Active transport of L-sorbose and 2-deoxy-D-galactose in Saccharomyces fragilis.

Sorbose and 2-deoxy-D-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars. The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-D-glucose and chemiosmotic coupling in the case of sorbose and 2-deoxy-D-galactose. The differences between the two mechanisms are discussed. Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport. The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

Transport-associated phosphorylation of 2-deoxy-d-glucose in Saccharomyces fragilis

2-Deoxy-d-glucose transport and metabolism was studied in Saccharomyces fragilis. Inside the cells four phosphorylated and three non-phosphorylated derivatives were found and identified. Accumulation of phosphorylated 2-deoxyglucose derivatives was balanced by a concomitant decrease of cellular ATP, orthophosphate and polyphosphates.The free sugar was concentrated against a concentration gradient, contradicting facilitated diffusion. Pulse labeling experiments revealed transport-associated phosphorylation.Theoretical considerations and analysis of the effects of iodoacetate showed that an intracellular hexokinase activity was not involved in 2-deoxyglucose phosphorylation, although this sugar is a good substrate for the enzyme in in vitro experiments.     

Renal sodium-d-glucose cotransport system. Involvement of tyrosine residues in sodium-transporter interaction

Using brush-border membrane vesicles isolated from calf kidney cortex the effect of tyrosine-reactive reagents on sodium-dependent d-glucose transport was investigated. Treatment of the membranes for 60 min with NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), N-acetylimidazole or tetranitromethane decreased d-glucose uptake 50, 70 and 40%, respectively. Tracer exchange experiments revealed that the inhibition of transport is due to a direct modification of the sodium-d-glucose cotransport system. The modification by NBD-Cl decreases the apparent V_max of the transport system with respect to its interaction with sodium. In addition, the rate of inactivation of the transport system by NBD-Cl is reduced in the presence of high concentrations of sodium. The results indicate that tyrosine residues play an essential role in sodium-d-glucose cotransport and are probably involved in the binding and/or transport of sodium by the sodium-d-glucose cotransport system.     

The influence of uncouplers on facilitated diffusion of sorbose in Saccharomyces cerevisiae

Sorbose uptake in Saccharomyces cerevisiae, strain Delft 1, proceeds via mediated passive transport. In the cell sorbose is distributed in at least two compartments. Efflux studies showed that sorbose uptake in one of these compartments is not readily reversible. Uncouplers of oxidative phosphorylation inhibit both transport velocity and steady-state uptake level. It could be shown that these two effects are caused by different modes of action of the uncouplers. None of these two effects could be ascribed to changes of the electrochemical H⁺ gradient or of the intracellular pH. It is suggested that the inhibition of uptake velocity is caused by binding of the uncoupler to the sorbose translocator, thus lowering the transport activity. The uncoupler binding site is probably located at the intracellular fragment of the carrier. The second effect, reduction of the steady-state uptake level, is probably due to blocking of sorbose influx into the compartment that exhibits poor reversibility.     

A simple laboratory exercise illustrating active transport in yeast cells

A simple laboratory class illustrating the chemiosmotic principles of active transport in yeast cells is described. The energy coupling mechanism of active α-glucoside uptake by Saccharomyces cerevisiae cells is easily demonstrated with a colorimetric transport assay using very simple equipment. The experiment can stimulate a range of tutorial discussion topics relevant to yeast sugar metabolism and biotechnology.     

Kinetic analysis of simultaneously occurring proton-sorbose symport and passive sorbose transport in Saccharomyces fragilis

Sorbose transport in Saccharomyces fragilis takes place both via an active sugar-H⁺ symport system and via facilitated diffusion.To establish whether the two modes of transport proceed via the same transporter or via two different carriers, the kinetic consequences of both models were investigated. The kinetic equations for initial transport were derived for three possible reaction sequences with respect to sugar and H⁺ binding to the symport carrier: random binding and obligatory ordered binding with either sugar or H⁺ binding first, yielding six sets of kinetic parameters.Analysis of experimental data of sorbose transport in S. fragilis showed the existence of separate carriers for active, sorbose-H⁺ symport and facilitated diffusion. Furthermore, it could be concluded that the symport carrier shows random binding of sugar and H⁺.In recent literature, a similar combination of active and passive sugar transport in Rhodotorula gracilis and Chlorella vulgaris was interpreted as two modes of action of the same carrier, viz., active symport via the protonated, and facilitated diffusion via the unprotonated carrier. Analysis of the experimental data according to the criteria presented in this paper showed, however, that this supposition is untenable and that two different carriers must also be involved in these micro-organisms.     

