Konrad Bloch

Inhibition of fatty acid synthetases by the antibiotic cerulenin.

Abstract The antibiotic cerulenin is a potent and apparently non-competitive inhibitor of fatty acid synthetase systems isolated from various microorganisms and from rat liver. Cerulenin specifically blocks the activity of β-keto acyl thioester synthetase (condensing enzyme). This effect may account for the inhibition of overall fatty acid synthesis by the antibiotic.

Sterol molecule: structure, biosynthesis, and function

This review briefly summarizes key researches on the structure of the sterol molecule from its very beginnings to the definitive elucidation in 1932. Cholesterol biosynthesis treated in somewhat greater detail covers the period from the 1930s to the 1960s. As a historic contribution, it presents researches previously published in numerous books, reviews, and original papers. The selection of topics, dictated by limits of time and space, is necessarily arbitrary and a personal choice. Readers of this journal will be familiar with the relevant chemical structures. Structural formulas are therefore omitted.

Fatty Acyl-Coenzyme A Elongation in Brain of Normal and Quaking Mice

Microsomal enzyme systems from mouse brain that catalyze, respectively, the elongation of palmitoyl-coenzyme A (palmitoyl-CoA), stearoyl-CoA, or arachidyl-CoA appear and reach maximal activity at different times after birth of the animal. A specific C20-CoA elongating system exists in mouse brain in addition to the previously recognized C16-CoA and Cl8-CoA elongating enzymes. The C20-CoA elongation system is severely reduced in the mutant quaking mouse.

Fatty acid synthetases from Euglena gracilis

Abstract Additional properties of two previously described (Delo, J., Ernst-Fonberg, M. L., and Bloch, K. (1971) Arch. Biochem. Biophys. 143, 384; Ernst-Fonberg, M. L., and Bloch, K. (1971) Arch. Biochem. Biophys. 143, 392) fatty acid synthetases (I and II) from Euglena gracilis are reported. The algal organism is now shown to contain another fatty acid synthetase (III). This system is ACP-dependent and elongates acyl-CoA derivatives from C10 to C18 to longer chain ACP thioesters. Synthetase III occurs in soluble form both in dark- and light-grown cells and in the latter is localized in the chloroplast. Evidence is presented that the two ACP-dependent synthetases (II and III) from Euglena operate in conjunction to furnish the fatty acids required for chloroplast membranes.

Inhibition of Lipid Synthesis in Escherichia coli Cells by the Antibiotic Cerulenin

The antibiotic cerulenin markedly inhibits the growth of Escherichia coli. The effects of the antibiotic on cellular syntheses were studied by measuring the incorporation of labeled precursors into lipids and macromolecules. During the first 40 min after the addition of cerulenin to a culture of growing cells, lipid synthesis was inhibited more than 90% and ribonucleic acid and deoxyribonucleic acid synthesis about 25%, whereas protein synthesis was not affected. At later periods after cerulenin addition (1 to 2 h), the inhibition of cell growth and of lipid and protein synthesis was complete. Upon removal of cerulenin from the culture, growth was restored and lipid synthesis resumed more rapidly than did the synthesis of protein. Addition of both palmitate and oleate, but not of either fatty acid alone, reversed the inhibition of growth by cerulenin. These findings support the conclusion that the antibiotic effects of cerulenin are due to a specific inhibition of fatty acid synthesis.

[10] Fatty acid synthases from Mycobacterium phlei

Publisher Summary This chapter discusses the fatty acid synthases from Mycobacterium phlei ( M. phlei ). The synthesis of long-chain fatty acids is catalyzed in biologic systems by enzyme systems of two types. Those designated as Type I synthases are multienzyme complexes in which all the component catalytic activities and acyl carrier protein (ACP) form tight aggregates of high molecular weight. They occur in yeast, all animal tissues, and in a few bacterial species. By contrast, fatty acid synthases of Type II consist of individual, separable enzymes and require for activity the external addition of ACP. Type II systems are found in most bacteria and in plants. Synthase is assayed by measuring the incorporation of [2- 14 C]malonyl-CoA into pentane-soluble acidic products. One unit of enzymatic activity is defined as the amount of enzyme required to incorporate 1 nmole of malonate into fatty acids per minute. For the purification of the synthase, all steps are carried out at 0–4°. Protein is determined spectrophotometrically at 260 and 280 nm. Buffers contain 1 m M DTT and 1 m M ethylenediaminetetraacetic acid (EDTA) and are adjusted to pH 7.0. Eighty grams of M. phlei cells are suspended in 300 ml of 0.1 M potassium phosphate buffer. The cells are broken by passage through a French pressure cell operated at 8000 psi and the disrupted cells centrifuged at 17,000 g for 20 minutes. The resulting supernatant is centrifuged at 105,000 g for 90 minutes and then brought to 35% saturation with ammonium sulfate.

Comparative properties of 2,3-oxidosqualene-lanosterol cyclase from yeast and liver

Abstract In yeast the enzyme 2,3-oxidosqualene-lanosterol cyclase (cyclase) is found in the soluble fraction of cell homogenates. The corresponding enzyme from liver is associated with the microsomes. The soluble yeast cyclase is optimally active in solutions of low ionic strength and stimulated by Triton X-100. By contrast, the detergent-solubilized liver cyclase requires deoxycholate and high ionic strength media for optimal activity. After acetone precipitation yeast cyclase is no longer soluble though it still responds to Triton X-100. Acetone-precipitated liver cyclase is rendered soluble by deoxycholate.

A new assay for fatty acid desaturation.

Abstract The enzymic formation of unsaturated fatty acids from their saturated homologs is ordinarily assayed by gas-liquid chromatography or by chromatography of the complexes formed between the olefinic acids and mercuric acetate or silver nitrate. In the present paper a rapid, sensitive assay is described. It is based on the release of tritium as tritiated water from a substrate specifically labeled at the positions to be desaturated. The method circumvents the time consuming operations (saponification, extraction, esterification, and chromatography) of the older procedures. A similar principle has been utilized for assaying aromatic hydroxylation (1). Both CoA and acyl carrier protein (ACP) fatty acid thioesters are precipitable by acid and can be collected on a Millipore filter which is conveniently attached to a syringe. The tritiated water filtrate is collected and an aliquot counted. The sensitivity of the method extends to the picomole range and is limited only by the specific activity of the substrate.

The metabolism of nicotinamide-N-oxide.

Abstract 1. 1. Nicotinamide-N-oxide is formed from nicotinamide, in vivo , by rats. 2. 2.Hog-liver homogenate reduces nicotinamide-N-oxide to nicotinamide.

Fatty acid synthetase activity in Mycobacterium smegmatis Characterization of the acyl carrier protein-dependent elongating system

Mycobacterium smegmatis extracts contain two fatty acyl synthetase systems (Brindley, D.N., Matsumura, S. and Bloch, K. (1966) Nature 224, 666-669). One is the extensively studied multienzyme complex, (molecular weight 1.39 - 10(6)) which produces shorter C16 and C18) and longer (C24 and higher) fatty acids in a bimodal pattern. The second synthetase is acyl carrier-protein (ACP) dependent and elongates the CoA derivatives of C12 and longer chains. In contrast to the type I synthetase which also extends long fatty acyl chains, the ACP-dependent system produces homologous fatty acids up to 30 carbon atoms long in approximately equal proportions. Other properties which distinguish the ACP-dependent system from the multienzyme complex include the resistance to high concentrations of palmitoyl-CoA and to low ionic strength and the lack of stimulation by mycobacterial polysaccharides. The possibility that the two fatty acid synthetases are complimentary in their function is discussed.