Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo.In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of the binding for thiamine was 29 nM which is about six fold lower than the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and $\text{O-}\text{benzoylthiamine}$ disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R₂) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 0.17μM at optimal pH 5.0 which is almost the same with the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and $\text{O-}\text{benzoylthiamine}$ disulfide. In addition, it was found that membrane fraction prepared frtom PT-R₂ has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae.These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.     

Thiamine transport in thiamine-deficient rats role of the unstirred water layer

As part or a systematic study of alcoholism and thiamine absorption, the effect of diet-induced thiamine deficiency and the role of the unstirred water layer on thiamine transport were investigated. Using ³H-labeled dextran as a marker of adherent mucosal volume, jejunal uptake of ¹⁴C-labeled thiamine hydrochloride was measured, in vitro, in thiamine-deficient rats and pair-fed controls. Uptake of low thiamine concentrations (0.2 and 0.5 μM) was greater in the thiamine-deficient rats thatn in the controls. In contrast, uptake rates for high thiamine concentrations (20 and 50 μM) were similar in both groups. While $\text{1J}{}_{\max }$ was unaltered, $\text{1K}{}_{\mathrm{m}}$ was decreased in thiamine deficiency, suggesting a decrease in unstirred water layer thickness. Accordingly, the thickness of the water layer was measured in both groups of animals and correlated with $\text{1J}{}_{\max }$ and $\text{1K}{}_{\mathrm{m}}$ under unstirred and st irred conditions. Without stirring, there was no difference in $\text{1J}{}_{\max }$ between the two groups. In contrast, both $\text{1K}{}_{\mathrm{m}}$ and the water layer were reduced in the thiamine-deficient rats. With stirring, $\text{1J}{}_{\max }$ was not affected, but both $\text{1K}{}_{\mathrm{m}}$ and the water layer thickness were reduced to similar values in both groups. Reversal of thiamine deficiency resulted in the return of thiamine uptake and the unstirred water layer thickness to control values. These data support the concept of a dual system of thiamine transport and emphasize the role of the unstirred water layer as an important determinant of transport kinetics not only under physiologic situations but also in diet-induced rat thiamine deficiency, a model for a clinical pathological state. The decrease in the unstirred water layer thickness in thiamine deficiency may be also viewed as a possible adaptive mechanism to facilitate absorption of meager supplies of thiamine.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Sucrose transport by Streptococcus mutans. Evidence for multiple transport systems

The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are $\text{phospho}\text{enol}\text{pyruvate-dependent}$ phosphotransferases with relatively low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. For strain 6715-13 mutant 33, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 6.25·10^?5 M, 2.4·10^?4 M, and 3.0·10^?3 M, respectively; for strain NCTC-10449, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 7.1·10^?5 M, 2.5·10^?4 M and 3.3·10^?3 M, respectively. The two lower $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) systems, but still has the lowest affinity (highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress $\text{phospho}\text{enol}\text{pyruvate-dependent}$ sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth.     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.     

Inhibition of anion transport in human red blood cells by 5,5′-dithiobis(2-nitrobenzoic acid)

The uptake of [³²P]phosphate into human red blood cells was inhibited ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.6}\text{mM}$) by the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7}\text{mM}$). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[¹⁴C]-benzamido-4′-aminostilbene-2,2′-disulfonate, a potent inhibitor of anion transport ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1?2 μ}\text{M}$), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.     

An allosteric pore model for sugar transport in human erythrocytes

Glucose transport in human erythrocytes is characterized by a marked asymmetry in the $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for entry and for exit. In addition, they show a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and a high $\text{V}$ for equilibrium exchange but low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and high $\text{V}$ transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Transport of [14C]Gly-Pro in a proline peptidase mutant of Salmonella typhimurium

The transport of [¹⁴C]Gly-Pro was examined using a mutant of Salmonella typhimurium (strain TN87) deficient in an X-Pro dipeptidase and an X-Pro-Y iminopeptidase. The dipeptide was taken up by one saturable transport system having a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of $\text{5.3 · 10}{}^{\mathrm{?7}}\text{M}$ and a $\text{V}$ of 1.4 nmol/mg dry wt cell per min. The uptake of Gly-Pro was not inhibited by amino acids or tripeptides and the transport system exhibited a rather broad side chain specificity for dipeptides. Dipeptides containing hydrophobic residues were the most potent inhibitors of this dipeptide transport system exhibiting $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values between 10^?8 and 10^?7 M. In contrast, dipeptides containing glycine residues were particularly weak inhibitors. Finally, Gly-Pro was found to be in the intact form inside the cell and was concentrated more than 1000-fold.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.     

