pH Of Incubation Medium Research Articles

Isolation, properties and role in progesterone biosynthesis of cytosolic malic enzyme from human term placenta.

Cytosolic malic enzyme (L-malate: NADP oxidoreductase decarboxylating, EC I. I. I.40) has been isolated and purified from postmitochondrial supernatant of human term placenta by ammonium sulphate fractionation, chromatography on diethylaminoethyl- (DEAE-)cellulose, Sepharose 6B, ADP-Sepharose 4B and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis (PAGE). The specific activity of the purified enzyme was 24.0 mumol X min-1 X mg-1 protein, which corresponds to about 7500-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be about 250,000. Sodium dodecyl sulphate- (SDS-)PAGE showed one polypeptide band of molecular weight 63,000. It appears that the native protein is a tetramer composed of subunits of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of pyruvate and bicarbonate at about 80 per cent of the rate of the forward reaction. The optimum pH for the carboxylation reaction was pH 7.3, and that for the decarboxylation reaction varied with malate concentration. The Km values, determined at pH 7.2, for malate, NADP+, Mn2+, and Mg2+ were 81 microM, 10 microM, 2.5 microM and 0.6 mM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 4 mM, 25 microM and 20 mM, respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 mM NAD+ at approximately 80 per cent of the maximum rate with NADP+. It exhibited oxaloacetate decarboxylase activity at about 10 per cent of the rate of oxidative decarboxylation of malate (with NADP+ as coenzyme) and pyruvate reductase activity at about 4 per cent of the rate of oxidative decarboxylation of malate with NADP+. The oxidative decarboxylation of malate was inhibited by malate at lower values of pH of incubation medium. This inhibition gradually decreased as the pH of the incubation medium increased. No inhibition was observed at pH 8.2. The addition of purified cytoplasmic malic enzyme, pyruvate, bicarbonate and NADPH generating system (consisting of NADP+, glucose 6-phosphate, glucose 6-phosphate dehydrogenase) stimulated about twofold progesterone biosynthesis by the isolated human placental mitochondria. This stimulation was abolished by arsenite and fluorocitrate. A possible role for the cytosolic malic enzyme in the regulation of progesterone biosynthesis in human placenta is discussed.

Red cell pH of lamprey (Lampetra fluviatilis) is actively regulated

The red cell pH of lamprey (Lampetra fluviatilis) was measured using the DMO method (based on the passive distribution of the wead acid, DMO, across the red cell membrane). The measured red cell pH was higher than the pH of the incubation medium throughout the pH range (7.2–8.2) studied, and higher than the red cell pH calculated from the chloride distribution ratio. Treatment of cells with the metabolic inhibitors 2,4-dinitrophenol or KCN caused a drop in the red cell pH to values lower than the pH of incubation medium, and abolished the difference between the measured red cell pH and the pH calculated from the chloride distribution ratio. These data strongly suggest that the proton gradient across lamprey red cell membrane is actively maintained. Acid extrusion from lamprey red cells may require sodium, as indicated by the observation that when choline was substituted for sodium in the incubation medium, the intracellular pH decreased significantly.

Isolation and Germination type of protoplasts of spores and hyphae of Pyricularia oryzae.

Isolation and culture conditions of protoplasts from spores and hyphae of Pyricularia oryzae were studied. The combination of zymolyase and β-glucuronidase and that of driselase and cellulase were effective for the isolation of viable protoplasts from spores and hyphae of P. oryzae, respectively. A majority of protoplasts thus obtained had the potential to germinate and reverse. Protoplasts germinated in two different types. One is direct reversion to hyphae from regenerated mother cells (hyphal type germination) and another is formation of budded cells (budding type germination). The germination type of protoplasts was significantly influenced by pH and presence of agar in the medium. In Czapek-yeast extract liquid medium (CzYLM), the frequency of hyphal type germination was maximum at pH7.0. About 70% of hyphal protoplasts and about 50% of spore protoplasts germinated in direct reversion to hyphae in CzYLM at pH7.0. In contrast, the frequency of budding type germination was minimum at pH7. Presence of agar in the medium increased the frequency of hyphal type germination. The intensity of fluorescence by a calcofluor white of regenerated mother cells and budded cells increased with increase of pH of incubation medium while that of reversional hyphae was not influenced by pH. Thus we suggest that cell wall regeneration process was one of the factors involved in determination of germination type. This is the first report on the successful isolation of abundant viable protoplasts from spores of P. oryzae.

