Mild Base Hydrolysis Research Articles

Lipoteichoic acid from Listeria monocytogenes.

A lipoteichoic acid (LTA) was extracted from Listeria monocytogenes (serotype 1) by phenol-water partition and isolated by gel-filtration chromatography. The LTA exhibited amphiphilic properties by changes in gel-filtration mobility in the presence of detergent buffers and after mild base hydrolysis. In a hemagglutination assay, Listeria LTA bound antibody prepared against a known LTA from Streptococcus spp. Listeria LTA inhibited the binding of anti-LTA antibody to a Lactobacillus LTA in a hemagglutination inhibition assay. The Listeria LTA contained glucose, galactose, fatty acids, glycerol, and phosphate with molar ratios of 0.05, 0.07, 0.21, 0.94, and 1.0 to phosphate, respectively. Adjacent glycerols were linked between the C-1 and C-3 positions by phosphodiesters (structural type 1). The average chain length was 19 +/- 2 (standard deviation) glycerol-phosphate repeating units. Approximately one glycosyl side chain was present per LTA molecule. The side chain was a galactose-containing disaccharide. The lipid portion of the LTA was a galactose- and glucose-containing glycolipid which may have been a phosphoglycolipid, but the structure was not confirmed. Major fatty acids of LTA and the glycolipid were 17:anteiso, 15:anteiso, 16:iso, 16:n, and 18:n. L. monocytogenes contained cell wall products typical of gram-positive bacteria which is in contrast to the reports by others of the presence of lipopolysaccharides from L. monocytogenes.

The discovery of a lipid-linked glucuronide and its synthesis by chicken liver.

Upon incubation with uridine diphosphate-[14C]glucuronic acid, membrane fractions from adult and phenobarbital-induced embryonic liver synthesize a single glucuronide, which is soluble in chloroform:methanol (2:1). The compound is completely hydrolyzed and glucuronic acid released by either mild acid or beta-glucuronidase, whereas mild base hydrolysis results in a mixture of glucuronic acid and glucuronic acid-1,2-cyclic phosphate. These data and the behavior of the lipid-linked glucuronide on DEAE-cellulose chromatography indicate that the compound contains a monophosphate diester of glucuronic acid, which is beta-linked to a lipid. The synthesis of the lipid-linked glucuronide in uninduced normal embryonic liver is very low (5-15 pmol product/mg/5 min) at all developmental ages up to hatching, but the introduction of phenobarbital into the air space of a 9-10-day-old embryo causes a premature increase of activity (75-150 pmol products/mg/5 min) within 7 days. The glucuronyltransferase in adult and induced embryonic liver has a Km for UDPGlcUA of 0.17 x 10(-3) M and a broad pH optimum between pH 6 and 7. Glucuronic acid is released from the lipid-linked glucuronide by a beta-glucuronidase in liver that is active at neutral pH and is not inhibited by saccharolactone. This glycosidase activity appears, therefore, to be distinct from the previously characterized lysosomal beta-glucuronidase. Fractionation of adult chicken liver membranes by differential centrifugation indicates that over 70% of the glucuronyltransferase is associated with the nuclear and mitochondrial fractions. The endogenous beta-glucuronidase capable of hydrolyzing the lipid-linked glucuronide was not separated from the glucuronyl-transferase activity during fractionation. The data available suggests that the lipid-linked glucuronide is involved directly in the generation of free glucuronic acid for further metabolism.

A novel ring cleavage reaction of oxazolines

An oxazoline can be cleanly converted to the parent carboxylic acid by treatment with NaOCl, followed by mild basic hydrolysis of the intermediate ester product(s).

Properties of a bactericidal extract fromBrucella abortus

A water-soluble bactericidal fraction was obtained by extractingBrucella abortus with aqueous methanol. Bactericidal activity was directed against the Gram-positive organism tested (Bacillus subtilis, Streptococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis), but not against the Gram-negative organisms tested (Escherichia coli and aPseudomonas sp.). Mild base hydrolysis, but not mild acid hydrolysis, potentiated the bactericidal activity of the extract. Vigorous acid hydrolysis destroyed the activity. Periodate oxidation and hydrolysis with glycosidases did not affect the activity, while hydrolysis with pronase decreased it by 15–20%. Bioactivity was retained by Sephadex gels, silica gel, and a cation exchange resin, but not by an anion exchange resin. The fact that the activity was precipitable with Fe3+, bound to a cation exchanger, and was extractable with ethanol and methanol suggests that the bioactive substance contains in addition to carbohydrate and lipid a cationic moiety, possibly a peptide.