Phosphate transport in Neurospora: Kinetic characterization of a constitutive,low-affinity transport system

Log-phase cells of Neurospora crassa, grown in standard minimal medium, possess an energy-dependent transport system for inorganic phosphate, with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ (at pH 5.8) of 0.123 mM and a J_max of 1.64 mmoles/l cell water per min. Like the PO₄^3? transport system in yeast, the Neurospora system is stimulated by high intracellular K⁺. In addition, it is inhibited by high extracellular salt concentrations, an effect which may be related to the known depolarization of the Neurospora plasma membrane at high salt concentrations.The most striking property of the system is its strong dependence upon the extracellular pH. From pH 4.0 to pH 7.3, the J_max remains essentially constant but the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increases nearly 400-fold, from 0.01 to 3.62 mM. The increase cannot be accounted for by a single system with a preference for H₂PO₄^? (which would show only a 3-fold increase in apparent $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ over this pH range) nor by two systems with different affinities and pH optima (which would display nonlinear double-reciprocal plots at intermediate pH values). It can be explained, however, by a model in which OH^? or H⁺ is assumed to act as a modifier of the transport system, altering its affinity for substrate.     

Effects of 5-hydroxylysine on acetylene reduction and NH4+-assimilation in the cyanobacterium Anabaena cylindrica.

5-hydroxylysine, an analogue of glutamate and lysine, causes $\text{NH}{}_4{}^{\mathrm{+}}$ production by N₂-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of $\text{NH}{}_4{}^{\mathrm{+}}$ on heterocyst production and C₂H₂ reduction and in $\text{NH}{}_4{}^{\mathrm{+}}\text{-grown}$ cultures results in heterocyst synthesis and in C₂H₂ reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary $\text{NH}{}_4{}^{\mathrm{+}}$ assimilation in A. cylindrica, that $\text{NH}{}_4{}^{\mathrm{+}}$ alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

Sulphate transport by rat ileum. Effect of molybdate and other anions

Kinetic constants for SO₄^2? transport by upper and lower rat ileum in vitro have been determined by computer fitting of rate vs concentration data obtained using the everted sac technique. MoO₄^2? inhibition of this transport is competitive, and kinetic constants for the inhibition were similarly determined. Transport is also inhibited by the anions WO₄^2?, S₂O₃^2? and SeO₄^2?, in the order $\text{S}{}_2\text{O}{}_3{}^{\mathrm{2?}}\text{> SeO}{}_4{}^{\mathrm{2?}}\text{≧ MoO}{}_4{}^{\mathrm{2?}}\text{> WO}{}_4{}^{\mathrm{2?}}$. These anions have no effect on the transport of l-valine. Low SO₄^2? transport rates were observed in sacs from animals fed a high-molybdenum diet. The significance of the results with respect to the problem of molybdate toxicity in animals is discussed, and related to the known protective effect of SO₄^2?.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

A comparison of the effects of ouabain and 2-deoxy-d-glucose on the thermodynamic variables of the frog skin

Previous studies support the validity of a linear thermodynamic formalism relating the rates of active Na⁺ transport and oxygen consumption J_r to the electrical potential difference ΔΨ an the affinity α (negative free energy) of the metabolic driving reaction. The formulation was further tested in paired control and experimental hemiskins by the use of two inhibitors of Na⁺ transport. Ouabain, a specific inhibitor of the Na⁺ pump, might be expected to diminish the dependence of J_r on ΔΨ without affecting α, whereas 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, should be expected to diminish α. Both inhibitors were used at concentrations adequate to depress Na⁺ transport (i.e. short-circuit current J_o) to some 50°_o of control level. Measurements were made of I_o and $\text{d}\text{J}{}_{\mathrm{<mtext>r</mtext>}}\text{d}\text{(ΔΨ)}$, and the apparent value of the affinity α_app was calculated according to the thermodynamic formulation. Ouabain depressed $\text{d}\text{J}{}_{\mathrm{<mtext>r</mtext>}}\text{d}\text{(ΔΨ)}$ without affecting α_app whereas 2-deoxy-d-glucose depressed α_app without affecting $\text{d}\text{J}{}_{\mathrm{<mtext>r</mtext>}}\text{d}\text{(αΨ)}$. The demonstration of these effects indicates the utility of the formalism.