Betaine Fermentation and Oxidation by Marine Desulfuromonas Strains

Two bacterial strains were dominant in anaerobic enrichment cultures with betaine (N,N,N-trimethylglycine) as a substrate and intertidal mud as an inoculum. One was a coccoid bacterium which was a trimethylamine (TMA)-fermenting methanogen similar to Methanococcoides methylutens. The other strain, a rod-shaped, gram-negative, motile bacterium, fermented betaine. On the basis of its ability to oxidize acetate and ethanol to CO₂ with sulfur as an electron acceptor, its inability to reduce sulfate and sulfite, its morphology, the presence of c-type cytochromes, and other characteristics, the isolated strain PM1 was identified as Desulfuromonas acetoxidans. Although only malate and fumarate were known as substrates for fermentative growth of this species, the type strain (DSM 684) also fermented betaine. Strain PM1 grew with a doubling time of 9.5 h at 30°C on betaine and produced approximately 1 mol of TMA per mol of betaine, 0.75 mol of acetate, and presumably CO₂ as fermentation products but only in the presence of selenite (100 nM). In this fermentation, betaine is probably reductively cleaved to TMA and acetate, and part of the acetate is then oxidized to CO₂ to provide the reducing equivalents for the initial cleavage reaction. In the presence of sulfur, betaine was converted to TMA and presumably CO₂ with the formation of sulfide; then, only traces of acetate were produced.     

Reductive cleavage of sarcosine and betaine by Eubacterium acidaminophilum via enzyme systems different from glycine reductase

The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO₂ or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [⁷⁵Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.Abbreviations DTE dithioerythritol - TES N-Tris (hydroxymethyl) methyl-2-amino-ethane sulfonic acid     

Glycine Betaine as a Direct Substrate for Methanogens (Methanococcoides spp.)

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.     

Anaerobic c(1) metabolism of the o-methyl-C-labeled substituent of vanillate

The O-methyl substituents of aromatic compounds constitute a C(1) growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C(1) substrate was determined by C radiotracer techniques. O-[methyl-C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C(1) substrate. The data showed that for every O-methyl carbon converted to [C]acetate, two were oxidized to CO(2). Quantitation of the carbon recovered in the two products, acetate and CO(2), indicated that acetate was formed in part by the fixation of unlabeled CO(2). The specific activity of C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO(2) and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-C]vanillate by strain TH-001 can be described as follows: 3CH(3)OC(7)H(5)O(3) + CO(2) + 4H(2)O --> CH(3)COOH + 2CO(2) + 10H + 10e + 3HOC(7)H(5)O(3).     

Metabolism of Trimethylamine, Choline, and Glycine Betaine by Sulfate-Reducing and Methanogenic Bacteria in Marine Sediments

The response of methanogenesis and sulfate reduction to trimethylamine, choline, and glycine betaine was examined in surface sediments from the intertidal region of Lowes Cove, Maine. Addition of these substrates markedly stimulated methanogenesis in the presence of active sulfate reduction, whereas addition of other substrates, including glucose, acetate, and glycine, had no effect on methane production. Sulfate reduction was stimulated simultaneously with methanogenesis by the various quaternary amines and all other substrates examined. Incubation of exogenous trimethylamine, choline, or glycine betaine with either bromoethane sulfonic acid or sodium molybdate was used to establish pathways of degradation of the substrates. Methanogenesis dominated the metabolism of trimethylamine, although limited nonmethanogenic activity, perhaps by sulfate-reducing bacteria, was observed. Acetate was oxidized primarily by sulfate reducers. Both choline and glycine betaine were fermented stoichiometrically to acetate and trimethylamine; apparently, neither substrate could be utilized directly by methanogens or sulfate reducers, and the activities of fermenters, methanogens, and sulfate reducers were all required to effect complete mineralization. These observations support the hypothesis that the presence of quaternary amines can mediate the coexistence of sulfate reduction and methanogenesis in marine surface sediments; they also implicate methanogens in the nitrogen cycle of marine sediments containing quaternary amines.     

