Characterization of a vanadate-based transition-state-analogue complex of phosphoglucomutase by kinetic and equilibrium binding studies. Mechanistic implications

The inhibitor complex produced by the binding of alpha-D-glucose 1-phosphate 6-vanadate to the dephospho form of muscle phosphoglucomutase exhibits an unusually small dissociation constant: about 15 fM for the Mg2+ enzyme at pH 7.4, when calculated in terms of the tetraanion. Such tight binding suggests that the enzyme/vanadate/glucose phosphate complex mimics a state that at least approaches the transition state for (PO3-) transfer in the normal enzymic reaction. This hypothesis also is supported by the observation that replacement of Mg2+, the normal metal ion activator, by Li+, a poor activator, substantially reduces the binding constant for the glucose phosphate/vanadate mixed diester. Other indicators that support this hypothesis are described. One is the derived equilibrium constant for replacement of a PO4(2-) group in bound glucose bisphosphate by VO4(2-): 3 x 10(6) when the replaced group is the phosphate at the (PO3-) transfer site of the Mg2+ enzyme--in contrast to about 10 for the same replacement (of PO4(2-) by VO4(2-)) in an aqueous solution of a phosphate ester. Another is the greatly decreased rate at which Mg2+ dissociates from the glucose phosphate/vanadate complex of the enzyme, relative to the rate at which it dissociates from the corresponding bisphosphate complex (rate ratio less than or equal to 3 x 10(-4)), presumably because Mg2+ binds more tightly to the glucose phosphate/vanadate complex than to the corresponding bisphosphate complex. This apparent increase in Mg2+ binding occurs in spite of what appears to be a reduced charge density at the bound vanadate grouping, relative to the bound phosphate grouping, and in spite of the somewhat weaker binding of Mg2+ by dianionic vanadate than by the phosphate dianion. Although a direct assessment of the binding constant for Mg2+ was not possible, the equilibrium constant for Mg2+/Li+ exchange could be evaluated for the complexes of dephospho enzyme with glucose bisphosphate or glucose 1-phosphate 6-vanadate. The results suggest that the glucose phosphate/vanadate complex of the Mg2+ enzyme mimics a state about halfway between the ground state and the transition state for (PO3-) transfer. This estimate also is in accord with the binding of glucose phosphate/vanadate relative to that expected for transition-state binding of glucose bisphosphate. A possible scenario for the (PO3-) transfer catalyzed by the Mg2+ form of phosphoglucomutase is discussed, on the basis of these observations, together with possible reasons why the bound vanadate group appears to mimic an intermediate state for (PO3-) transfer rather than the ground state for phosphate binding.     

Different sensitivity of cross-bridges in the muscle fiber sarcomere in Ca2+-activation

The investigation of transient kinetics of force development by glycerinated rabbit m. psoas fibres at consecutive pCa stepwise drops in the presence of 0.1 mM Na3VO4 was carried out. Stationary content of trapped vanadate in the fibres at low levels of Ca2+-activation was deduced to be attained slowly as compared to the initial rate of force development and vanadate trapping. This result is considered an indication of the fact, that not all cross-bridges can be activated by turns at low levels of Ca2+-activation.     

Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.     

Is the change in intracellular pH during fatigue large enough to be the main cause of fatigue?

The intracellular pH of frog sartorius muscles exposed to an extracellular pH 8.0 (25 mM HCO3-, 1% CO2) was 6.9-7.1. Following a fatiguing stimulation period (one tetanic contraction per second for 3 min), the intracellular pH was 6.5-6.7. When similar experiments were repeated with frog sartorius muscles exposed to pH 6.4 (2mM HCO3-, 1% CO2), the intracellular pH was 6.8-6.9 at rest and 6.3-6.4 following fatigue. So, in both experiments the intracellular pH decreased by 0.4-0.5 pH unit during fatigue. When the CO2 concentration of the bathing solution was increased from 1 to 30%, the intracellular pH of resting muscles decreased from 7.0 to 6.2-6.3. Although the effect of CO2 on the intracellular pH was greater than the fatigue effect, the decrease in tetanic force with CO2 was less than 40%, while during fatigue the tetanic force decreased by at least 70%. Therefore in frog sartorius muscle the decrease in tetanic force during fatigue exceeds the decrease that is expected from just a change in intracellular pH.     

