The absorption of protons with specific amino acids and carbohydrates by yeast

1. Proton uptake in the presence of various amino acids was studied in washed yeast suspensions containing deoxyglucose and antimycin to inhibit energy metabolism. A series of mutant strains of Saccharomyces cerevisiae with defective amino acid permeases was used. The fast absorption of glycine, l-citrulline and l-methionine through the general amino acid permease was associated with the uptake of about 2 extra equivalents of protons per mol of amino acid absorbed, whereas the slower absorption of l-methionine, l-proline and, possibly, l-arginine through their specific permeases was associated with about 1 proton equivalent. l-Canavanine and l-lysine were also absorbed with 1-2 equivalents of protons. 2. A strain of Saccharomyces carlsbergensis behaved similarly with these amino acids. 3. Preparations of the latter yeast grown with maltose subsequently absorbed it with 2-3 equivalents of protons. The accelerated rate of proton uptake increased up to a maximum value with the maltose concentration (K(m)=1.6mm). The uptake of protons was also faster in the presence of alpha-methylglucoside and sucrose, but not in the presence of glucose, galactose or 2-deoxyglucose. All of these compounds except the last could cause acid formation. The uptake of protons induced by maltose, alpha-methylglucoside and sucrose was not observed when the yeast was grown with glucose, although acid was then formed both from sucrose and glucose. 4. A strain of Saccharomyces fragilis that both fermented and formed acid from lactose absorbed extra protons in the presence of lactose. 5. The observations show that protons were co-substrates in the systems transporting the amino acids and certain of the carbohydrates.     

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.     

The stoicheiometry of the absorption of protons with phosphate and L-glutamate by yeasts of the genus Saccharomyces.

1. A study was made of the pH changes occurring when 0.1-4 mumol of glutamate, phosphate and certain phosphate esters was added at about pH 4.8 to washed cell preparations (50 mg dry wt.) of strains of Saccharomyces. The system also contained deoxyglucose and antimycin to inhibit energy metabolism and so prevent proton ejection from the yeast. 2. A strain of Sacc. carlsbergensis was grown in a chemostat with a limiting supply of phosphate in order to enhance the subsequent rate of phosphate transfer into the yeast. These preparations absorbed 0.2 mumol of phosphate with about 3 equiv. of protons/mol of phosphate. The charge balance was maintained by the efflux of 2 equiv. of K-+ from the yeast. 3. Larger amounts of phosphate were absorbed with fewer proton equivalents. 4. Arsenate and phosphate caused similar pH changes. 5. Glucose 6-phosphate, ATP and certain order phosphate esters each initiated a rise in pH, possibly because hydrolytic extracellular enzymes released phosphate that was subsequently absorbed. 6. Four strains of yeast were grown with glutamate as principal source of nitrogen. Each absorbed extra protons in the presence of L-glutamate. 7. One of them, a strain of Sacc. cerevisiae, absorbed 0.2 mumol of glutamate with 3equiv. of protons/mol of glutamate, and in these circumstances 1-2 equiv. of K-+ left the yeast cells. 8. The role of ionic gradients in the transport of these anions is discussed.     

13C nuclear magnetic resonance study of trehalose mobilization in yeast spores.

Using high-resolution 13C nuclear magnetic resonance, we examined the mobilization of endogenous trehalose in suspensions of yeast asci. Sporulation of yeast cells in [1-13C]acetate resulted in incorporation of label into the C-3 and C-4 positions of trehalose within the asci. During germination of these asci with [1-13C]glucose, the consumption of both endogenous trehalose and exogenous glucose were followed simultaneously by 13C nuclear magnetic resonance, as was the formation of glycerol and ethanol, their glycolytic and products. Time courses for carbohydrate consumption indicated that trehalose, although it decreased to 25% of its initial value upon germination, was not preferentially catabolized and did not provide the primary energy supply for germination with glucose. The ratio of trehalose to glucose catabolized was 0.09. Exogenous glucose levels appeared to regulate trehalose mobilization since trehalose was only consumed when sufficiently high levels (more than 2 mM) of glucose were present. Upon glucose depletion newly synthesized [1-13C]trehalose was observed. Nuclear magnetic resonance spectra of extracts confirmed the trehalose peak assignments and showed products of [1-13C]glucose catabolism. In addition by quantitating trehalose consumption and 2-deoxyglucose incorporation in dormant yeast asci, we found that 3.8 +/- 0.l4 molecules of 2-deoxyglucose were incorporated for each trehalose molecule consumed. Trehalose can therefore function as a carbohydrate source for ATP formation during dormancy.     

Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.     

Last Step in the Conversion of Trehalose to Glycogen: A MYCOBACTERIAL ENZYME THAT TRANSFERS MALTOSE FROM MALTOSE 1-PHOSPHATE TO GLYCOGEN

We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [¹⁴C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [¹⁴C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the ¹⁴C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 μmol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named α1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [¹⁴C]maltose-1-P, but not [¹⁴C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.     

The putative electrogenic nitrate-proton symport of the yeast Candida utilis. Comparison with the systems absorbing glucose or lactate.

Strain N.C.Y.C. 193 of Candida utilis was grown aerobically at 30 degrees C with nitrate as limiting nutrient in a chemostat. The washed yeast cells depleted of ATP absorbed up to 5 nmol of nitrate/mg dry wt. of yeast. At pH 4-6, extra protons and nitrate entered the yeast cells together, in a ratio of about 2:1. Charge balance was maintained by an outflow of about 1 equiv. of K+. Nitrate stimulated the uptake of about 1 proton equivalent during glycolysis or aerobic energy metabolism. Studies with 3,3'-dipropylthiadicarbocyanine indicated that the proton-linked absorption of nitrate, amino acids or glucose depolarized the yeast cells. Proton uptake along with lactate led neither to net expulsion of K+ nor to membrane depolarization.     

The concentration of glycine by preparations of the yeast Saccharomyces Carlsbergensis depleted of adenosine triphosphate: Effects of proton gradients and uncoupling agents.

1. At pH 4.5 and 30degreesC, yeast preparations depleted of ATP in the presence of antimycin and deoxyglucose spontaneously lost K+, gaining roughly an equivalent amount of H+. 2. Five proton conductors including azide and 2,4-dinitrophenol accelerated this process, as did [14C]glycine, which was absorbed with two extra equivalents of H+. 3. The rate of glycine uptake at pH 4.5 diminished fourfold when cellular K+ fell by 20%. 4. The distribution of [14C]propionate indicated that the intracellular pH fell from 6.2 to 5.7 when the cellular content of K+ fell by 30%. 5. Glycine uptake from a 5 muM solution was about 400 times faster at pH 4.5 than it was at pH 7.4 with 100mM-KC1 present ostensibly to lower the membrane potential. 6. Yeast preparations containing 2mM-[14C]glycine absorbed a further amount from a 0.1 muM solution at pH 4.5. After about 10 min a net movement of [14C]glycine out of the yeast occurred. The ratio of the cellular [14Ia1glycine concentration to the concentration outside the yeast reached 4 X 10(4) in these assays, whereas at pH 7.4 in the presence of 100mM-KC1 it did not exceed 15 in 3h. Dimitrophenol lowered the accumulation ratio at pH 4.5, apparently by causing proton conduction. 7. The observations are consistent with the notion that glycine uptake is driven by a proton symport mechanism. 8. Possible factors governing the strikingly low rate of glycine efflux as opposed to its optimum rate of influx are discussed.     

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.     

Glycogen synthesis in the fungus Neurospora crassa.

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [14C]glucose from ADP-[14C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose : glycogen glucosyltransferase as compared with those of the already known UDP glucose : glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-14C]glucose or ADP-[14C]glucose released all the radioactivity as maltose after alpha or beta amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[14C]glucose was the substrate but the stimulation was much greater with ADP-[14C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent &on the presence of a 'primer' of the alpha 1 leads to 4 glucan type.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells.

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Strøm, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

The intrinsic as opposed to the apparent stoichiometry of the glycine-proton symport of the yeast Saccharomyces carlsbergensis.

