Ultrastructural Comparison of the Vacuolar and Mitochondrial H+-ATPases of Daucus carota

Eukaryotic vacuolar H⁺-ATPases (V-ATPases) are related to the F₀F₁-ATPases of chloroplasts and mitochondria and are believed to be organized into peripheral and integral membrane complexes. Vacuolar membranes isolated from purified carrot (Daucus carota) root vacuoles were observed to be coated with F₁-like particles after negative staining with phosphotungstic acid. The F₁-like particles formed typical “ball and stalk” structures, about 9.4 nm in diameter and 13.6 nm in height. The head portion frequently had a characteristic bifurcation or cleft at the apex and appeared to be composed of subunits. Such “V₁” complexes were frequently associated with smaller stalked particles emerging near the base. In contrast, negatively-stained carrot mitochondrial F₁ complexes averaged 8.7 nm in diameter and 11.7 nm in height. The head groups of the mitochondrial F₁s were nearly always spherical, and had no other smaller structures associated with them. The V₁ complexes of carrot are thus similar in form to the V₁ complexes of Neurospora (Bowman et al. J. Biol. Chem. 264 (1989) 15606–15612).     

Boron is required for the stimulation of the ferricyanide-induced proton release by auxins in suspension-cultured cells of Daucus carota and Lycopersicon esculentum

Boron deficiency reduces the ferricyanide-induced net proton release of suspension-cultured carrot ( Daucus carota L.) and tomato ( Lycopersicon esculentum Mill.) cells by more than 50%. This effect is reversed within 60 to 90 min by the addition of B. Vanadate (400 μ M ) completely suppresses the proton release, indicating an ATPasedriven process. The differences between B treatments do not appear when auxins are omitted from the experimental solution, but can be observed within less than 30 min after the addition of auxin to auxin-deficient cell cultures. This suggests, that an adequate supply of B is required for the auxin action to take place. The results are discussed with respect to the primary functions of B in membranes and transport processes, and its possible influence on auxin-induced metabolic events.     

Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts

A xyloglucan oligosaccharide was isolated from cell walls of Daucus carota L. suspension-cultured cells. From analytical data (gel-permeation chromatography, thin-layer chromatography, monosaccharide analysis, methylation analysis) it can be concluded that this oligosaccharide preparation consists mainly of a nonasaccharide known as XG9 (Glc₄Xyl₃GalFuc). This nonasaccharide showed excellent “anti-auxin” properties in the pea-stem bioassay, with 80% inhibition of 2,4-dichlorophenoxyacetic acid (2,4-D)-induced longitudinal growth of etiolated pea stem segments at concentrations of 1-0.1 nM. Applied in nanomolar concentrations to protoplasts regenerating in a medium containing 4.52 μM 2,4-D, the nonasaccharide influenced the viability of the protoplasts and the activities of glycan synthases in vitro. The effects were similar to those achieved by the omission of 2,4-D from the regeneration medium. The composition of the regenerated cell wall was not changed significantly by the use of 2,4-D-depleted medium or the addition of XG9 to 2,4-D-containing medium.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Drought stress stimulates p-nitrophenyl phosphate hydrolysis rate of the plasma membrane H+-ATPase from wheat leaves

The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H⁺-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The K_m for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H⁺-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H⁺-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H⁺-ATPase, therefore increasing the catalytic activity of the phosphatase domain.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction.     

In situMechanical Testing of Fully Hydrated Carrots (Daucus carota) in the Environmental SEM

We demonstrate the use of the Environmental Scanning ElectronMicroscope (ESEM) forin situobservation of mechanical testson carrot (Daucus carota)parenchymal tissue. The ESEM toleratesseveral Torr of water vapour in the specimen chamber, thus allowingfully hydrated specimens to be examined at high resolution,but without preparation. Three tests were performed, involvingslicing, tension and compression. The manner in which stresswas distributed in the tissue, crack propagation and cell walldistortion were all observed in real time.Copyright 1998 Annalsof Botany Company. Environmental scanning electron microscopy, carrot,Daucus carota,mechanical testing.     

Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential

An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S- Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.     

Studies on the origin of totipotent cells in explants of Daucus carota L.