Phorbol esters inhibit gluconeogenesis in canine renal proximal tubular segments

Gluconeogenesis is a major metabolic function of the renal proximal tubular cell. To characterize the regulation of this process in proximal tubule, glucose production from gluconeogenic precursors was measured in proximal tubular segments prepared from dog kidney. Production of glucose was a linear function of time for up to 120 min of incubation at 37 degrees C under a variety of conditions. Lowering the pH of incubation media from 7.5 to 7.0 increased glucose synthesis. Production of glucose was inhibited by 3-mercaptopicolinate. Incubation of proximal tubular segments with insulin diminished synthesis of glucose. Incubation of segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in decreased production of glucose. This effect was not observed following incubation with the inactive phorbol ester 4 alpha-phorbol. Changes in glucose synthesis could not be attributed to alterations in cell viability or in rates of glucose oxidation induced by experimental maneuvers. Our findings confirm the usefulness of proximal tubular segments for characterization of metabolic processes in this portion of the nephron. The experimental results are consistent with a role for protein kinase C in the control of gluconeogenesis in proximal tubule.

Ingestion of Dye by the Sexes of Trichostrongylus colubriformis

Trichostrongylus colubriformis ingested the fluorescent dye, Rhodamine B, while feeding in vivo and in vitro. Ingestion by females of T. colubriformis in vivo significantly exceeded that of males from 15 to 30 days of infection. However, the rate of ingestion declined linearly in both sexes as the helminths' age increased. Uptake of dye in vitro was dependent on time and concentration. Ingestion by the sexes of T. colubriformis in vitro increased significantly with elevated temperature, length of time that the worms were outside the host, decreasing concentrations of salt in the incubation medium and acidic pH's. In vitro ingestion was similar in light or dark conditions. Saturation of the medium with oxygen or nitrogen/carbon dioxide significantly increased or decreased, respectively, ingestion by helminths. Uptake of dye by T. colubriformis decreased significantly in groups when nematodes were increased from 250 to 1,000 worms of a single sex in 2 ml of medium. In females there was a similar decrease in single- or mixed-sex groups containing more than 500 worms while ingestion by males in mixed-sex groups was significantly less than in groups containing only males.

Dormancy in red rice. IV. Response of unimbibed and imbibing seeds to nitrogen dioxide

Nitrogen dioxide at 4 to 8 ml 1−1 breaks dormancy of dehulled, unimbibed seeds of red rice (Oryza sativa L.) with minimum exposure times to the gas of 20 min or less. The response is independent of incubation medium pH following treatment. Germination is complete within 2 days but proceeds 4 to 6 h more slowly than non‐dormant controls. The magnitude of response at a constant NO2 dose is dependent upon initial seed moisture. There was less than 40% germination at 9.6% initial moisture while germination was 95% at 12% initial moisture. NO2 treatment also breaks dormancy of imbibing, dehulled seeds, but the response is dependent upon the pH of the medium. Dormancy is broken at pHs close to the pKa of nitrous acid. Intact, unimbibed or imbibing seeds respond to NO2 treatment only if partially dry‐afterripened prior to exposure.

Modulation of N-hydroxyparacetamol cytotoxicity in suspensions of isolated rat hepatocytes.

