Plasma membrane ATPase of red beet forms a phosphorylated intermediate

When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [γ-³²P] ATP, 40 micromolar MgSO₄ at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: IV. N,N'-Dicyclohexylcarbodiimide Binding and Inhibition of the Plasma Membrane H-ATPase

The molecular weight and isoelectric point of the plasma membrane H⁺-ATPase from red beet storage tissue were determined using N,N′-dicyclohexylcarbodiimide (DCCD) and a H⁺-ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [¹⁴C]-DCCD at 0°C, a single 97,000 dalton protein was visualized on a fluorograph of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [¹⁴C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H⁺-ATPase. An antibody raised against the plasma membrane H⁺-ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H⁺-ATPase to be 6.5.     

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

The GTP-driven component of Ca²⁺ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca²⁺-translocating ATPase and assess its utility as a probe for this transport system. Uptake of ⁴⁵Ca²⁺ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca²⁺ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca²⁺-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of ⁴⁵Ca²⁺-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven ⁴⁵Ca²⁺ uptake represents the capacity of the plasma membrane Ca²⁺-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-³²P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca²⁺-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca²⁺-ATPases present in animal cells.     

Target molecular size of the red beet plasma membrane ATPase

Radiation inactivation of the red beet (Beta vulgaris L.) plasma membrane ATPase was carried out using γ-ray radiation from a ¹³⁷Cs source. Inactivation of vanadate-sensitive ATPase activity by γ-ray radiation followed an exponential decline with increasing total dose, indicating a single target size calculated to have a molecular weight of about 228,000. Since the catalytic subunit of the red beet plasma membrane ATPase has been demonstrated to have a molecular weight of about 100,000 by dodecyl-sulfate gel electrophoresis following ³²P-phosphorylation, it is suggested that the native enzyme may exist, at least, as a dimer of catalytic subunits.     

Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([γ-³²P]ATP and [5′,8-³H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg²⁺-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg²⁺ increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.     

Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate

The chemical nature of the phosphoryl enzyme linkage of the electrogenic proton-translocating ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the plasma membrane of Neurospora has been identified as a mixed anhydride between phosphate and the beta-carboxyl group of an aspartic acid residue in the polypeptide chain. Incubation of isolated Neurospora plasma membrane vesicles containing 32P-labeled ATPase in buffers of increasing pH followed by analysis of the hydrolysis products yielded a pH versus hydrolysis profile characteristic of an acyl phosphate linkage. Reaction of labeled membranes with hydroxylamine at pH 5.3 also released [32P]i from the ATPase. Amino acid analyses of the Na[3H]BH4 reduction products obtained from membranes containing phosphorylated and dephosphorylated ATPase identified [3H]homoserine, the expected reduction product of beta-aspartyl phosphate, as the only additional tritiated reduction product in the samples from phosphorylated membranes. Tritium was not found in alpha-amino-delta-hydroxyvaleric acid, the reduction product of gamma-glutamyl phosphate, nor in proline, the degradation product of alpha-amino-delta-hydroxyvaleric acid. These results indicate that the phosphorylated intermediate of the Neurospora plasma membrane ATPase is a beta-aspartyl phosphate identical with that already known to exist in the Na+:K+- and Ca2+-translocating ATPases of animal cell origin. A common model for the mechanisms of all 3 ion-translocating ATPases is presented.     

Evidence for the formation of a gamma-phosphorylated glutamyl residue in the Escherichia coli acetate kinase reaction

Studies of phosphorylated $\text{Escherichia coli}$ acetate kinase, which has been reduced with [³H]sodium borohydride and subjected to acid hydrolysis, suggest that [³H] α-amino-δ-hydroxyvaleric acid is formed with a 20–25% overall yield. This finding is compatible with the formation of a γ-phosphorylated glutamyl residue upon incubation of enzyme with acetyl-P.     

Altered Na+ and Li+ Homeostasis in Saccharomyces cerevisiae Cells Expressing the Bacterial Cation Antiporter NhaA

The bacterial Na⁺(Li⁺)/H⁺ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na⁺, Li⁺) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H⁺ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na⁺, Li⁺) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H⁺ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase.     

Phosphorylation by Inorganic Phosphate of the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.)