The uptake and fate of vanadyl ion in ascidian blood cells and a detailed hypothesis for the mechanism and location of biological vanadium reduction. A visible and X-ray absorption spectroscopic study

Vanadium K-edge X-ray absorption spectroscopy (XAS) has been used to track the uptake and fate of VO(2+) ion in blood cells from Ascidia ceratodes, following exposure to dithiothreitol (DTT) or to DTT plus VO(2+). The full range of endogenous vanadium was queried by fitting the XAS of blood cells with the XAS spectra of model vanadium complexes. In cells exposed only to DTT, approximately 0.4% of a new V(III) species was found in a site similar to Na[V(edta)(H(2)O)]. With exposure to DTT and VO(2+), average intracellular [VO(aq)](2+) increased from 3% to 5%, and 6% of a new complexed form of vanadyl ion appeared evidencing a ligand array similar to [VO(edta)](2-). At the same time, the relative ratio of blood cell [V(H(2)O)(6)](3+) increased at the expense of [V(H(2)O)(5)(SO(4))](+) in a manner consistent with a significant increase in endogenous acidity. In new UV/Visible experiments, VO(2+) could be reduced to 7-coordinate [V(nta)(H(2)O)(3)] or [V(nta)(ida)](2-) with cysteine methyl ester in pH 6.5 solution. Ascorbate reduced [VO(edta)](2-) to 7-coordinate [V(edta)(H(2)O)](-), while [VO(trdta)](2-) was unreactive. These results corroborate the finding that the reductive EMF of VO(2+) is increased by the availability of a 7-coordinate V(III) product. Finally, a new and complete hypothesis is proposed for an ascidian vanadate reductase. The structure of the enzyme active site, the vanadate-vanadyl-vanadic reduction mechanism, the cellular locale, and elements of the regulatory machinery governing the biological reduction of vanadate and vanadyl ion by ascidians are all predicted. Together these constitute the new field of vanadium redox enzymology.     

(Model) studies on vanadate-dependent bromo/iodoperoxidase from Ascophyllum nodosum. VO2+ is not incorporated into the active site.

Vanadate-dependent peroxidase A.n.I, the main isoenzyme (M(r) = 100 kDa) from the seaweed, Ascophyllum nodosum, contains 2 V per enzyme molecule (as shown by ICP-MS metal analysis) after complete reconstitution with vanadate (V), possibly distributed in a 1:1 ratio between the surface and active site. VO2+ is only weakly associated to the surface of A.n.I. There is no transport channel for VO2+. The EPR spectrum of the reduced holoenzyme is anisotropic (axial) already at room temperature, with EPR parameters similar to those of VO2+ complexes of small model peptides such as Ala-His, Gly-Tyr, Gly-Ser, Gly-Glu, Ser-Gly and Phe-Glu. The complex formation between Ala-His and H2VO4- in water has also been investigated (by 51V NMR); the formation constant at pH 7.2 amounts to 266(28) M-1.     

Cofactor and substrate binding to vanadium chloroperoxidase determined by UV-VIS spectroscopy and evidence for high affinity for pervanadate

The vanadate cofactor in vanadium chloroperoxidase has been studied using UV-VIS absorption spectroscopy. A band is present in the near-UV that is red-shifted as compared to free vanadate and shifts in both position and intensity upon change in pH. Mutation of vanadate binding residues has a clear effect on the spectrum. Substrate-induced spectral effects allow direct measurement of separate kinetics steps for the first time for vanadium haloperoxidases. A peroxo intermediate is formed upon addition of H(2)O(2), which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stable at pH 5.0, which is the optimal pH for activity. Upon addition of halides to the peroxo form, the native spectrum is re-formed as a result of halide oxidation. Stopped-flow experiments show that H(2)O(2) binding and Cl(-) oxidation occur on the millisecond to second time scale. These data suggest that the oxidation of Cl(-) to HOCl occurs in at least two steps. In the presence of H(2)O(2), the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H(2)O(2). This is attributed to the uptake of pervanadate by the apo-enzyme. Human glucose-6-phosphatase, which is evolutionarily related to vanadium chloroperoxidase, is also likely to have a higher affinity for pervanadate than vanadate. This could explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.     