Composition, Variation, and Dynamics of Major Osmotic Solutes in Methanohalophilus Strain FDF1

Methanohalophilus strain FDF1, a member of the halophilic genus of methanogens, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol, trimethylamine, and dimethyl sulfide as substrates for methanogenesis. It produces the osmolytes glycine betaine, beta-glutamine, and N-acetyl-beta-lysine with increasing external NaCl, but the relative ratio of these zwitterions depends primarily on the methanogenic substrate and less on the external osmolarity. When the cells are grown on methanol in defined medium, accumulation of glycine betaine predominates over the other zwitterionic solutes. The cells also synthesized a carbohydrate which was not detected in cells grown on trimethylamine. This negatively charged compound, identified as alpha-glucosylglycerate from the C and H chemical shifts, does not act as an osmoregulatory solute in the salt range 1.4 to 2.7 M in this methanogen as evidenced by its invariant intracellular concentration. CH(3)OH-pulse/CH(3)OH-chase experiments were used to determine half-lifes for these organic solute pools in the cells. l-alpha-Glutamate showed a rapid loss of heavy isotope, indicating that l-alpha-glutamate functions as a biosynthetic intermediate in these cells. Measurable turnover rates for both beta-glutamine, which acts as an osmolyte, and alpha-glucosylglycerate suggest that they function as metabolic intermediates as well. Molecules which function solely as osmolytes (glycine betaine and N-acetyl-beta-lysine) showed a slower turnover consistent with their roles as osmotic solutes in Methanohalophilus strain FDF1.     

Formation of N,N-Dimethylglycine, Acetic Acid, and Butyric Acid from Betaine by Eubacterium limosum

Two bacterial strains that grow anaerobically on betaine were isolated from enrichment cultures and identified as strains of Eubacterium limosum. In a mineral medium supplemented with yeast extract and Casitone, the doubling time of E. limosum strain 11A on betaine was 6 h at 37°C. The molar growth yield amounted to 9 g of dry cell mass per mol. Betaine was fermented in accordance with the following equation: 7 betaine + 2 CO₂ → 7 N,N-dimethylglycine + 1.5 acetate + 1.5 butyrate. E. limosum also grew on methanol and choline. The former was converted to acetate and butyrate, and the latter was converted to N,N-dimethylethanolamine, acetate, and butyrate. The conditions for the quantitative determination of N,N-dimethylglycine by capillary tube isotachophoresis have been determined.     

Methylammonium transport in Anacystis nidulans R-2

Methylammonium was taken up rapidly by illuminated cells of Anacystis nidulans R-2, leading to internal concentrations of 1.3 +/- 0.1 mM within 1 min, and a gradient of up to 200 between the cells and medium. Accumulation of 14CH3NH3+ required at least 5 mM NaCl, but the uptake rate was independent of medium pH between 6.5 and 9. The kinetics of uptake could be resolved into an initial fast phase lasting less than 1 min (approximate Km, 7.2 microM; Vmax, 12.5 nmol min-1 mg of protein-1 at 15 degrees C). A second, slower phase associated with product formation was eliminated by preincubation with methionine sulfoximine, a specific inhibitor of glutamine synthetase; the rapid phase was unaffected by this treatment. Ammonium ions competed with 14CH3NH3+ for entry, and addition of 5 microM NH4+ or 100 microM CH3NH3+ released 14CH3NH3+ accumulated during the rapid phase of entry. Small additions of NH4+ made at the same time as additions of 14CH3NH3+ delayed the start of radioactivity uptake by a time which corresponded accurately with the period needed for the complete removal of the added NH4+. The effects of inhibitors on accumulation and carbocyanine dye fluorescence suggest that ATP-dependent membrane potential was needed to drive 14CH3NH3+ transport. Spheroplasts were as active as whole cells in accumulating NH4+ and 14CH3NH3+, indicating that soluble periplasmic components are not involved in the translocation. Some significant differences between the translocation of 14CH3NH3 and that of NH4+ were observed: growth with NH4+ in place of NO3- repressed 14CH3NH3+ accumulation ability without affecting the NH4+ uptake rate Na+ was not required for NH4+ uptake, and concentration of KCl inhibitory with 14C3NH3+ did not reduce NH4+ uptake.     