The phosphorylation of plasma membrane proteins from red beet (Beta vulgaris L.) by radioactive inorganic phosphate was studied. Only few proteins were phosphorylated, among them was one polypeptide with an apparent molecular weight of about 100,000. The phosphorylation of this protein was decreased when orthovanadate was present in the reaction mixture, or when the phosphorylated protein was treated with hydroxylamine. These facts suggest that this protein is a transport ATPase which is phosphorylated in a carboxyl group during the catalytic cycle. This protein was identified immunologically as the plasma membrane H⁺-ATPase. The phosphorylation level of this enzyme was enhanced by dimethyl sulfoxide, whereas potassium ions did not have a significant effect on this level unless ATP was present. ATP stimulated the phosphorylation by inorganic phosphate. This stimulation was more apparent in the presence of potassium ions.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.     

N-acetyl-D-galactosaminyl-β-(1→4)-D-galactose: A terminal non-reducing structure in human blood group Sda-active Tamm-Horsfall urinary glycoprotein

Human Sd^a-active Tamm-Horsfall urinary glycoprotein labelled with galactose oxidase and tritiated sodium borohydride was found to contain both galactose and N-acetylgalactosamine as [³H]-labelled terminal non-reducing sugars. Fragmentation of the macromolecule achieved by hydrazinolysis and acid hydrolysis was followed by fractionation of the degradation products by gel filtration, ion exchange and paper chromatography. A major product was a disaccharide which contained unlabelled galactose and [³H]-labelled N-acetylgalactosamine. Sugar analysis, sodium borohydride reduction, methylation analysis and enzymic degradation enabled the structure N-acetyl-D-galactosaminyl-β-(1→4)-D-galactose to be assigned to the disaccharide.     

Role of magnesium in the plasma membrane ATPase of red beet

The phosphorylation technique was used to assess the role of Mg in the red beet (Beta vulgaris L.) plasma membrane ATPase. When an excess of ethylenediaminetetraacetate (Tris salt, pH 6.5) was added to phosphorylation reactions at steady-state, the phosphorylation level declined exponentially and the rate constant for dephosphorylation was similar to that observed when phosphorylation reactions were chased with unlabeled ATP. When KCl was included with the EDTA chase, a 2.4-fold increase in the turnover of the phosphoenzyme was observed. Thus, the formation of the phosphorylated intermediate but not its breakdown requires free Mg to be present. When an excess of unlabeled ATP containing MgSO₄ was added to plasma membranes incubated for 20 seconds with [γ-³²P]ATP in the absence of MgSO₄, a burst of phosphorylation was observed that declined exponentially. The rate constant for this decline was similar to that observed for phosphoenzyme turnover after initial labeling in the presence of MgSO₄. Extrapolation of this kinetic plot to zero time indicated that ATP binding can occur when MgSO₄ is absent. It is proposed that Mg has a specific role in the transphosphorylation reaction of the terminal phosphate group of ATP to the enzyme.     

An evaluation of techniques for labelling the surface proteins of cultured mammalian cells

We have evaluated four techniques for labelling the surface proteins of cultured mammalian cells. The techniques are: (a) the lactoperoxide system; (b) the pyridoxal phosphate-[³H]borohydride system; (c) the [³H]4,4′-diisothiocyano-2,2′-dihydrostilbene disulfonate system and (d) the galactose oxidase-[³H]borohydride system. The subcellular distribution of radiolabel produced by these techniques has been evaluated by authoradiography at the light microscope level and by cellular fractionation. We find that while all four systems label the surface membranes in the majority of the cell population, they also heavily label internal sites in a small subpopulation of nonviable cells. The contribution of the internally labelled cells to further biochemical analysis may represent a severe problem in investigations which rely solely on surface labels for the study of plasma membrane organization     

H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase

Microsomal membranes isolated from red beet (Beta vulgaris L.) storage tissue were found to contain high levels of ionophore-stimulated ATPase activity. The distribution of this ATPase activity on a continuous sucrose gradient showed a low density peak (1.09 grams per cubic centimeter) that was stimulated over 400% by gramicidin and coincided with a peak of NO₃⁻-sensitive ATPase activity. At higher densities (1.16-1.18 grams per cubic centimeter) a shoulder of gramicidin-stimulated ATPase that coincided with a peak of vanadate-sensitive ATPase was apparent. A discontinuous sucrose gradient of 16/26/34/40% sucrose (w/w) was effective in routinely separating the NO₃⁻-sensitive ATPase (16/26% interface) from the vanadate-sensitive ATPase (34/40% interface). Both membrane fractions were shown to catalyze ATP-dependent H⁺ transport, with the transport process showing the same differential sensitivity to NO₃⁻ and vanadate as the ATPase activity.