Interaction between polyhydroxy compounds and vanadate ions: electrophoresis and composition of complexes

Several polyhydroxy compounds form anionic complexes with vanadate ions. The V/polyol ratio in several complexes has been determined. In the pH range 5–9.5, the complexing agent is likely to be a metavanadate ion, (VO)_n^n-. Electrophoresis in sodium metavanadate solution is particularly useful for the separation of hexitols, hex-2-uloses, and several disaccharides.     

Effect of pH on the metabolism and fertility of turkey spermatozoa

Oxygen uptake during a 4-h incubation period at 37 degrees C, and the motility of the spermatozoa before and after incubation, increased significantly with increasing pH from 6.3 to 8.8. No interaction between buffer and pH was noticed. In a second series of experiments on the aerobic metabolism of turkey spermatozoa, the effect of the pHs 6.8, 7.3 and 7.8 was studied. Fructose was formed from glucose without regard to the pH of the medium. The glucose consumption, i.e. the glucose disappearance minus fructose formation, the lactic acid accumulation, and the oxidation of glucose and of other substances, were higher, although not always statistically, at pH 7.8 than at pH 6.8. The percentage of fertile eggs during the 3rd week of collection after insemination with fresh semen diluted in the pH 7.8 medium was significantly lower than that with semen diluted in the pH 6.8 or 7.3 media. After 4 h of storage at 15 degrees C, the decrease in the fertility of spermatozoa in the high pH medium was apparent from the 1st week of collection.     

Vanadate and phosphate ions reduce tension and increase cross-bridge kinetics in chemically skinned heart muscle

Tension development, immediate stiffness and ATPase of chemically skinned myocardial strips were measured in solutions with varying concentrations of phosphate (Pi) or vanadate (predominantly H2VO4 at pH 7) ion. Vanadate and Pi decreased stiffness in proportion to tension. The results show that, like Pi, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the Pi effect. Both Pi and vanadate increased the energy cost of isometric tension maintenance (that is, the ratio of ATPase to tension) and increased the velocity of delayed tension development following quick stretch of the chemically skinned myocardial strips. The results also show that changes in the rate of rise of delayed tension during stretch activation probably reflect changes in the kinetics of the biochemical cycle of the cross-bridges.     

Interaction of RNase A with VO3- and VO2+ ions. Metal ion binding mode and protein secondary structure

Some of vanadyl complexes have shown potential to inhibit RNase activity by acting as transition state analogue, while at the same time not inhibiting DNase. To gain an insight into the interaction of protein with vanadate (VO3-) and vanadyl (VO2+) ions, the present study was designed to examine the binding of ribonuclase A (RNase A) with NaVO3 and VOSO4 in aqueous solution at physiological pH with metal ion concentrations of 0.001 mM to 1 mM, and protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second derivative resolution enhancement were used to determine the cation binding mode, association constant and the protein secondary structure in the presence of vanadate and vanadyl ions in aqueous solution. Spectroscopic results show that an indirect metal ion interaction occurs with the polypeptide C = O, C-N (via H2O) with overall binding constants of K(VO3-) = 3.93x10(2) M(-1) and K(VO2+) = 4.20x10(3) M(-1). At high metal ion concentrations, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 23-24%; beta-sheet (pleated and anti) 50% (free enzyme) to 64-66% and turn 21% (free enzyme) to 10-12% in the metal-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of high metal ion concentration.     

Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.     

Activation of serine sulphydrase from baker's yeast (Saccharomyces cerevisiae) by vanadate

Vanadate stimulates the liberation of H2S from cysteine in intact cells of baker's yeast (Saccharomyces cerevisiae) with a maximal increase of 60% at 10 microM NH4VO3. Protein separation from crude yeast extract yielded two active protein fractions which were found to catalyze the degradation of cysteine to H2S, pyruvate and ammonia or H2S and serine, respectively, thus characterizing them as cysteine desulphydrase and serine sulphydrase. Only the latter enzyme was found to be activated by vanadate, showing optimal enhancement of about 100% at 10 microM NH4VO3.     

Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment.

We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).     