Effect of mal secco toxin on lemon leaf cells

Leaves of Rough lemon (Citrus jambhiri) were inoculated withPhoma tracheiphila or treated with mal secco toxin. The ultrastructural changes in toxin-treated leaves resembled closely those infected by the pathogen, being characterized by separation of the plasmalemma from the cell wall, vesiculation of plasmalemma, disruption of the chloroplast membrane, disorganization of thylakoids, and increase in size and number of osmiophilic droplets in the chloroplasts. The rate of photo-synthetic CO2 fixation by mesophyll protoplasts was reduced in the presence of the toxin. The toxin induced agglutination of mesophyll protoplasts of lemon only in the presence of an antiserum to the toxin. Agglutination of protoplasts was independent of the biological activity of the toxin and was inhibited by Dmannose or after mild base hydrolysis of the toxin.

Analysis of human urine for a metabolite of endrin by chemical oxidation and gas-liquid chromatography as an indicator of exposure to endrin.

A method for determining residues of the β-glucuronide of anti-12-hydroxyendrin in urine is presented. Samples are treated with sodium metaperiodate followed by mild base hydrolysis. The anti-12-hydroxyendrin is determined by gas-liquid chromatography with electron-capture detection. Chemical confirmation techniques are described. Results obtained from analyses of urine samples from workers at an endrin manufacturing plant are included.

Serum stimulated DNA synthesis in mouse embryo fibroblasts: Synergistic enhancement by staphylococcal lipoteichoic acid

Lipoteichoic acid (LTA) isolated from Staphylococcus aureus was found to exert a synergistic effect with calf serum to enhance DNA synthesis and growth of mouse embryo fibroblasts. LTA alone had no effect on DNA synthesis or cell multiplication. Mild base hydrolysis of LTA, which separates the molecule into its hydrophilic and hydrophobic moieties, completely destroyed its biological activity in this regard. Results are presented which suggest that LTA may enhance serum-induced DNA synthesis through interaction with cell surface components. The data indicate that LTA may prove to be a useful tool with which to probe cell membrane parameters involved in the regulation of cell proliferation.

Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis.

The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity.

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80°C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem. Biophys. Res. Commun. 63, 875–880). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB 3H 4. The polysaccharide was only slightly degraded (2%) by jack bean α-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymer was also detected in the cell “periplasm” and was secreted from cells grown in defined media during the period of decelerating growth.

Preparation, properties and biological activities of succinyl derivatives of vitamin B 12

Three new derivatives of vitamin B 12, O 2′-succinyl-, O 5′-succinyl-, and O 2′. O 5′—disuccinyl-vitamin B 12, whose α-ribose moieties of the nucleotide ligand are succinylated, were prepared by reaction of the vitamin with succinic anhydride. The first succinylation took place rapidly and almost predominantly on 5′-OH of α-ribose, and the second succinylation much more slowly on 2′-OH. From the behaviors in paper electrophoresis and the lability to CN − of cobalt-base bond of O 2′-succinylated vitamin B 12 derivatives, it was suggested that the terminal COOH of the O 2′-succinyl group forms an inner salt with the imidazole nucleus of 5,6-dimethylbenzimidazole. Monosuccinyl- and disuccinyl-vitamin B 12 were easily reconverted into vitamin B 12 by mild acid or base hydrolysis. Heating at 130° for 5 min also led to the complete severance of the succinyl group of O 5′-succinyl-vitamin B 12. None of the three succinyl derivatives inhibited the diol dehydrase reaction when added with coenzyme B 12, suggesting that the ability to bind to the apoenzyme is strongly diminished or almost lost by succinylation on 2′- or 5′-OH of α-ribose. None of the succinyl vitamin B 12 compounds showed either biological activity or anti-vitamin B 12 activity when tested with Escherichia coli 215, a methionine-B 12 auxotroph. None of them significantly inhibited [ 3H] vitamin B 12 uptake by E. coli 215 cells. This observation implies that succinyl derivatives of vitamin B 12 are hardly incorporated into the cells of E. coli.