Characterization of the lower density ATPase (16/26% interface) indicated that it was highly stimulated by gramicidin, inhibited by KNO₃, stimulated by anions (Cl⁻ > Br⁻ > acetate > HCO₃⁻ > SO₄²⁻), and largely insensitive to monovalent cations. These characteristics are very similar to those reported for tonoplast ATPase activity and a tonoplast origin for the low density membrane vesicles was supported by comparison with isolated red beet vacuoles. The membranes isolated from the vacuole preparation were found to possess an ATPase with characteristics identical to those of the low density membrane vesicles, and were shown to have a peak density of 1.09 grams per cubic centimeter. Furthermore, following osmotic lysis the vacuolar membranes apparently resealed and ATP-dependent H⁺ transport could be demonstrated in these vacuole-derived membrane vesicles. This report, thus, strongly supports a tonoplast origin for the low density, anion-sensitive H⁺-ATPase and further indicates the presence of a higher density, vanadate-sensitive, H⁺-ATPase in the red beet microsomal membrane fraction, which is presumably of plasma membrane origin.

     

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding

We have isolated the plasma membrane H⁺−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H⁺-ATPase, thus activating H⁺ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H⁺-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H⁺-ATPase.     

Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP-Pyridoxal: Evidence for an Active-Site Amino Group

UDP-pyridoxal competitively inhibits the Ca²⁺-, cellobiose-activated (1→3)-β-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K_i of 3.8 ± 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V_max of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 ± 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (1→3)-β-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca²⁺ and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1→3)-β-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[³H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca²⁺ and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP-[³H]pyridoxal in the presence of Mg²⁺ and copurifies with (1→3)-β-glucan synthase activity.     

Inhibition and Ultraviolet-Induced Chemical Modification of UDP-Glucose:(1,3)-beta-Glucan (Callose) Synthase by Chlorpromazine : Mechanism of Chlorpromazine Binding to the Plant Plasma Membrane

UDP-glucose:(1,3)-β-glucan (callose) synthase (CS) from storage tissue of red beet (Beta vulgaris L.) was strongly inhibited by the phenothiazine drug chlorpromazine (CPZ). In the absence of ultraviolet irradiation, CPZ was a noncompetitive inhibitor with 50% inhibitory concentration values for plasma membrane and solubilized CS of 100 and 90 μm, respectively. Both the Ca²⁺- and Mg²⁺- stimulated components of CS activity were affected. CPZ inhibition was partially alleviated at saturating levels of Ca²⁺, but not Mg²⁺, suggesting that CPZ interferes with the Ca²⁺-binding site of CS. Binding experiments with [¹⁴C]CPZ, however, showed strong non-specific partitioning of CPZ into the plasma membrane, providing evidence that perturbation of the membrane environment is probably the predominant mode of inhibition. Ultraviolet irradiation at 254 nm markedly enhanced CPZ inhibition, with complete activity loss following exposure to 4 μm CPZ for 2 min. Inhibition followed a pseudo-first order mechanism with at least three CPZ binding sites per CS complex. Under these conditions, [³H]CPZ was covalently incorporated into plasma membrane preparations by a free radical mechanism; however, polypeptide labeling profiles showed labeling to be largely nonspecific, with many polypeptides labeled even at [³H]CPZ levels as low as 1 μm, and with boiled membranes. Although CPZ is one of the most potent known inhibitors of CS, its use as a photolabel will require a homogeneous CS complex or establishment of conditions that protect against the interaction of CPZ with specific binding sites located on various polypeptide components of the CS complex.     

Inhibition of α-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na⁺-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport.The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. ³²P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the ³²P from [γ-³²P]ATP is incorporated into protein phosphomonoester linkages.     

Effects of vanadate on the plasma membrane ATPase of red beet and corn

The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K_i for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K⁺, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K_m and V_max of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats.     

Affinity labeling of yeast mitochondrial adenosine triphosphatase by reduction with [3H]borohydride.

Oligomycin-sensitive adenosine triphosphatase (ATPase) has been purified in large yields from yeast mitochondria by a procedure employing Sepharose 6B chromatography. The nature of the oligomycin binding site in this purified preparation has been studied by an affinity labeling technique in which oligomycin binding to the ATPase complex was followed by reduction of the complex with sodium [³H]borohydride. A major incorporation of label into protein with a molecular weight near 8000 was noted. This incorporation is dependent on the presence of oligomycin, is blocked by dicyclohexylcar-bodiimide, and is altered by mutations conferring oligomycin resistance to the ATPase. The evidence suggests that the low molecular weight proteolipid component of the ATPase complex is the site of oligomycin binding.