In an attempt to identify the origin of cells capable of generatingsomatic embryos, hypocotyl explants from carrot seedlings werecultured in the presence of auxin. The various tissues respondedin different ways. The external layers (epidermis, corticalparenchyma) expanded, whereas the provascular cells dividedand expanded. Hence only the latter cells can generate celllines and somatic embryos. A cyto-histological analysis showedthat pro-embryogenic masses, from which embryos develop in theabsence of auxin, are generated from the same provascular cellswhich, after a proper exposure to auxin, undergo an asymmetricdivision. Key words: Somatic embryogenesis, pro-embryogenic masses, Daucus carota, totipotency     

Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.     

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm^?1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

     

Coincidence of H+ binding and Ca2+ dissociation in the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis

H+ and Ca2+ concentration changes in the reaction medium following MgATP addition at pH 6.0 were determined with the partially purified Ca-ATPase from sarcoplasmic reticulum vesicles in the presence of 25-50 microM CaCl2 and 5 mM MgCl2 at 4 degrees C. Previously, we showed a sequential occurrence of H+ binding and H+ dissociation in the Ca-ATPase during ATP hydrolysis and further suggested that the H+ binding takes place inside the vesicles (Yamaguchi, M., and Kanazawa, T. (1984) J. Biol. Chem. 259, 9526-9531). The present results demonstrate that the H+ binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-insensitive form in the catalytic cycle of ATP hydrolysis. As KCl decreased in the medium, the extent of the H+ binding increased almost proportionately with the extent of either the Ca2+ dissociation or the accumulation of ADP-insensitive EP. Both the H+ binding and the Ca2+ dissociation were prevented by a modification of the specific SH group of the enzyme essential for the conversion of ADP-sensitive EP to ADP-insensitive EP. In the late stage of the reaction, H+ dissociation from the enzyme occurred coincidently with Ca2+ binding to the dephosphoenzyme which was formed by EP decomposition. These results are consistent with the possibility that the H+ ejection during the Ca2+ uptake with the intact vesicles previously shown by several investigators takes place through a Ca2+/H+ exchange directly mediated by the membrane-bound Ca-ATPase.     

Expression of the Daucus carota somatic embryogenesis receptor kinase (DcSERK) protein in insect cells

The Daucus carota somatic embryogenesis receptor kinase (DcSERK) gene serves as marker to monitor the transition from somatic into embryogenic plant cells. To determine the intrinsic biochemical properties of the DcSERK protein, a predicted transmembrane receptor, the kinase domain was expressed as a 40-kDa his-tag fusion protein in the baculovirus insect cell system. The kinase domain fusion protein was able to autophosphorylate in vitro. Phosphoamino acid analysis of the autophosphorylated DcSERK protein revealed that it was autophosphorylated on serine and threonine residues. This is the first evidence of the biochemical characterization of a transmembrane receptor kinase from embryogenic plant cell cultures.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome.

PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.     

Diferric transferrin reduction stimulates the Na+/H+ antiport of HeLa cells

Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.     

Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c' is not essential for assembly but is for functional coupling of the enzyme.     

Kinetic effects of Ca2+ and Mg2+ on ATP hydrolysis by the purified ATP diphosphohydrolase

ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is sensitive to divalent cations. In this paper, we investigated the dependence of ATP hydrolysis on the concentration of free Mg2+ and Ca2+ and the cation ATP complexes. The enzyme was isolated from porcine zymogen granule membranes, solubilized in Triton X-100, and purified on a 5'-AMP-Sepharose 4B affinity column resulting in a 1500-fold purification. Free unprotonated ATP4- was hydrolyzed in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When hydrolysis rate was measured at different concentrations of the cation-ATP complex at constant free cation concentrations, normal hyperbolic curves were obtained. In CaCl2, both Kapp and Vapp increased as free Ca2+ increased from 25 to 1000 microM. In MgCl2, Kapp increased and Vapp decreased as free Mg2+ increased from 25 to 500 microM. From the rapid equilibrium rate equation, Ks and Vmax values of the substrates were calculated. We found that free ATP4-, Ca-ATP2-, and Mg-ATP2- are substrates and free cations do not bind the enzyme.