Purification and Properties of Fatty Acyl Thioesterase I from Escherichia coli

Abstract Fatty acyl thioesterase activity in crude Escherichia coli extracts consists of two activities separable by gel filtration. The two enzymes are designated thioesterases I and II from the order in which they are eluted from Sephadex G-100. Thioesterase I, after 8400-fold purification, was homogeneous as judged by acrylamide gel electrophoresis and constant specific activity of the enzyme peak on DEAE-Sephadex. The native enzyme is a tetramer with a molecular weight of 122,000 and a subunit molecular weight of 30,000. Saturated and unsaturated fatty acyl-CoA thioesters of chain length C14 to C18 were the most active substrates with Km values of 4 to 6 µm and turnover numbers of 18,000 and 27,000 per min, respectively. Neither acetyl-CoA nor oxygen esters were hydrolyzed at detectable rates. Of the many active site reagents tested only photoactivated methylene blue and iodoacetamide inhibited thioesterase I. Acid hydrolysis of thioesterase I inhibited with [14C]iodoacetamide yielded [14C]glycolic acid as the major radioactive product, while mild base hydrolysis yielded mainly [14C]-glycolamide. The evidence presented indicates that iodoacetamide inhibits thioesterase I by esterifying an essential carboxyl group.

Antimetabolites produced by microorganisms. VI. L-N 5 -(1-iminoethyl) ornithine.

A new antimetabolite of arginine and metabolically related compounds was isolated from a fermentation broth produced by an unclassified Streptomycete. The structure was shown to be L-N5-(1-iminoethyl) ornithine and the compound was synthesized by reaction of the copper complex of L-ornithine with ethyl acetimidate. Mild base hydrolysis of the compound gave the expected products, L-ornithine and L-N5-acetylornithine and, in addition, a rearranged product, L-N2-acetylornithine. Arginase hydrolyzed the antimetabolite to L-ornithine.

Acetyl transport mechanisms. Involvement of N-acetyl aspartic acid in de novo fatty acid biosynthesis in the developing rat brain.

The incorporation into fatty acids of N-acetyl aspartic acid, labeled in the acetyl group, was investigated during brain growth and development. Parasagittal injection of the acetyl amino acid into rats of various ages resulted in maximum incorporation just before and during myelination, a period in which the endogenous N-acetyl aspartic acid increases rapidly. The results indicate that the only barrier to the uptake of this compound is at the blood-brain barrier. Gas liquid chromatographic analysis of the fatty acid methyl esters isolated by mild base hydrolysis showed that the labeled fatty acids were primarily the C16 and C18 long-chain acids. These results indicate that N-acetyl aspartic acid is actively involved in de novo fatty acid synthesis throughout growth and development of the brain. It is suggested that, in the brain, N-acetyl aspartic acid is a constituent of the acetyl transport system for extra-mitochondrial fatty acid synthesis.

Analogs of corticosteroids

1. The behavior of stereoisomeric epoxides of 1-acetyl-4-t-butylcyclohexene-1 under conditions of acetolysis was studied. 2. The 2-acetate of lα-acetyl-4β-t-butylcyclohexane-1β,2α-diol is formed exclusively by axial attack of acetic acid at the C2-reaction center of 1α-acetyl-1β,2β-epoxy-4β-t-butylcyclohexane. 3. In the acetolysis of 1β-acetyl-1α,2α-epoxy-4β-t-butylcyclohexane is formed the 2-acetate of 1,β-acetyl-4β-t-butylcyclohexane-1α,2β-diol, probably by axial attack at the C2 in a “half-tub” conformation and the 1-acetate of 1α-acetyl-4β-t-butylcyclohexane-1β,2α-diol by axial attack at the Cl in the “halfchair” conformation. 4. The complex course of acetolysis of 1β-acetyl-1α,2α-epoxy-4β-t-butylcyclohexane at a higher temperature is caused by partial isomerization of the product of trans-diaxial opening — the tertiary monoacetate of the trans-diol. 5. The 1,2-diacetates of four isomeric 1-acetyl-4-t-butylcyclohexane-1,2-diols were synthesized, and their behavior under conditions of mild basic hydrolysis was examined. 6. The secondary acetoxy group of the diacetate saponifies more easily than the tertiary. During saponification of the diacetates of the ketodiols, a migration of the acyl from the tertiary position to the secondary by an orthoester mechanism is observed.

Properties of structural proteins of bovine platelets

Bovine platelet ghosts were found to consist largely of protein with a small amount of protein-bound carbohydrate. A full spectrum of amino acids was obtained, and the N-terminal amino acid proved to be serine. Partial solubilization of platelet ghosts was accomplished by a mild basic hydrolysis. The bonds broken in this procedure were found to be ester-like, as shown by a positive hydroxamic acid reaction. A water-soluble DNP-fraction was isolated by mild basic hydrolysis of DNP-platelet ghosts. Ultracentrifugal analysis revealed that this fraction consisted of at least three components.