Oxidative DNA damage induced by aminoacetone, an amino acid metabolite

We investigated DNA damage induced by aminoacetone, a metabolite of threonine and glycine. Pulsed-field gel electrophoresis revealed that aminoacetone caused cellular DNA cleavage. Aminoacetone increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human cultured cells in a dose-dependent manner. The formation of 8-oxodG in calf thymus DNA increased due to aminoacetone only in the presence of Cu(II). DNA ladder formation was observed at higher concentrations of aminoacetone than those causing DNA cleavage. Flow cytometry showed that aminoacetone enhanced the generation of hydrogen peroxide (H2O2) in cultured cells. Aminoacetone caused damage to 32P-5'-end-labeled DNA fragments, obtained from the human c-Ha-ras-1 and p53 genes, at cytosine and thymine residues in the presence of Cu(II). Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 and Cu(I) were involved. Analysis of the products generated from aminoacetone revealed that aminoacetone underwent Cu(II)-mediated autoxidation in two different pathways: the major pathway in which methylglyoxal and NH+4 are generated and the minor pathway in which 2,5-dimethylpyrazine is formed through condensation of two molecules of aminoacetone. These findings suggest that H2O2 generated by the autoxidation of aminoacetone reacts with Cu(I) to form reactive species capable of causing oxidative DNA damage.     

A novel HPLC procedure for detection and quantification of aminoacetone, a precursor of methylglyoxal, in biological samples

Increase in methylglyoxal is thought to be involved in different pathological conditions. Deamination of aminoacetone by semicarbazide-sensitive amine oxidase (SSAO) leads to production of methylglyoxal. We have synthesized aminoacetone and developed a novel HPLC procedure for its quantitative determination. The urinary excretion of aminoacetone is approximately 20-30 microg/mouse/day, and the concentration is about 0.5 microg/g in mouse liver and small intestine. SSAO inhibitor increases aminoacetone levels in both tissues and urines. Results confirm that aminoacetone is an endogenous substrate for SSAO. However, data also indicate that deamination is not the only catabolic pathway for aminoacetone.     

Microbial metabolism of amino ketones: l-1-Aminopropan-2-ol dehydrogenase and l-threonine dehydrogenase in Escherichia coli

1. A wide range of intermediary metabolites and substrate analogues have no effect on the oxidation of dl-1-aminopropan-2-ol to aminoacetone by washed-cell suspensions of Escherichia coli. Only dl-2-hydroxy-2-phenylethylamine, dl-1,3-diaminopropan-2-ol, dl-serine and l-1-(3,4-dihydroxyphenyl)-2-aminoethanol act as inhibitors. 2. Dialysed cell-free extracts of E. coli exhibit an NAD(+)-dependent dl-1-aminopropan-2-ol-dehydrogenase activity of approx. 8mmumoles of aminoacetone formed/mg. of protein/min. at the pH optimum of approx. 10. The K(m) values for the coenzyme and dl-amino alcohol are approx. 0.4 and 10.0mm respectively. A smaller peak of activity occurs at pH7.0-7.2, the K(m) for NAD(+) at pH7 being approx. 0.05mm. 3. Enzyme activity in cell-free extracts is inhibited by dl-2-hydroxy-2-phenylethylamine, dl-1-aminopropane-2,3-diol and dl-serine. dl-Phenylserine and dl-1-aminobutan-2-ol are oxidized to compounds reacting as amino ketones. 4. In fresh cell-free extracts l(+)-1-aminopropan-2-ol preparations are oxidized more rapidly than racemic or laevo-rotatory material, the d(-)-enantiomorph appearing to act as a competitive inhibitor. The K(m) for l(+)-1-aminopropan-2-ol appears to be approx. 1.5mm when highly resolved substrate preparations are used, either in the free base form or as the l(+)-tartrate salt. 5. l(+)-1-Aminopropan-2-ol dehydrogenase is a labile enzyme, and in appropriately treated extracts activity towards the d-enantiomorph is detectable and relatively higher than that towards the l-enantiomorph. 6. Optimum activity of l-threonine-dehydrogenase in cell-free extracts is exhibited at pH9.6 in the presence of NAD(+). The K(m) values for coenzyme and amino acid substrate are approx. 0.08 and 5.0mm respectively. This enzyme is distinct from 1-aminopropan-2-ol dehydrogenases on the basis of kinetic evidence, and the separation of activities by gel filtration. 7. Both l-threonine and dl-1-aminopropan-2-ol dehydrogenases are markedly inhibited by 8-hydroxyquinoline and p-chloromercuribenzoate, but only slightly by other chelating and thiol reagents. 8. E. coli is incapable of growth on simple synthetic media, containing a variety of carbon sources, when dl-1-aminopropan-2-ol is supplied as the sole source of nitrogen. It appears unlikely that the micro-organism can deaminate aminoacetone. 9. The metabolic roles of l-threonine dehydrogenase, aminoacetone and 1-aminopropan-2-ol dehydrogenases are discussed.     

