Superoxide-dependence of the short chain sugars-induced mutagenesis

Short chain sugars such as glycolaldehyde are produced at the initial stages of nonenzymatic glycosylation. Because their carbonyl groups cannot be blocked by cyclization, such compounds tautomerize to enediols, which are prone to autoxidation. Superoxide radical serves as an initiator and a propagator of this autoxidation. The biological importance of the involvement of superoxide in sugar autoxidation in vivo was examined using superoxide dismutase (SOD)-deficient and SOD-replete strains of Escherichia coli. Glycolaldehyde, glyceraldehyde, and dihydroxyacetone greatly enhanced the mutation rates in SOD-deficient E. coli. The effect was oxygen-dependent and was suppressed by SOD or by a SOD mimetic. The mutagenic effect of glycolaldehyde coincided with intracellular accumulation of glyoxal, a product of glycolaldehyde autoxidation.     

Metabolism of glycerol by mature boar spermatozoa.

Mature boar spermatozoa oxidized glycerol to carbon dioxide in the absence of any detectable activity of glycerol kinase. With triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase inhibited by the presence of 3-chloro-1-hydroxypropanone (CHOP), dihydroxyacetone phosphate accumulated in incubates when glycerol-3-phosphate was the substrate, but not when it was glycerol. Both dihydroxyacetone and glyceraldehyde could be used as substrates; in the presence of CHOP, dihydroxyacetone phosphate and fructose-1,6-bisphosphate accumulated when dihydroxyacetone was the substrate, but not when it was glyceraldehyde. The metabolic pathways glycerol----glyceraldehyde----glyceraldehyde 3-phosphate and dihydroxyacetone----dihydroxyacetone phosphate have been shown to operate in these cells.     

Isolation and characterization of crystalline methylglyoxal synthetase from Proteus vulgaris.

Methylglyoxal synthetase, which catalyzes the conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate, has been isolated and crystalized in good yields from Proteus vulgaris. The enzyme was shown to be homogeneous by a variety of criteria and was found to be a dimer (Mr = 135,000; s20,w = 7.2 S) composed of two apparently identical catalytic and physical properties and their interconvertible nature suggest that they do not represent true isozymes. The enzyme is specific for dihydroxyacetone phosphate and does not form methylglyoxal from glyceraldehyde 3-phophate, glyceraldehyde, or dihydroxyacetone. Nonphosphorylated analogs are neither substrates nor competive inhibitors, but a variety of phosphorylated analogs are competitive with respect to dihydroxyacetone phosphate. The enzyme is inhibited by inorganic orthophosphate in a complex manner which is overcome by dihydroxyacetone phosphate in a signoidal manner     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva

An NADP⁺-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella.     

Correlation between the release of sugars and uronic acid and free oil recovery following enzymatic digestion of oil seed cell walls

Aqueous extraction of sunflower seeds and rapeseeds with mixtures of hydrolytic enzymes were carried out in 2-L reactor and uronic acid and reducing sugar concentrations were measured during digestion. The goal of the study was to determine if a correlation between those concentrations and free oil exists that would allow predicting free oil release from aqueous solution measurements. Similar concentrations of 4.5 and 4.7 g/L of reducing sugars and 43 and 55 g/L of uronic acid were found for sunflower and rapeseeds, respectively. The corresponding yields of free oil from the two seeds were 83% and 16% of total oil, and this difference was due to the formation of an emulsion in the rapeseed dispersion. Therefore, measurement of reducing sugars or uronic acid concentrations are not sufficient to predict a release of free oil from all oil seeds.     

