Juichi Awaya

Inhibition of fatty acid synthesis by the antibiotic cerulenin: Specific inactivation of β-ketoacyl-acyl carrier protein synthetase

Abstract Cerulenin, (2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide, an antibiotic isolated from the culture filtrate of Cephalosporium caerulens, inhibits the fatty acid synthetase of Escherichia coli by specifically inhibiting β-ketoacyl-acyl carrier protein synthetase, the enzyme which catalyzes the condensation reaction of fatty acid biosynthesis. Although cerulenin and tetrahydrocerulenin inhibited both β-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase, neither dihydrocerulenin nor hexahydrocerulenin, analogs which lack the epoxide ring, inhibited these enzyme systems. Both β-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase were protected to the same extent against inhibition by cerulenin by prior incubation with acetyl-acyi carrier protein. The acyl group of this thioester is bound at the fatty acyi site of β-ketoacyl-acyl carrier protein synthetase. Detailed studies with homogeneous β-ketoacyl-acyl carrier protein synthetase indicated that cerulenin inhibited model reactions representing both the fatty acyl transacylase and the malonyl-acyl carrier protein decarboxylase components of the β-ketoacyl-acyl carrier protein synthetase reaction. Inhibition of β-ketoacyl-acyl carrier protein synthetase by cerulenin was irreversible, and it was associated with the binding of approximately 1 mole of inhibitor per mole of enzyme when inhibition approached 100 %.

Calculated two-dimensional sugar map of pyridylaminated oligosacchardies: Elucidation of the jack bean α-mannosidase digestion pathway of Man9GlcNAc2

We have developed a two-dimensional (2-D) mapping of pyridylaminated oligosaccharides as an aid to structural determination of glycoprotein-derived oligosaccharides. Using the available data of reverse-phase HPLC of pyridylamino-oligosaccharides, this was further extended to parameterization of unit contribution by each sugar component, which allows the prediction of possible structures from the elution volume. We have extended this approach to the data obtained with amide-silica HPLC column to obtain a calculated 2-D mapping technique for the oligomannose-type oligosaccharides (M-series). In this method, the elution volumes of all possible pyridylamino-oligosaccharides up to the size of Glc1Man9GlcNAc2 (50 in total) are calculated from the established UC values to construct a 2-D map. To test the validity of the calculated 2-D map, the structures of intermediate PA-oligosaccharides generated during the alpha-mannosidase (jack bean) digestion of Man9GlcNAc2 (porcine thyroglobulin) were analyzed to establish the digestion pathway. The validity of this approach is substantiated by an independent deduction of the intermediate structures based on structural relationships and the coincidence of elution volumes. Our results agree well with the recently published digestion pathway of Man5GlcNAc2 by the same enzyme and that of Man9GlcNAc2 by lysosomal alpha-mannosidase.

Substitution of cellular fatty acids in yeast cells by the antibiotic cerulenin and exogenous fatty acids

Cell growth of Saccharomyces cerevisiae ATCC 12341 inhibited by the antibiotic cerulenin, a specific inhibitor of fatty acid and sterol syntheses, was reversed by various exogenous fatty acids. Myristic acid (14 : 0), pentadecanoic acid (15 : 0), palmitic acid (16 : 0), and oleic acid (18 : 1) reversed effectively the growth inhibition by cerulenin. When these cells were reversed by adding pentadecanoic acid, over 90% of native even-numbered fatty acids was substituted by odd-numbered fatty acids. Those in the cells reversed by adding oleic acid were almost all unsaturated fatty acids. Cerulenin did not inhibit either elongation or desaturation systems in S. cerevisiae.

Determination of monosaccharides and sugar alcoholsin tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection

A sensitive and simple high-performance liquid chromatographic method has been developed to determine the concentration of monosaccharides and sugar alcohols in animal tissues. Five neutral monosaccharides (D-glucose, D-galactose, D-mannose, D-fructose, and D-ribose) and three neutral sugar alcohols (myo-inositol, glycerol, and D-sorbitol) predominate in the renal cortices and sciatic nerves of rats. These monosaccharides and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak C18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca2+ and Pb2+ cation-exchange columns, eluted with deionized distilled water at 80 degrees C, and detected using integrated pulsed amperometry. About 10 pmol of each sugar was detectable with a signal-to-noise ratio of 10:1. D-Glucose, D-fructose, D-sorbitol, and D-mannose were higher in both the renal and sciatic tissues of diabetic rats than in those of normal animals. D-Ribose and glycerol were higher in the renal cortex of diabetic animals.

Cerulenin resistance in a cerulenin-producing fungus. Isolation of cerulenin insensitive fatty acid synthetase.

Abstract Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens , is a potent inhibitor of fatty acid synthetase systems. This antibiotic specifically blocks the activity of β-ketoacyl thioester synthetase (condensing enzyme). The mechanism of the resistance of C. caerulens to cerulenin was investigated. The rate of growth in medium containing up to 100 gmg/ml cerulenin was as rapid as that in cerulenin-free medium. At a cerulenin concentration of 300 μg/ml, the rate of growth was still more than half that of the control. The addition of cerulenin (200 μg/ml) to a culture of growing cells has almost no effect on the incorporation of [ 14 C ]acetate into cellular lipids. Fatty acid synthetase was purified from C. caerulens to homogeneity. Properties of this fatty acid synthetase were almost the same as those of yeast fatty acid synthetase except for the sensitivity to cerulenin. C. caerulens synthetase is much less sensitive to cerulenin than fatty acid synthetases from other sources. These findings suggested that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus.

Target of inhibition by the anti-lipogenic antibiotic cerulenin of sterol synthesis in yeast

Abstract The antibiotic cerulenin inhibited the incorporation of 14 C-acetyl-CoA by 67% at a concentration of 9 × 10 −6 M but not that of 14 C-HMG-CoA into the non-saponifiable fraction in a cell-free extract of Saccharomyces cerevisiae . Cerulenin markedly inhibited the activity of partially purified HMG-CoA synthase. No inhibition of acetoacetyl-CoA thiolase activity was observed in the same preparation of HMG-CoA synthase. Therefore, cerulenin inhibition of overall sterol synthesis may be accounted for by the specific inhibition of HMG-CoA synthase activity.

Pyrindicin, a New Alkaloid from a Streptomyces Strain

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD50 of the hydrochloride (ip, in mice) was 87 mg/kg.