L-amino acid dehydrogenases in Bacillus subtilis spores

The presence of two kinds of l-amino acid dehydrogenase in resting spores of Bacillus subtilis was indicated. One of them was l-alanine dehydrogenase, which used only l-alanine as a substrate, and the other was nonspecific dehydrogenase, which used l-valine, l-isoleucine, l-leucine, and l-alanine (slightly) as substrates. Several properties of these dehydrogenases were compared.     

Oxidation of Betaine Aldehyde by Betaine Dehydrogenase from Spinach Leaves

Betaine content in leaves of fifteen plant species was determined. The results showed higher betaine levels in those salt-, drought-, and chilling-resistant species. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 ) was isolated and partially purified from spinach leaves. Some properties of this enzyme were studied. BADH was precipitated by 60% saturation of (NH4)2SO4. Its activity was not detected in 70% saturation of (NH4)2SO4. BADH has two isoenzymes. The activity of BADH was quite stable below –80℃. It was inhibited by 0.125–1.0 mol/L NaG1 or KC1 but not by Mn2+ and Mo6+, and slightly increased by Mg2+.     

Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp. by cyanide.

NaCN was tested for its inhibitory effects on growth of and metabolism by Methanosarcina barkeri 227. NaCN (10 microM) inhibited catabolism of acetate methyl groups to CH4 and CO2 but did not inhibit methanogenesis from methanol, CO2, methylamine, or trimethylamine. NaCN also inhibited the assimilation of methanol or CO2 (as the sole carbon source) into cell carbon and stimulated the assimilation of acetate. These results suggest that inhibition by NaCN was a result of its action as an inhibitor of in vivo CO dehydrogenase. The results also implicate CO dehydrogenase in the oxidation of acetate but not methanol methyl groups to CO2.     

Substrate inhibition in the hydrolysis of N-acylglycine esters by carboxypeptidase A

The rates of hydrolysis of a series of 21 N-acylglycine esters (YCONHCH2CO2CH(CH2CH3)CO2H (2)) by bovine pancreatic carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) have been studied over the substrate concentration range 10(-4)-10(-1) M at pH 7.5, 25 degrees C, ionic strength 0.5. All substrates display substrate inhibition except Y = CH3, CH3CH2 and (CH3)3C for which normal Michaelis-Menten kinetics are observed. In all cases substrate inhibition is consistent with the formation of an ES2 complex and parameters for the second-degree rate equation v/E = (kapp2 S + kapp3 S2/KappSS)/(KappS + S + S2/KappSS) have been evaluated. For a series of eight aliphatic groups varying in size between Y = CH3 and Y = cyclo-C6H11 the following linear correlations were observed: -log KappS = 0.82 pi + 1.32 and log kapp2/KappS = 0.71 pi + 5.81 (pi is Hansch's hydrophobicity parameter). Aryl and aralkyl Y moieties deviate from these correlation lines. KappSS also depends on the hydrophobicity of Y but no quantitative correlation is obvious. Thus the Y unit of 2 is involved in a hydrophobic interaction with the enzyme when 2 binds at both the catalytically productive and inhibitor sites. Parameters for the enzymic hydrolysis of the esters YCONHCH2CO2CH(CH2CH(CH3)2)CO2H (3) (Y = C6H5(CH2)n (n = 0, 1, 2)) are also presented. Pronounced nonproductive 1: 1 enzyme.substrate complex formation is observed for each of 2: Y = C6H5(CH2)n (n = 2, 3) and 3: Y = C6H5(CH2)2. Hippurate anion is shown to be an uncompetitive inhibitor (Ki = 12 mM) for the hydrolysis of 2: Y = (CH3)3C. Data are now available which can only be interpreted in terms of at least three enzymic sites being available for hydrophobic interactions with ester substrate molecules.     

Metabolism of H2-CO2, methanol, and glucose by Butyribacterium methylotrophicum.