The autoxidation of glyceraldehyde and other simple monosaccharides under physiological conditions catalysed by buffer ions

Glyceraldehyde and other simple monosaccharides autoxidise under physiological conditions generating 1-hydroxyalkyl (carbon-centred) free radicals and intermediates of dioxygen reduction: superoxide, hydrogen peroxide and hydroxyl radicals. The major glyceraldehyde-derived product is the alpha-ketoaldehyde, hydroxypyruvaldehyde. Close similarities between the temperature dependence of the kinetics of glyceraldehyde autoxidation and glyceraldehyde enolisation to an ene-diol indicates that enolisation is the rate-determining step in the autoxidative process. Inspection of a wide range of carbonyl compounds showed that the monosaccharide moiety -CH(OH)-C- is conserved in carbonyl compounds reactive towards autoxidation, indicating that the ability to form an ene-diol is a prerequisite to monosaccharide autoxidation. The ene-diol intermediate autoxidises rapidly to the products: hydrogen peroxide, water and alpha-ketoaldehydes: beta-hydroxypyruvaldehyde is produced from glyceraldehyde and dihydroxyacetone, glyoxal from glycolaldehyde autoxidation. Ene-diol autoxidation is catalysed by hydrogen peroxide and trace metal ion contaminants; removal of either of these factors sufficiently retards ene-diol autoxidation such that ene-diol autoxidation rather than enolisation becomes the rate determining step in the overall autoxidative process. Under enolisation control, the rate of monosaccharide autoxidation is influenced by pH and the buffer system used for pH control.     

Enzymes involved in the formation of glycerol 3-phosphate and the by-products dihydroxyacetone and glycerol in Zymomonas mobilis

Abstract In Zymomonas mobilis a novel pathway for the formation of glycerol 3-phosphate was identified by enzymatic studies and nuclear magnetic resonance spectroscopy. This pathway branches off from the Entner-Doudoroff pathway at the intermediate glyceraldehyde 3-phosphate and proceedes via dihydroxyacetone phosphate, dihydroxyacetone, glycerol to glycerol 3-phosphate. The reaction sequence is catalyzed by the enzymes triosephosphate isomerase (0.4 U (mg protein)⁻¹), dihydroxyacetone phosphatase (0.31 U (mg protein)⁻¹), dihydroxyacetone reductase (0.25 U (mg protein)⁻¹), and glycerokinase (0.08 mU (mg protein)⁻¹), respectively. The action of a postulated aldolase catalyzing the cleavage of fructose 6-phosphate to dihydroxyacetone and glyceraldehyde 3-phosphate could be excluded.     

Characteristics of Effective and Host Specific Ineffective Strains of Rhizobium japonicum

The formation of dimers in the initial stage of methyl linoleate (ML) autoxidation was demonstrated. The oxidation profile of freshly prepared ML was followed by TLC during autoxidation by aeration at 30°C for 192 hr. After 24 hr of autoxidation, the peroxide value of ML was still 0.6, and two unknown polar spots appeared besides intact ML and methyl linoleate hydroperoxides (MLHPO). These two spots were identified as dimers by successive gel and high performance liquid Chromatographic separations and by molecular weight determination. The ratio of dimers/MLHPO reached a maximum (0.74) after 96 hr of autoxidation. This result indicates that the formation of dimers in the initial stage of autoxidation was slightly less than that of MLHPO. The dimers were linked through ?C?O?O?C? bonds and contained hydroperoxy and/or carbonyl groups and conjugated dienes.     

Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation

The aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC₅₀ around 200 μM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC₅₀ around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance.     

Purification and Characterization of Two Dihydroxyacetone Kinases from Schizosaccharomyces pombe IFO 0354

Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schizosaccharomyces pombe IFO 0354. They were immunologically different from each other. Although both of the enzymes had some affinity for glycerol and dl-glyceraldehyde in addition to dihydroxyacetone and glyceraldehyde, V(infmax) values for dihydroxyacetone were much higher than those for glycerol and dl-glyceraldehyde. On the basis of the K(infm) values of both enzymes for dihydroxyacetone, DHAK II plays a more important role than DHAK I in dissimilation of glycerol via dihydroxyacetone.     