The effect of inhibitors of toxicity of N-hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of paracetamol, was studied in isolated rat hepatocytes. Additions of ascorbate, menadione, thiol-containing amino acids and glutathione (GSH) led to an increased stability of N-OH-pHAA, reduced the covalent binding of N-OH-pHAA to cellular protein and decreased GSH depletion caused by N-OH-pHAA. Two to three hours elapsed after a 30 min. exposure of the cells to N-OH-pHAA before the cells responded with increased cell permeability. Ascorbate, acetylcysteine, GSH and promethazine were capable of inhibiting this second phase of N-OH-pHAA cytotoxicity in addition to their effects during the initial exposure phase. In contrast, the anti-oxidant tocopherole and phenacetin wee only effective during the second phase. Increasing the incubation medium pH during the second phase of N-OH-pHAA mediated cellular damage resulted in decreases in cytotoxicity. Lipid peroxidation, as measured by accumulation of thiobarbituric acid reactive metabolites, did not seem to be directly correlated with cytotoxicity, since cysteamine or higher concentrations of N-OH-pHAA inhibited lipid peroxidation without decreasing cellular damage.

Metabolic acidosis suppresses 25-hydroxyvitamin in D3-1alpha-hydroxylase in the rat kidney. Distinct site and mechanism of action.

Effect of metabolic acidosis on two distinct 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase) systems was studied in the kidneys of vitamin D-deficient rats; one is localized in the proximal convoluted tubule (PCT), is activated in vitamin D deficiency, and is regulated primarily by parathyroid hormone (PTH) via cyclic AMP; the other is localized in the proximal straight tubule (PST), is latent in vitamin D deficiency, and is selectively stimulated by calcitonin via a cyclic AMP-independent mechanism. The 1alpha-hydroxylase activities were measured in the PCT and PST microdissected from the kidney of vitamin D-deficient rats with or without metabolic acidosis of varying duration. The 1alpha-hydroxylase activity decreased in the PCT from 0.74+/-0.07 fmol/mm per h to 0.24+/-0.02 at day 3 of metabolic acidosis without a further decline at day 7. Neither metabolic acidosis of 16 h duration nor reduction of the incubation medium pH from 7.4 to 7.0 affected the enzyme activity in the PCT. To examine the underlying mechanism for the suppression of 1alpha-hydroxylase activity, PTH, cyclic AMP, or calcitonin was given to rats with metabolic acidosis of 3 d duration. Although PTH failed to augment the suppressed 1alpha-hydroxylase activity in the PCT, cyclic AMP restored it to the level of control rats. The 1alpha-hydroxylase activity in the PST remained undetectable in control rats and in acidotic rats with or without PTH or cyclic AMP treatments. However, calcitonin stimulated the 1alpha-hydroxylase activity in the PST equally from undetectable to 0.75+/-0.09 fmol/mm per h in control and to 0.78+/-0.10 in acidotic rats. The data suggests that metabolic acidosis suppresses 1alpha-hydroxylase only in the PCT by inhibiting PTH-dependent adenylate cyclase, and that cellular events beyond cyclic AMP in the PCT and the events responsive to calcitonin in the PST are unaffected. The results show the definite advantage of using defined single nephron segments to study the hormonal and ionic control of the 1alpha-hydroxylase system in the kidney.

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na +-free, Na + and K +-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers ( p-CMBS, 4-CMB and DIP 2+) and ATP. It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K +. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na +/K + ratio, both effects were partially overcome by GSH. After treatment by DIP 2+, Na +/K + ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K + and accumulation of Na + Here neither effect could be reversed by GSH; consequently, high cellular amounts of K + and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake. It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.

Effect of pH and divalent cations on the release and re-entry of RNA in isolated nuclei

Release of prelabeled RNA from isolated nuclei is inhibited by divalent cations and low pH [l-3]. This phenomenon is still poorly understood. It is believed that divalent cations act through binding nucleoside triphosphates which supply energy for RNA transport [4-61. Divalent cations may also interact with components of nuclear envelope and alter its structure. As for incubation medium pH, the mechanism of its influence on RNA release from isolated nuclei has not been considered before. We demonstrate here that low pH of incubation buffer causes re-entry of in vitro released RNA into nuclei. Divalent cations do not exhibit such effect.