The fermentative metabolism of Butyribacterium methylotrophicum grown on either H2-CO2, methanol, glucose, or CO is described. The following reaction stoichiometries were obtained: 1.00 H2 + 0.52 CO2 leads to 0.22 acetate + 0.06 cell C; 1 methanol + 0.18 CO2 + 0.01 acetate leads to 0.24 butyrate + 0.29 cell C; and 1.00 glucose leads to 0.31 CO2 + 1.59 acetate + 0.21 butyrate + 0.13 H2 + 1.58 cell C. Cell yields of 1.7 g (dry weight) per mol of H2, 8.2 g (dry weight) per mol of methanol, 42.7 g (dry weight) per mol of glucose, and 3.0 g (dry weight) per mol of CO were obtained from linear plots of cell synthesis and substrate consumption. Doubling times of 9.0, 9.0, and 3 to 4 h were observed during batch growth on H2-CO2, methanol, and glucose, respectively. Indicative of a growth factor limitation, glucose fermentation in defined medium displayed a lower cell synthesis efficiency than when yeast extract (0.05%) was present. B. methylotrophicum fermentation displayed atypically high substrate/cell carbon synthesis conversion ratios for an anaerobe, as greater than 24% of the carbon was assimilated into cells during growth on methanol or glucose. The data indicate that B. methylotrophicum conserves carbon-bound electrons during growth on single-carbon or multicarbon substrates.     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

己酸菌LⅡ己酸发酵过程中生理及代谢特点

在了解了产己酸细菌ＬＩＩ的发酵最适条件的基础上,进一步对己酸发酵过程中的生理及代谢特性作了较详细的分析。结果表明,在己酸发酵过程中除产生正常代谢产物己酸外,还产生丁酸、乙酸、戊酸等其它酸类和甲烷,氢气和ＣＯ_２等气体。用１—^１３Ｃ－乙酸钠和未标记的乙醇作为底物发酵,用色质联用仪（ＧＣ／ＭＳ）分析,结果表明：在己酸合成中碳的来源为乙醇和乙酸,中间代谢产物为丁酸。     

Prebiotic synthesis in atmospheres containing CH4, CO,and CO2

The prebiotic synthesis of organic compounds using a spark discharge on various simulated primitive earth atmospheres at 25 degrees C has been studied. Methane mixtures contained H2 + CH4 + H2O + N2 + NH3 with H2/CH4 molar ratios from 0 to 4 and pNH3 = 0.1 torr. A similar set of experiments without added NH3 was performed. The yields of amino acids (1.2 to 4.7% based on the carbon) are approximately independent of the H2/CH4 ratio and whether NH3 was present, and a wide variety of amino acids are obtained. Mixtures of H2 + CO + H2O + N2 and H2 + CO2 + H2O + N2, with and without added NH3, all gave about 2% yields of amino acids at H2/CO and H2/CO2 ratios of 2 to 4. For a H2/CO2 ratio of 0, the yield of amino acids is extremely low (10(-3)%). Glycine is almost the only amino acid produced from CO and CO2 model atmospheres. These results show that the maximum yield is about the same for the three carbon sources at high H2/carbon ratios, but that CH4 is superior at low H2/carbon ratios. In addition, CH4 gives a much greater variety of amino acids than either CO or CO2. If it is assumed that an abundance of amino acids more complex than glycine was required for the origin of life, then these results indicate the requirement for CH4 in the primitive atmosphere.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [¹⁴C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.     

Effects of organic solvents, methylamines, and urea on the affinity for Pi of the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. In a previous report (de Meis, L., Alves, E., and Martins, O.B. (1980) Biochemistry 19, 4252-4261), it was shown that organic solvent such as dimethyl sulfoxide and glycerol cause a decrease in the apparent Km for Pi. In this report it is shown that a similar effect is obtained with the methylamines glycine betaine and trimethylamine N-oxide. The apparent Km value for Pi in totally aqueous medium and in the presence of either 6.4 M glycerol, 1.4 M dimethyl sulfoxide, 0.4 M trimethylamine N-oxide, or 1 M glycine betaine were found to be respectively 2.85, 0.52, 0.52, 0.81, and 0.93 mM at pH 6.2 and greater than 10.0, 1.08, 2.53, 3.05, and 2.05 mM at pH 7.5. In contrast to the effect of methylamines, urea caused an increase in the apparent Km for Pi. When mixed in the appropriate concentration ratio, the effect of either organic solvent or methylamines is cancelled by urea.