Microbial metabolism of amino ketones. Aminoacetone formation from 1-aminopropan-2-ol by a dehydrogenase in Escherichia coli

1. Washed-cell suspensions of Escherichia coli, incubated at the optimum pH of 6.4 and with a saturating substrate concentration of approx. 10mm, convert dl-1-aminopropan-2-ol into aminoacetone at a rate of approx. 4.0mmumoles/mg. dry wt. of cells/min. at 30 degrees . 2. Mg(2+), Mn(2+), Co(2+), Zn(2+), Ca(2+), K(+) and NH(4) (+), as sulphates, and EDTA have no effect on this rate, although Cu(2+) inhibits and Fe(2+) activates to some extent. 3. Conditions of growth markedly affect the rate of aminoacetone production by cell suspensions. 4. Dialysed cell-free extracts of E. coli exhibit 1-aminopropan-2-ol-dehydrogenase activity, the enzyme having optimum activity at pH7.0, a requirement for NAD(+) and K(+), and a K(m) for the amino alcohol substrate of 0.8mm, calculated for a single enantiomorph. 5. Under optimum conditions 1-aminopropan-2-ol dehydrogenase forms aminoacetone at rate of approx. 3.0mmumoles/mg. of protein/min. at 37 degrees . The enzyme is only slightly inhibited by dl-3-hydroxybutyrate and dl-2-hydroxy-2-phenylethyl-amine. 6. l-Threonine-dehydrogenase activity is exhibited by both whole cells and cell-free extracts. Whole cells produce aminoacetone from l-threonine more slowly than they do from dl-1-aminopropan-2-ol, whereas the situation is reversed in cell-free extracts. Both kinetic evidence, and the fact that synthesis of 1-aminopropan-2-ol dehydrogenase, but not of threonine dehydrogenase, is repressed by compounds such as glucose and pyruvate, provide evidence that the amino alcohol is oxidized by a specific enyme. 7. The metabolic role of 1-aminopropan-2-ol dehydrogenase is discussed.     

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal,a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH₄ ⁺ ion, and H₂O₂ coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O₂ ^•− and HO^• radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5′-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E₀ values = −0.51 and −1.0 V) to ferricytochrome c (E₀ = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH₄ ⁺ ion. In the presence of oxygen, aminoacetone enoyl and O₂ ^•− radicals propagate aminoacetone oxidation to methylglyoxal and H₂O₂. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.     

Differential rates of protein synthesis in vitro and RNA contents in rat heart ventricular and atrial muscle.

The rates of protein synthesis in perfused rat heart ventricular or atrial muscle were measured by incorporation of [U-14C]phenylalanine in the presence of the remaining plasma amino acids. Atrial protein-synthesis rates were about twice the ventricular rates. Atrial RNA contents were also about twice the ventricular contents. Thus the efficiencies of protein synthesis (protein-synthesis rate/RNA) in the two compartments were similar. There were marked differences in ventricular and atrial RNA contents during the course of rat growth. Atrial RNA content was always greater than ventricular content and declined more slowly during growth, producing a 2-fold change in atrial/ventricular RNA-content ratio between the 88 g and 370 g rat groups.     