Effects of anoxia and low pH on organic ion transport and electrolyte distribution in harbor seal (Phoca vitulina) kidney slices

The present study was undertaken to examine the effects of anoxia and/or low pH on active organic ion transport and tissue water and electrolyte distribution in freshly prepared cortical slices from harbor seal and rat kidneys.p-Aminohippurate (PAH) and tetraethylammonium (TEA) were used as a representative organic anion and cation, respectively. The slice uptake of PAH was not influenced by the incubation medium pH over a range of 5.0–7.5 in the harbor seal while it was very sensitive to the medium pH in the rat, decreasing significantly with reductions in pH (Fig. 1). Although the TEA uptake decreased in anoxia in both species, it decreased considerably less in the harbor seal than in the rat (40% vs 74%) (Fig. 2). However, in contrast to the PAH transport system, the TEA uptake decreased significantly at low pH in both species. The magnitudes of increase in intracellular Na and of decrease in intracellular K in anoxia were consistently less in the harbor seal than in the rat, especially at low pH (Fig. 3). However, the pH dependence of Na−K-ATPase activity of the crude renal cortical homogenate was not different between the two species (Fig. 4). These results, together with the previous finding on the anoxic tolerance of the renal organic anion transport system, indicate that (1) the overall anoxic tolerance of active renal transport systems for both organic anions and cations is greater in the harbor seal than in the rat, and (2) the renal organic anion (but not cation) transport system is resistant to low pH in the harbor seal.

A simple system for the presumptive identification ofCandida albicansand differentiation of strains within the species

A system of 10 agar plate tests allowed presumptive identification of Candida albicans and differentiation of up to 512 strain types within the species. The yeast isolates were tested for acid and salt tolerance, proteinase production, resistance to 5-fluorocytosine and safranine, and assimilation of urea, sorbose, citrate and glycine. Media were inoculated semiquantitatively with a multiple-pronged device so that 55 yeasts and 5 reference strains could be printed on each plate at once. The results for the 9 strain differentiation tests were arranged in 3 groups to allow simple designation of types by 3-digit numbers. Mouth and vaginal samples from 85 patients and healthy volunteers yielded to 45 different strain types, of which types 153, 157 and 357 were the most prevalent. The reproducibility of the system was good, if attention was paid to the precision which the media were prepared and inoculated. The effect of test variables, including incubation temperature, medium pH and inoculum size, was assessed. Work is presently in progress to extend the system for presumptive identification of other clinically important Candida spp. and differentiation of their strain types.

Response of the rat and dog kidney to H + concentration in vitro—A comparative study with slices and tubules

1. 1. The present study reevaluates the metabolic response of rat and dog renal cortical tissue to changes in H + concentration of incubation medium and compares the response of kidney slices to (hat of isolated tubules. 2. 2. In the rat, acidification (pH 7.1) depresses glutamate accumulation and enhances gluconeogenesis with both slices and tubules, but glutamine extraction and ammonia production increase significantly only with tubules. 3. 3. Alkalinization (pH 7.7) depresses glucose and ammonia production and enhances glutamate accumulation with both slices and tubules but glutamine extraction is also slightly stimulated with tubules only. 4. 4. Thus, in the rat, variations of incubation medium pH modulates (a) glutamate utilization and (b) glutamine transport from the medium to the mitochondria, the second effect being apparent with tubules only. The different response observed with slices and tubules is attributed to a difference of permeability for glutamine between these two preparations. 5. 5. The intracellular metabolite profile of tubules rapidly separated from the incubation medium after 30 min suggests that in vitro acidosis increases and alkalosis decreases glutamate utilization and ammonia production probably by affecting the activity of the alpha-ketoglutarate dehydrogenase complex. 6. 6. In the dog, no effect of H + concentration on glutamine transport or utilization can be seen. However, H + concentration modifies glutamate accumulation and stimulates gluconeogenesis (from glutamate and endogenous substrates). 7. 7. Both effects are in accord with observations in the rat and may involve similar mechanisms.