Inhibition of oxidation of unsaturated fatty acid methyl esters by essential oils

The essential oils from 16 various spice plants were studied as natural antioxidants for the inhibition of autooxidation of polyunsaturated fatty acids methyl esters isolated from linseed oil. The content of methyl oleate, methyl linoleate, and methyl linolenoate after 1, 2, and 4 months of autooxidation were used as criteria to estimate the antioxidant efficiencies of essential oils. In 4 months, 92% of the methyl linolenoate and 79% of the methyl linoleate were oxidized in a control sample of a model system. It was found that the most effective antioxidants were essential oils from clove bud, cinnamon leaves, and oregano. They inhibited autooxidation of methyl linolenoate by 76–85%. The antioxidant properties of these essential oils were due to phenols— eugenol, carvacrol, and thymol. Essential oil from coriander did not contain phenols, but it inhibited methyl linolenoate oxidation by 38%. Essential oils from thyme, savory, mace, lemon, and tea tree inhibited methyl linolenoate oxidation by 17–24%. The other essential oils had no antioxidant properties.     

Gylcerol-3-phosphate shuttle and its function in intermediary metabolism of hamster brown-adipose tissue.

1. Brown adipose tissue of the hamster possesses high specific activities of soluble, cytoplasmic NAD-linked, as well as mitochondrial flavin-coupled, glycerol-3-phosphate dehydrogenases. The ratio of the two enzyme activities is high (close to 1), when compared with other tissues of the hamster. 2. In the presence of rotenone, NADH is oxidised very poorly by homogenates of brown adipose tissue. A high rate of oxidation is obtained upon further addition of dihydroxyacetone phosphate, which itself is negligible oxidised. When followed fluorimetrically glycerol 3-phosphate can also be observed to induce NADH oxidation, but only after a significant lag time. Similar results are obtained with isolated mitochondria plus high-speed supernatant. With high-speed supernatant alone, only dihydroxyacetone phosphate has any effect, whereas with isolated mitochondria neither dihydroxyacetone phosphate nor glycerol 3-phosphate induce any NADH disappearance. 3. Respiration induced by NADH plus dihydroxyacetone phosphate in homogenates equals 56% of the respiration induced by glycerol 3-phosphate alone. 4. Respiration induced by NADH plus dihydroxyacetone phosphate, as well as that induced by glycerol 3-phosphate, is inhibited by the same concentrations of inhibitors as are required for inhibition of the mitochondrial dehydrogenase i.e. EDTA, long-chain unsaturated fatty acids, long-chain fatty acyl CoA esters. 5. In isolated brown adipocytes in the presence of rotenone, norepinephrine significantly inhibits respiration induced by glycerol 3-phosphate. 6. The results obtained are discussed with respect to the role of glycerol 3-phosphate as an electron sink for cytosolic reducing equivalents to maintain a low level of extramitochondrial NADH. A means of maintaining a level of glycerol 3-phosphate adequate for triglyceride synthesis is also considered.     

Purification and properties of glycerol:NADP+ 2-oxidoreductase from Schizosaccharomyces pombe

Glycerol:NADP+ 2-oxidoreductase (EC 1.1.1.156) was isolated from Schizosaccharomyces pombe, purified and characterized. It had an Mr of 57,000, and SDS-PAGE revealed two polypeptides, of Mr 25,000 and 30,000. Its coenzyme requirement was satisfied exclusively by NADP. The pH optimum for glycerol oxidation was 9.5, for dihydroxyacetone reduction 6.0. Rates of oxidation with some structurally related diols were three- to six-fold lower than for glycerol, while glyceraldehyde and other carbonyl compounds showed negligible rates of reduction. Neither monovalent nor divalent cations activated the enzyme. Apparent Km and Vmax values were determined. The enzyme is similar to glycerol dehydrogenases isolated from Mucor javanicus and from Dunaliella parva but differs considerably from the glycerol:NAD+ 2-oxidoreductase of S. pombe.     

Biocatalytic and fermentative production of useful chemicals by processes using methylotrophs

Abstract Processes for production of glycerol, aldehydes and polyols by using the methylotrophic function of yeasts were developed. The unique metabolism of glycerol involving a new enzyme, dihydroxyacetone reductase, was applied to the glycerol production employing a glycerol-negative mutant. Alcohol oxidase activity was improved and used for production of aldehydes from corresponding alcohols. NADH generation during methanol oxidation was coupled with hydrogenation of sugars to produce sorbitol, iditol and xylitol.     