Immunological studies of the mechanism of anaemia in experimental Trypanosama evansi infection in rats

Pathological changes in the blood of rats acutely infected with Trypanosoma evansi and the probable mechanism of the accompanying anaemia, were investigated. A severe anaemia, together with rreticulocytosis and hepato-splenomegaly, were regularly observed. Histological examination of the liver, spleen and bone-marrow confirmed the increasedin erythropoietic activity that the observed anaemia was due to increasedextravascular destruction of erythrocytes rather than by inhibition of haemopoietic activity. All the infected rats showed significant immune responses to the infecting trypanosome peak agglutinin titres occurring 10–12 days after injection, coincidentally with maximun destruction of erythrocytes. Serological examination of sera and erythrocytes from all infected and control rats did not reveal the presence of either circulating or adsorbed erythrocyte auto--antibodies. Furthermore, there was no in vivo trypanosomal antigen coating of the erythrocytes from either infected or multiple antigen-injected rats. Repeated intraveoous injections into rats of more than 100 μg per g body weight of soluble T. evansi antigen resulted in moderately severe, probably antibody-mediated, haemolytic anaemia. It is considered that an immunologically-mediated mechanism may be responsible for the development of the anaemia accompanying T. evansi infection.     

Survival of freeze-dried bacteria.

The aim of this study was to investigate the survival of freeze-dried bacterial species stored at the International Patent Organism Depository (IPOD) and to elucidate the characteristics affecting survival. Bacterial strains were freeze-dried, sealed in ampoules under a vacuum (<1 Pa), and stored in the dark at 5 degrees C. The survival of a variety of species following storage for up to 20 years was analyzed. The survival of freeze-dried species was analyzed in terms of two stages, freeze-drying and storing. Nonmotile genera showed relatively high survival after freeze-drying. Motile genera with peritrichous flagella showed low survival rates after freeze-drying. Vibrio and Aeromonas, which produce numerous flagella, showed very low survival rates. In Lactobacillus, non-trehalose-fermenting species showed better survival rates after freeze-drying than did fermenting species, and those species with teichoic acid in the cell wall showed lower survival rates during storage than species with teichoic acid in the cell membrane. Human pathogenic species of Corynebacterium, Bacillus, Streptococcus, and Klebsiella showed lower survival rates during storage than nonpathogenic species within the same genus. Among Pseudomonas species, P. chlororaphis, the only species tested that forms levan from sucrose, showed the lowest survival rate during storage in the genus. Survival rates of Gram-negative species during storage tended to be lower than those of Gram-positive species, though Chryseobacterium meningosepticum had stable survival during storage. The conclusion is that smooth cell surfaces (i.e., no flagella) and lack of trehalose outside the cytoplasm improved survival rates after freeze-drying. Because desiccation is important for survival during storage, the presence of extracellular polysaccharides or teichoic acids is disadvantageous for long-term survival. The lower survival rates of freeze-dried Gram-negative bacteria compared with those of Gram-positive bacteria may be attributed to the thinner peptidoglycan layer and the presence of lipopolysaccharides on the cell wall in the former species.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.     

Influence of drying treatment on three Sargassum species

The effects were investigated of two different drying treatments (oven- andfreeze-drying) on the proximate composition, amino acid profile and somephysico-chemical properties of three subtropical brown seaweeds, Sargassum hemiphyllum, S. henslowianum and S. patens. Therewere significant differences (p < 0.05, two-way ANOVA,Tukey-HSD) in the ash, crude lipid and moisture contents of the threespecies treated by the two drying methods. The amount of total aminoacids in the oven-dried seaweed samples was significantly (p <0.05, two-way ANOVA, Tukey-HSD) lower than that of the freeze-driedones. However, there were no significant differences on the amount oftotal essential amino acids and individual amino acid between the oven- andfreeze-dried brown seaweeds. Physico-chemical properties includingswelling, water holding and oil holding capacity of the freeze-dried Sargassum species were significantly (p < 0.05, two-wayANOVA, Tukey-HSD) higher than those of the oven-dried seaweedsamples. This indicated that freeze-dried seaweeds had greater potential tobe used as food ingredients in formulated food products than oven-driedones.     