Ammoniagenesis in kidney cortex mitochondria of the rat: role of the mitochondrial dicarboxylate anion transporter.

Since glutamine enters rat kidney mitochondria without exchange for an anion, the exit of its carbon skeleton must involve the dicarboxylate anion transporter (malate - inorganic phosphate) for ammoniagenesis to proceed. Therefore, this important mitochondrial anion transporter was studied in isolated renal cortex mitochondria. The phosphate concentration required for half-maximal rates of malate exit from renal mitochondria of normal rats was 1.0 mM. This value was not decreased in renal cortex mitochondria from rats with chronic metabolic acidosis. The maximum velocity of the dicarboxylate transporter was not increased in renal cortex mitochondria from these acidotic rats. These kinetic parameters were similar in liver mitochondria. There was no acute activation of the dicarboxylate carrier when the incubation medium pH lowered. Thus, there is no demonstrable activation of the dicarboxylate anion transporter in kidney cortex mitochondria of the rat with chronic metabolic acidosis. The significance of these results with respect to the regulation of renal ammoniagenesis is discussed.

Studies on carcinogenic azo dyes. VI. Effect of factors influencing drug metabolism on the NIH shift during hydroxylations of 4-dimethylamino-3'-methylazobenzene and 3-methylacetanilide.

The NIH shift during aryl hydroxylations of 4-dimethylamino-3'-methylazobenzene (3'-Me-DAB) and 3-methylacetanilide under various conditions has been investigated. 4-Hydroxy-3-methylacetanilide was obtained from 3-methylacetanilide (4-2H, or 4-3H) by rat in vivo metabolism or by incubation with hepatic microsomal preparations of rat, mouse, hamster, or rabbit, and 3'-Me-4'-hydroxy-DAB by incubation of 3'-Me-DAB (4'-3H, or 4'-8H) with the liver homogenates. Retention of heavy hydrogen in the hydroxylated products was determined by mass spectrometry or liquid scintillation counting. The degree of retention of isotopic hydrogen during hydroxylation of the labeled 3-methylacetanilide was affected by the pH of incubation media, the species variation, the sex of animal used as the source of microsomal preparations, and the pretreatment of animal with inducing agents. Pretreatment of rat or mouse with phenobarbital caused an increase in the retention of tritium in 4-hydroxy-3-methylacetanilide produced from 3-methylacetanilide-4-3H, while pretreatment with 3-methylcholanthrene or 3, 4-benzpyrene caused a decrease in the retention. Pretreatment of rat with the carcinogenic azo dye, 3'-Me-DAB, did not affect the NIH shift during the in vivo hydroxylation of 3-methylacetanilide-4-2H. The effect of induction on the retention of tritium or deuterium is unique to 3-methylacetanilide (4-2H, or 4-3H), and is not observed during hydroxylation of 3'-Me-DAB (4'-2H, or 4'-3H) in which the tritium retention is also independent on the pH of incubation media and the species.

A simple method for R banding of human chromosomes, showing a pH-dependent connection between R and G bands.

Incubation in an unbuffered solution of 1 M NaH2PO4 at 88°C (pH 4.0–4.5) causes R banding to appear on human chromosomes after subsequent staining in Giemsa dissolved in distilled water. G banding, results when the pH of the incubation medium is adjusted towards neutral.

The inhibitory effect of phenothiazine stains on rat and guinea-pig mast cell damage induced by antihistamines and compound 48-80.

