobutyric acid, L-leucine, and L-threonine), ATP, Mg²⁺ and an acylating system consisting of octanoyl CoA and an ammonium sulfate fraction of cell extracts are required.     

Partial purification and properties of acid sphingomyelinase from rat liver

Acid sphingomyelinase was purified approximately 5,200-fold from the mitochondria-lysosome-enriched particles of rat liver by sequential chromatography on DEAE-cellulose, octyl-Sepharose, Sephacryl S-300, Concanavalin A-Sepharose, and CM-cellulose. The specific activity of this highly purified enzyme was 3.2 mmol per hr per mg protein. The enzyme was active against 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, but bis-4-methylumbelliferyl-phosphate and bis-p-nitrophenyl-phosphate were poor substrates. The preparation was free of Mg2+-dependent neutral sphingomyelinase and eight lysosomal enzymes except for the trace amount of acid phosphatase and beta-galactosidase. Apparent molecular weight of the enzyme was 200,000, estimated by Sephadex G-200 filtration in 0.1% Triton X-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed three major bands corresponding to molecular weights of 45,600, 44,500, and 40,000 with several minor bands. Characterization of the enzyme revealed almost the same properties as those of human tissues reported by other investigators, including pH optimum, requirement of Triton X-100, effects of metal divalent cations, phosphate ion, EDTA, some thiol blocking reagents, and amphophilic drugs.     

Partial purification and characterization of a pyruvate dehydrogenase-complex-inactivating enzyme from rat liver.

An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.     

Partial purification and properties of the amino-terminal amino acid-acetylating enzyme from hen's oviduct

By using the synthetic peptide ACTH1-24 as a model substrate, an enzyme that may be involved in the amino-terminal acetylation of certain proteins and growing nascent polypeptide chains has been found in hen's oviduct. It was partially purified by a four-step procedure comprising extraction from the homogenates, ammonium sulfate fractionation, chromatography on a column of QAE-Sephadex A-50, and gel filtration on a Sepharose 6B column. An enzyme preparation purified about 40-fold from the homogenates transferred the acetyl group from acetyl coenzyme A preferentially to the amino-terminal amino acids of several ACTH-related peptides at an optimum pH of around 7.2. This occurred to different extents depending on the peptide length and on the nature of the amino-terminal residue. The molecular weight of the enzyme was estimated to be approximately 250,000 by gel filtration.     

Partial purification and some properties of a neutral sulfhydryl and an acid proteinase from Entamoeba histolytica.

The partial purification of two intracellular proteinases from the protozoan parasite Entamoeba histolytica is reported. One of these enzymes is an acid proteinase exhibiting maximum activity at pH 3.5 (hemoglobin substrate), is little affected by a range of inhibitors or activators, and is presumed to be similar to cathepsin D. Also present is a neutral proteinase exhibiting optimum activity at pH 6.0 (azocasein) but only poorly hydrolyzing either hemoglobin or serum albumen. This latter enzyme displayed no metal ion requirement, but was markedly inhibited by thiol-blocking agents and activated by free sulhydryl-containing compounds.     

Lipoxygenase from potato tubers. Partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid

A lipoxygenase (EC 1.13.1.13) was partially purified from potato tubers and was shown to differ from previously characterized soya-bean lipoxygenases in the positional specificity and pH characteristics of the oxygenation reaction. The potato enzyme converted linoleic acid almost exclusively (95%) into 9-d-hydroperoxyoctadeca-trans-10,cis-12-dienoic acid. The 13-hydroperoxy isomer was only a minor product (5%). Linolenic acid was an equally effective substrate, which was also oxygenated specifically at the 9-position. The enzyme had a pH optimum at 5.5-6.0 and was inactive at pH9.0. A half-maximal velocity was obtained at a linoleic acid concentration of 0.1mm. No inhibition was observed with EDTA (1mm) and cyanide (1mm) or with p-chloromercuribenzoate (0.2mm). Haemoproteins were not involved in the lipoxygenase activity. The molecular weight of the enzyme was estimated from gel filtration to be approx. 10(5). Preliminary evidence suggested that the enzyme oxygenated the n-10 position of fatty acids containing a penta(n-3, n-6)diene structure.     

Alkaline phosphatase of Thiobacillus thioparus. Partial purification and properties of the enzyme.

Soluble alkaline phosphatase from Thiobacillus thioparus cells was purified about 230-fold. The enzyme had a mol. wt. of 50 000 daltons, optimum pH at 10.5, and was heat-resistant in the presence of diethanolamine. Polyacrylamide-gel electrophoresis demonstrated contamination of the preparation with inactive proteins and the presence of two active bands. The enzyme activity was distinctly stimulated by increasing concentrations of Tris or diethanolamine. In the presence of glycine, 1 mM-Zn2+ enhanced the enzyme activity; in Tris or diethanolamine buffers the activity was stimulated by 1 mM-Mg2+ whereas Zn2+ had a strong inhibitory effect. Glycine at concentrations exceeding 25 mM also inhibited the enzyme. Specificity of the enzyme is fairly broad.     

Arachidonic acid 15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Partial purification and properties

Arachidonic acid 15-lipoxygenase was purified from rabbit peritoneal polymorphonuclear leukocytes. The enzyme was recovered in the cytosol fraction after sonication and purified about 250-fold by acetone precipitation, column chromatography on CM52, Sephadex G-150, and hydroxyapatite. The enzyme catalyzed the conversion of arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which then decomposed to a mixture of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), 15-keto-5,8,11,13-eicosatetraenoic acid, 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid. The enzyme was specific for oxygenation at carbon 15 of arachidonic acid. The apparent molecular weight of the enzyme was about 61,000 as measured by Sephadex G-150 gel filtration chromatography. The enzyme was sensitive to sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid. The enzyme activity was inhibited by eicosatetraynoic acid (ETYA) or 3-amino-1-(m-(trifluoromethyl)-phenyl)2-pyrazoline (BW755C), but not by indomethacin up to 200 micrograms/ml.