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40+/-0.070min(-1) and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using (1)H NMR, had a K(m) of 1.49+/-0.16mM and V(max) of 2.61+/-0.08micromolL(-1)min(-1). Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of approximately 10microM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were approximately 5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.     

Semicarbazide-sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a methylglyoxal-AGE: implications for vascular complications in diabetes

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes formation of methylglyoxal (MG) from aminoacetone; MG then reacts with proteins to form advanced glycation end products or AGEs. Because of its potential to generate MG, SSAO may contribute to AGE-associated vascular complications of aging and diabetes. We developed a method to measure SSAO activity in bovine aortic smooth muscle cells (BASMC) based on the oxidation of 2',7'-dichlorofluorescin by hydrogen peroxide and horseradish peroxidase. The SSAO activity was completely inhibited by 10 mM semicarbazide. Argpyrimidine is a readily detectable fluorescent product of the reaction between MG and arginine. Cell lysates incubated with aminoacetone formed argpyrimidine in a reaction that was inhibited by 20 mM semicarbazide. Immunostaining of tissue sections showed that aminoacetone-treated rats (normal as well as diabetic) formed more argpyrimidine in aortic smooth muscle than untreated controls. We believe that SSAO can enhance AGE synthesis in the macrovasculature of diabetic individuals by production of MG.     

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken

The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.     

Aberrant DNase I digestion kinetics of nucleosomal core particles from sea urchin sperm

The pancreatic deoxyribonuclease (DNase I) digestion rates at the susceptible sites on nucleosomal core particles from blastula, gastrula and sperm cells of the sea urchin, Parechinus angulosus, have been determined. Although there are differences in their isohistone composition, the rates of digestion are similar for both embryonic stages. The rates of digestion for sperm core particles are 3-5 times lower than for embryo core particles at the more, and up to 2.5 times lower at the less susceptible sites. An explanation for these differences could be sought in the sperm isohistones H2B which are characterized by N-terminal extensions of 20-25 amino acid residues.     

Replication of Epstein-Barr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells.

We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (less than or equal to 50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (greater than or equal to 300 particles per cell), a productive cycle was the rule.     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes.

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and nonagglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. This was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination. Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg2+, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca2+ (0.2 mM) had little effect on agglutinability, although high Ca2+ (5 mM) inhibited agglutinability of the resealed membranes somewhat. Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells. The agglutination of erythrocytes was not affected by cytochalasin B (40 mug/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes. It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca2+ or Mg2+ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitivie to vinblastine.     

Amino acid transport in normal and glutathione-deficient sheep erythrocytes.

1. Uptake rates for 23 amino acids were measured for both normal (high-GSH) and GSH-deficient (low-GSH) erythrocytes from Finnish Landrace sheep. 2. Compared with high-GSH cells, low-GSH cells had a markedly diminished permeability to D-alanine, L-alanine, alpha-amino-n-butyrate, valine, cysteine, serine, threonine, asparagine, lysine and ornithine. Smaller differences were observed for glycine and proline, whereas uptake of the other amino acids was not significantly different in the two cell types.     

The oxidation of aminoacetone by a species of Arthrobacter

1. A micro-organism similar to Arthrobacter globiformis has been isolated from sewage by elective growth on a medium containing l-threonine as sole source of carbon and nitrogen. 2. Washed cell suspensions of the organism catalyse the complete disappearance of aminoacetone from the medium and its almost complete oxidation. 3. In the presence of iodoacetate, aminoacetone disappearance is accompanied by the accumulation of methylglyoxal, about 70% of the aminoacetone removed being accounted for in this way. 4. It is suggested that the conversion of aminoacetone into methylglyoxal is catalysed by an amine oxidase.