1. Various ways of computing the proton stoichiometry of glycine absorption were examined in relation to the problem of distinguishing the proton flow (i) through the symport from the basal proton flow (ii) outside it. By depolarizing the plasma membrane, i will tend to inhibit ii. 2. A series of 23 yeast (Saccharomyces carlsbergensis) preparations grown with proline or glutamate were used, some of which were starved in the presence of glucose. Consequently, after ATP depletion, the rate of glycine uptake from a 0.2 mM solution varied through the series from 3 to 14 nmol.min-1.mg-1. Basal proton uptake in the absence of glycine was fairly constant at 3-4 nmol.min-1.mg-1. 3. After addition of glycine, the number of extra equivalents of protons entering the yeast with each amino acid equivalent in 30 s was 0.5 at the lowest rate of glycine absorption and 1.8 equivalents at the fastest rate. However, total proton absorption in 30 s increased in direct proportion to the amount of glycine absorbed. The proportionality factor, indicative of the carrier stoichiometry, was 2.25 +/- 0.13 (23) S.E.M. The effective basal proton uptake was negligibly small. 4. Progress of proton and glycine absorption by each yeast preparation in the period up to 180 s fitted the mathematical model described in the preceding paper by Eddy, Hopkins & Johnson [(1988) Biochem. J. 251, 111-114]. The analysis led to two estimates of the constant ratio of the inflow of protons to the inflow of glycine that would apply when the basal proton flow vanished. These further estimates of the carrier stoichiometry were also near 2, being 2.07 +/- 0.24 (6) and 2.22 +/- 0.07 (17).     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis.

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Str?m, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

Interaction of Maltose Transport with the Transport of Glucose and Galactosides

In cells of Escherichia coli possessing both maltose and galactoside permease, fluxes via one permease are independent of the substrate for the other permease. However, both fluxes are partially inhibited by glucose or alpha-methyl glucoside at low concentrations in cells grown on glucose. Neither maltose nor galactosides have an inhibitory effect on glucose permease function. These observations are consistent with the hypothesis that the number of glucose permease systems on the cell surface of such cells is much larger than the number for maltose or galactosides.     

Specific maltose labeling in the reducing glucose moiety.

Radioactive maltose with label in the reducing glucose moiety was prepared using a glucosyltransferase enzyme to catalyze exchange of [6-³H]glucose into unlabeled maltose. The enzyme was isolated from spinach by ammonium sulfate precipitation followed by DEAE column chromatography. A 77% yield of [6-³H]maltose was obtained after a reaction of 100 nmol of maltose with 0.0147 nmol of [6-³H]glucose was catalyzed by the most active column peak. The product was exclusively labeled in the reducing glucose moiety as indicated by the label occurring only in sorbitol following sodium borohydride reduction and sulfuric acid hydrolysis. Between 88.3 and 96.0% of the tritium in the synthesized preparation was present as [6-³H]maltose by Dowex 1-X4 chromatography. This column separates [6-³H]maltose-[U-¹⁴C]maltose mixtures and [6-³H]glucose-[U-¹⁴C]glucose mixtures apparently as a result of an isotope effect.     

Biosynthesis of a C21 steroid conjugate in an insect. The conversion of [14C]cholesterol to 5-[14C]pregnen-3 beta,20 beta-diol glucoside in the tobacco hornworm, Manduca sexta

Following injection into Manduca sexta (L.) female pupae (day 16), [14C]cholesterol was converted to a C21 steroid conjugate, 5-[14C]pregnen-3 beta,20 beta-diol glucoside. The conjugate was isolated from ovaries and eggs and contained three glucose units at least one of which is attached to C-20. The distribution of the other two glucose units remains to be determined. Other than the dealkylation of C-24 alkane or alkene substituents, side-chain cleavage of sterols is uncommon to insects. Here we report the first definitive proof of the biosynthesis of a C21 steroid conjugate from cholesterol in an insect species. The capability of M. sexta to so readily convert cholesterol to a C21 steroid suggests a physiological role for 5-pregnen-3 beta,20 beta-diol in this species.     

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco.

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.