Inhibition of (Na,K)-ATPase by tetravalent vanadium

Vanadyl, the tetravalent state of vanadium and a divalent cation, VO2+, was a relatively powerful inhibitor of highly purified membrane-bound sodium and potassium ion transport adenosine triphosphatase. The sensitivity of the ATPase activity to vanadyl characteristically correlated positively with the specific activity of the enzyme preparation. Inhibition ranged from nearly complete inhibition at less than 5 microM vanadyl for some of the purest fractions (specific activity approximately 45 mumol/min/mg of protein) to no observable inhibition at 300 microM vanadyl in one crude preparation of the enzyme with a specific activity of 10 mumol/min/mg of protein. The level of free vanadyl was reduced by incubation with these membranes, but this reduction was not sufficient to account for the low sensitivity to vanadyl observed in crude preparations. A reduction in specific activity by partial inactivation of a sensitive preparation by treatment with FeCl3 and ascorbate reduced its sensitivity to vanadyl. Anionic ligands of the enzyme, vanadate or ATP, increased the rate of recovery from inhibition after chelation of free vanadyl. At pH 6.1, the inhibition was characteristically fully reversible (t1/2 approximately 10 min), whereas at pH 8.1 it was stable for hours. The degree and stability of enzyme inhibition by vanadyl increased for several hours during incubation of the vanadyl-enzyme mixture, and at pH 6.1 the properties of the inhibitor itself also changed with time. Preincubation of the ion at that pH for 5 h before addition of the enzyme produced a more stable inhibition. The time- and pH-dependent changes in the degree and stability of enzyme inhibition probably relate to the complex chemistry of the vanadyl ion in solution.     

Different metal-binding properties of the two sites of human transferrin.

Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.     

Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.     

Effects of pH on the length-dependent twitch potentiation in skeletal muscle.

When muscle is elongated, there is a length dependence of twitch potentiation and an increased Ca(2+) sensitivity of the myofilaments. Changes in the charge potential of myofilaments, induced by a decrease in pH, are known to abolish the length dependence of Ca(2+) sensitivity. This study was aimed at testing the hypothesis that a decrease in pH, and the concomitant loss of length dependence of Ca(2+) sensitivity, depresses the length dependence of staircase potentiation. In vitro, isometric twitch contractions of fiber bundles dissected from the mouse extensor digitorum longus, performed before and after 10 s of 10-Hz stimulation (i.e., the staircase potentiation protocol) were analyzed at five different lengths, ranging from optimal length for maximal force production (L(o); = 12 +/- 0.7 mm) to L(o) + 1.2 mm (L(o) + 10%). These measurements were made at an extracellular pH of 6.6, 7.4, and 7.8 (pH changes induced by altering the CO(2) concentration of the bath solution). At pH 7.4 and 7.8, the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased fiber bundle length (r(2) = 0.95 and r(2) = 0.99, respectively). At pH 6.6, the length dependence of potentiation was abolished, and the slope of the length-potentiation relationship was not different from zero (r(2) = 0.05). The results of this study indicate that length dependence of potentiation in intact skeletal muscle is abolished by lowering the pH. Because decreasing the pH decreases Ca(2+) sensitivity and changes the charge potential of the filaments, the mechanism of length-dependent potentiation may be closely related to the length dependence of Ca(2+) sensitivity, and changes in the charge potential of the myofilaments may be important in regulating this relationship.     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.     

Low Temperature-Induced Cytoplasmic Acidosis in Cultured Mung Bean (Vigna radiata [L.] Wilczek) Cells

Cold-induced changes in vivo in the cytoplasmic pH of suspension-cultured cells of mung bean (Vigna radiata [L.] Wilczek) were investigated by fluorescence-ratio imaging cryomicroscopy with special reference to the variations in the chilling sensitivity of cells during the growth cycle. Because of the preferential localization of the fluorophore in the cytoplasm under specified conditions and the ideal response of fluorescence to pH, fluorescein diacetate allows measurements to be made of temporal changes in cytoplasmic pH at low temperature. A remarkable difference was demonstrated in the cold-induced changes in cytoplasmic pH between cells at the early and late stages of exponential growth. The cells at the early stage of exponential growth were most sensitive to chilling, and the cytoplasmic pH decreased dramatically within a short period of incubation at 0[deg]C, decreasing from 7.4 to 6.8 after 4 h and to 6.3 after 18 h. The cells at the late stage of exponential growth were chilling tolerant, and no significant decrease in the cytoplasmic pH was observed during the incubation at 0[deg]C for 24 h or even longer. From the results presented here, it appears that cold-induced cytoplasmic acidosis is characteristic of chilling-sensitive mung bean suspension-cultured cells.