1. Toluidine blue inhibits rat mesentery mast cell disruption induced by compound 48/80 and by the antihistamine drugs chlorcyclizine and diphenhydramine. 2. Glucose reverses the blocking action of toluidine blue. Calcium does not. 3. In the guinea-pig mesentery, mast cell damage induced by promethazine is inhibited by toluidine blue. Glucose does not reverse this blocking action. Damage by compound 48/80 is not inhibited. 4. Two other phenothiazine stains—methylene blue and thionin—also inhibit rat mesentery mast cell damage by compound 48/80. For its inhibiting action thionin requires a much lower concentration than the other two dyes. 5. The action of methylene blue and thionin on rat mast cells is influenced by the pH of the incubation medium. Much higher concentrations of these stains are needed to protect mast cells from the action of compound 48/80 when the pH is lowered from 8.4 to 7.0. Toluidine blue, however, is not influenced by pH changes. 6. “In vitro” staining of mast cells with methylene blue and thionin is also influenced by pH. At 8.4 the cells are well stained, while at 7.0 they are barely stained at all. 7. It is concluded that phenothiazine stains block mast cell damage by inhibiting cell metabolism. Therefore, besides its lytic action, antihistamines must also act through an energy-requiring process similar to that of compound 48/80. In the guinea-pig, promethazine damages mast cells through a mechanism different from that of compound 48/80, although both are non energy-requiring. Either in the rat or in the guinea-pig the phenothiazine stains do not seem to inhibit the action of mast cell damaging agents by blocking their access to the active cell sites.

Stimulation of renal gluconeogenesis in methylacetamide diabetes

Kidney cortex slices from rats poisoned with the diabetogenic drug N-monomethylacetamide (NMMAA) produced substantially more glucose from exogenous α-ketoglutarate (α-KG) than did slices from controls. The stimulation of gluconeogenesis was not due to enhancement of renal phosphoenolpyruvate carboxykinase activity, which was found to be similar in experimental and control rats. Despite acceleration of α-KG conversion to glucose, gluconeogenesis from glutamine was not stimulated. The gluconeogenic flux from α-KG, thus, was not rate-limiting for glutamine conversion into glucose. However, when the pH of incubation medium was lowered from 7.5 to 7.2, the rate of gluconeogenesis from glutamine increased together with that from α-KG in slices from both normal and poisoned rats. The data demonstrate major difference in the mechanism of stimulation of renal gluconeogenesis by NMMAA poisoning and by acidosis.

17α-HYDROXYSTEROID-OXIDOREDUCTASE ACTIVITY IN THE HUMAN FOETAL LIVER

Microsome and 105 000 × g supernatant preparations (in 0.25 M sucrose solution) from liver tissue obtained from 19-20 week old male foetuses have been incubated with 4-androstene-3,17-dione for 60 min at 37°C using an incubation medium (pH 7.5) supplemented with an NADPH-regenerating system (NADP-isocitrate-isocitrate dehydrogenase).

Fluoride inhibition of enolase activity in vivo and its relationship to the inhibition of glucose-6-P formation in Streptococcus salivarius

The intracellular concentrations of glucose-6-P, 2-P-glycerate, P-enolpyruvate, and pyruvate were determined in cells of Streptococcus salivarius metabolizing glucose in the presence and absence of NaF in an attempt to elucidate the mechanism of action of this inhibitor. The addition of 2.4 mm NaF to metabolizing whole cells resulted in a cessation of glucose uptake and an immediate decline in the cellular glucose-6-P content. A comparison of the 2-P-glycerate and P-enolpyruvate levels in treated and untreated cells indicated that fluoride had also inhibited intracellular enolase (EC 4.2.1.11) activity. These two apparently separate fluoride effects could not be separated regardless of the pH of the incubation medium (pH 7.2, 8.0, and 5.8), or the concentration of NaF added to the cells (final concentration, 2.4, 0.36, and 0.12 mm). The addition of 2.4 mm NaF to cells metabolizing intracellular glycogen at pH 7.2 also resulted in the inhibition of enolase activity; however, in this case, inhibition of cellular glucose-6-P synthesis was not observed. These results indicated that fluoride was directly involved in the inhibition of glucose-6-P formation from exogenous glucose. The possible relationship between the two fluoride-sensitive sites was made clear by the demonstration that the P-enolpyruvate-phosphotransferase system was involved in the transport of glucose into cells of S. salivarius. The evidence strongly suggests that the fluoride inhibition of enolase results in the inhibition of glucose transport by reducing the amount of P-enolpyruvate available for phosphorylation.