Lipid and lipopolysaccharide composition of Acholeplasma oculi.

The total lipid content of Acholeplasma oculi comprises 13.3% of the dry weight of the organism and is about equally distributed between the neutral lipids plus glycolipids and the phospholipids. The phospholipids were identified as phosphatidyl glycerol and diphosphatidyl glycerol. The glycolipid fraction contained O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol and O-alpha-D-glucopyranosyl-(1 leads to 2)-O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol. The neutral lipid contained pigmented carotenoids. Hot aqueous phenol extraction of lipid-extracted whole cells yielded a polymeric carbohydrate comprising 2.3% of the dry weight of the organism. The A. oculi lipopolysaccharide was found to contain only neutral sugars and no amino sugar, in contrast to other acholeplasmas. The neutral sugars consisted of fucose, galactose, and glucose in a ratio of 2:19:3.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Inhibition of triosephosphate isomerase in boar spermatozoa by (S)-3-chlorolactaldehyde

Mature epididymal boar spermatozoa converted fructose and glycerol to carbon dioxide but in the presence of 3-chloro-1-hydroxyacetone these oxidations were inhibited. When the substrate was fructose, there was an increase in the amounts of fructose 1,6-bisphosphate and dihydroxyacetone phosphate but these glycolytic intermediates did not accumulate when glycerol was the substrate. Examination of enzyme activities in mature boar spermatozoa incubated with 3-chloro-1-hydroxyacetone, which is metabolised in vitro to (S)-3-chlorolactaldehyde, confirmed that glyceraldehyde 3-phosphate dehydrogenase and triosephosphate isomerase were both inhibited to equivalent degrees by this metabolite.     

The Effect of Lipid Peroxide on the Lipid and Carbohydrate Metabolism in Rat Liver

Feeding tests were carried out on rats to clarify the mechanisms of fatty liver formation induced by autoxidized methyl linoleate. Lipid peroxides prepared by autoxidation of highly purified methyl linoleate were given orally to rats. Triglyceride and glycogen contents in liver were determined and enzyme activities including triglyceride synthetase and α-glycerophosphate dehydrogenase were also examined. The following results were obtained. 1. Triglyceride accumulation in rat liver fed autoxidized methyl linoleate was observed. 2. Increase in triglyceride content in rat liver was soon followed by the decrease of hepatic glycogen. 3. When rats were starved prior to introduction of autoxidized methyl linoleate, hepatic triglyceride accumulation did not occur. 4. The activities of α-glycerophosphate dehydrogenase and triglyceride synthetase in liver, and those of glutamic oxalacetic transaminase and leucine aminopeptidase in plasma were practically similar among the rats of test groups fed fresh or autoxidized methyl linoleate and the control fed diet without methyl linoleate. 5. The addition of l-carnitine which is a stimulator of fatty acid oxidation retarded the accumulation of the hepatic triglyceride mentioned above.     

Minimum requirements of n-3 and n-6 essential fatty acids for the function of the central nervous system and for the prevention of chronic disease.

General behavioral patterns of rats or mice fed 5 wt% safflower oil (75% linoleate [n-6] and less than 0.1% alpha-linolenate [n-3]) for two generations were significantly different from those of animals fed 5 wt% perilla oil (15% n-6 and 55% n-3). Also, brightness-discrimination learning ability and retinal function were higher in the perilla group than in the group fed 5 wt% soybean oil (53% n-6 and 4.7% n-3) or safflower oil, indicating that the requirement of n-3 for the maximum responses of the nervous system is above 0.6 en% when there is 6.8 en% linoleate n-6. Perilla oil has been found to be beneficial for the suppression of carcinogenesis, allergic hyperreactivity, thrombotic tendency, apoplexy, hypertension, and aging in animals, as compared with soybean oil and safflower oil. These results are against a lipid peroxide theory of aging, carcinogenesis, and chronic diseases. Animal experiments and epidemiological studies lead to a recommendation that the intake of n-6 should be decreased to as low as 2-4 en% and that of n-3 be increased to levels higher than linoleate n-6 for the prevention of chronic diseases prevailing in the industrialized countries.