Kinetics and mechanism of oxidation of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium

Kinetic data for the oxidations of d-fructose and l-sorbose by chromium(VI) and vanadium(V) in perchloric acid medium are reported. The addition of perchloric acid and sodium perchlorate increases the pseudo-first-order rate constants. Change of the reaction medium from water to deuterium oxide appreciably affects the rates of chromium(VI) oxidations, but does not affect those of vanadium(V) oxidations. The activation parameters are ΔH3 = 46.6 ±3.4 (fructose) and 50.6 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?105 ±11 (fructose) and ?100 ±20 (sorbose) J.deg^?1.mol^?1 for chromium(VI) oxidations, and, for the other reactions, ΔH3 = 53.2 ±4.2 (fructose) and 52.3 ±6.3 (sorbose) kJ.mol^?1, and ΔS3 = ?139.0 ±14 (fructose) and ?137 ±20 (sorbose) J.deg^?1.mol^?1. The kinetics of the oxidations of ketohexoses by chromium(VI) indicate no intermediate-complex formation, whereas those for vanadium(V) indicate the formation of a 1:1 intermediate complex between ketohexoses and vanadium(V).     

The influence of uncouplers on proton-sugar symport in Saccharomyces fragilis

Uncouplers of oxidative phosphorylation inhibit proton-sugar symport in Saccharomyces fragilis. However, they do not induce efflux of accumulated sugar. It is shown that the effect cannot be explained by uncoupler-induced alterations in the transmembrane potential or transmembrane pH difference. It is also indicated that a decrease in intracellular pH is not involved in inhibition of sugar transport. It is argued that inhibition of transport by uncouplers is most likely caused by a direct interaction with the translocator.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

Electron diffraction study on single crystals of l-type and dl-type lecithins

An electron diffraction study was carried out on thin single micro-crystals of l-type and dl-type dipalmitoyl lecithins grown in xylene suspensions and fine net patterns were obtained and the mechanism of the thermotropic phase transitions of them was clarified.From the apparent structure of diffraction patterns in low temperature, it is confirmed that the two dimensional lattices have p mm symmetry in l-type and in dl-type lecithins. Lattice parameters from the [001] projection are $\text{d}{}_{100}\text{= 9.9}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.8}\text{A}\text{?}$ in l-type, and $\text{d}{}_{100}\text{= 17.2}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.9}\text{A}\text{?}$ in dl-type.With anisotropic variation of dimensions along a and b axes, i.e. contraction for a and expansion for b, induced by temperature rise by electron irradiation during the observation, these diffraction patterns of the lattices of l-type and dl-type were transformed into those characterized by the six diffraction spots having nearly the same spacings. Four of them are observed on slightly outer and two are slightly inner positions as compared with their mean spacings of about (4.1 Å)^?1 in l-type and about (4.2 Å)^?1 in dl-type. The changes in the patterns observed indicate that at low temperatures the hydrocarbon chains are nearly perpendicular to the layer in dl-type lipid, and tilted with a more complicated packing in l-type ones. The dimension along a in dl-type is twice as large as that in l-type.     

Investigation of transport-associated phosphorylation of sugar in yeast mutants (snf3) lacking high-affinity glucose transport and in a mutant (fdp1) showing deficient regulation of initial sugar metabolism

Pool-labeling experiments with 2-deoxyglucose in derepressed cells of the yeastSaccharomyces cerevisiae confirmed the previously reported results pointing to the possible existence of transport-associated phosphorylation of sugar. In yeast mutants containing a disruption or an inactivating point mutation in thesnf3 gene, which codes for the high-affinity glucose carrier, no evidence for transport-associated phosphorylation of 2-deoxyglucose was observed. If transport-associated phosphorylation in yeast exists, it is apparently not mediated by the low-affinity glucose carrier. Mediation by the high-affinity carrier would fit with the known requirement of an active kinase for high-affinity sugar transport. A mixed type of uptake in cells having both carriers would explain many of the problems associated with the 2-deoxyglucose pool-labeling experiments. Since mutants that have only low-affinity glucose transport are not deficient in the glucose-induced RAS-mediated cAMP signal, transport-associated phosphorylation of glucose is not required for or involved in the induction of the signal. The yeastfdp mutant, which dies on media containing fermentable sugars because of overaccumulation of sugar phosphates, also did not show any evidence for the existence of transport-associated phosphorylation. The same was true for the double mutantfdp snf3. The latter also showed the typicalfdp phenotype, indicative that the lethality on media containing fermentable sugar is owing to aberrant regulation of low-affinity transport. The high protein kinase activity in thefdp mutant does not appear to be responsible for the absence of evidence for transport-associated phosphorylation, because another mutant with high protein kinase activity, thebcy mutant, displayed normal transport behavior.     

Phenylalanine ammonia-lyase: A model for the cooperativity kinetics induced by d- and l-phenylalanine

A preparation of l-phenylalanine ammonia-lyase (EC 4.1.3.5.) from soybean (Glycine max L. cv. Kanrich) showed negative cooperativity with respect to l-phenylalanine and competitive inhibition by d-phenylalanine. A two-protomer partially concerted model for inhibition kinetics is described. If cooperativity is associated with ligand binding but not k_cat, plots of v against log [S] at constant [I] are symmetrical. Such curves may be fitted by graphical or iterative least-squares methods. The experimental results conform to this restricted model. The three-substrate and three-inhibitor dissociation constants were estimated by a stepwise procedure. For substrate only the first and second dissociation constants were 12 and 78 μm, respectively, with a symmetry point value of 30.5 μm. To a first approximation, site occupancy determines the cooperativity. As d- and l-phenylalanine produce equivalent effects, they are assumed to pack into the same induced space. As ligand binding at one site has little influence on the relative d:l binding at the other and does not influence k_cat, cooperativity probably reflects changes in regions remote from the active site such as the interface between the protomers. The regulatory range in [S] of the enzyme in vivo may be indicated by the linearity range of the semilog plot for the isolated enzyme. The observed range corresponds to a 100-fold change in [S] compared to a 10-fold change for Michaelis-Menten kinetics.     

Transport of l-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of l-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris·HCl of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in part buffered saline. l-Methionine is actively transported in WI38 by saturable, chemically specific mechanisms which are temperature, pH and, in part, Na⁺ dependent, and are reactive with both l- and d-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent K_M of 21.7 μM and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent K_M of 547 μM and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na⁺- free media or after treatment with ouabain suggested that system 1 is Na⁺ independent and that system 2 is Na⁺ dependent. Preloading of cells with unlabeled l-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled l- or d-methionine or l-phenylalanine, but not in the presence of l-arginine. l-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

A kinetic analysis of d-xylose transport in rhodotorula glutinis

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for d-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low K_m carrier was repressed in rapidly growing cells and depressed by starvation of the cells.Several hexoses were observed to inhibit d-xylose transport. In the studies reported here, the inhibitions produced by d-galactose and 2-deoxy-d-glucose were examined in some detail in order to define the interactions of these sugars with the d-xylose carriers. 2-Deoxy-d-glucose competitively inhibited both of the d-xylose carriers. In contrast, only the low-K_m carrier was competitively inhibited by d-galactose.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/d-glucose cotransport system

To characterize further the Na⁺/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na⁺/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na⁺/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).     

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.     

Immunochemical studies on L-rhamno-D-mannans of sporothrix schenckii and related fungi by use of rabbit and human antisera

Antisera were prepared in rabbits against the human pathogenic yeast Sporothrix schenckii (strain 1099.12) grown at two different temperatures (25° and 37°). Precipitation and inhibition data showed that the former serum had a specificity directed against α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-D-Man-(1→ determinants, whereas the latter had a broad specificity in which α-L-rhamnosyl or α-L-Rhap-(1→3)-D-Man-was the immunodominant structure. These results are consistent with data on the structures of the L-rhamno-D-mannans isolated from the organism grown at the two different temperatures. Human sera from patients with sporotrichosis were shown to have different specificities resembling the specificities developed in the rabbits. The rabbit antisera were also used to examine the cross-reactivity with L-rhamno-D-mannans from species of the genus Ceratocystis, which is reputed to include the ascigerous (perfect) state of S. schenckii. Polysaccharides from four species of Ceratocystis grown at 25° reacted with the antisera in a manner resembling that of the L-rhamno-D-mannan from S. schenckii grown at 37°. This is in accord with earlier data that showed that only S. schenckii, of the species studied, produces a polysaccharide with large amounts of α-L-Rhap-(1→2)-α-L-Rhap-(1→ side-chains when grown at 25°.     

β-d-xylosidase from Bacillus pumilus prl b12: hydrolysis of aryl β-d-xylopyranosides

The influence of substituents on the binding and hydrolysis of several substituted β-d-xylopyranosides by β-d-xylosidase Bacillus pumilus PRL B12 has been investigated. From a comparison of the inhibition constants of 1-thio-β-d-xylopyranosides with the apparent Michaelis-Menten constants of the substrates, it followed that the latter constants are good approximations of the true equilibrium constants. The influence of the substituent on the rate and activation parameters is small. The results are in agreement with, but do not prove, a one-step mechanism without the formation of a glycosyl-enzyme intermediate.