Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

PQQ and quinoprotein enzymes in microbial oxidations

Abstract Pyrroloquinoline quinone (PQQ) is found in a wide range of microorganisms, and several bacteria even excrete this compound into their culture medium when grown on alcohols. The existence of different classes of quinoprotein (PQQ-containing) enzymes is now well established (alcohol dehydrogenases, aldose (glucose) dehydrogenases, amine dehydrogenases and amine oxidases) while several other enzymes are suspected to be quinoproteins. In addition, many bacteria produce a quinoprotein apoenzyme, e.g., Escherichia coli and Pseudomonas testosteroni , producing glucose and ethanol dehydrogenase apoenzyme, respectively. It is unclear why these bacteria do not produce the holoenzyme form, but the apoenzymes have the ability to become functional, as was shown when the organisms were provided with PQQ. With this approach it could be demonstrated that E. coli has a non-phosphorylative route of glucose dissimilation via gluconate. Also, results with mixed cultures indicate that PQQ is a growth factor for certain bacteria under certain conditions. Despite the relatively high redox potential of the PQQ/PQQH₂ couple, quinoproteins transfer electrons to a variety of natural electron acceptors. Depending on the type of quinoprotein enzyme, the following components of the respiratory chain appear to be active: cytochrome c (sometimes with a copper protein as an intermediate), cytochrome b , and NADH dehydrogenase. PQQ is not restricted to a particular group of organisms, and reactions catalysed by quinoproteins can also be performed by NAD(P)-dependent or flavoprotein enzymes. Thus, these observations do not provide arguments for the view that quinoproteins have a unique role in microbial oxidations. Further comparative studies on oxidoreductases are necessary to reveal the special features of this novel group of enzymes.     

On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein

Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.     

Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae

Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO₂ with H₂ or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F₄₂₀-nonreactive hydrogenase, NADP⁺: F₄₂₀ oxidoreductase, NADP⁺-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F₄₂₀ dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F₄₂₀-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F₄₂₀ to provide electrons for biosynthesis, was formulated.     

Non-coordinated synthesis of glucose dehydrogenase and its prosthetic group PQQ in Acinetobacter and Pseudomonas species

Abstract The activity of pyrrolo-quinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) was determined in Acinetobacter and Pseudomonas species, grown under different conditions. In Acinetobacter lwoffi which, in contrast to Acinetobacter calcoaceticus , is unable to oxidize glucose to gluconic acid, the absence of GDH activity was not due to the absence of GDH protein (apoenzyme) but to the absence of its prosthetic group, PQQ. GDH activity could be restored by addition of PQQ to cell suspensions. Taxonomic implication of these results are discussed. Pseudomonas aeruginosa , strain PAO1 is known to contain active GDH when grown aerobically on glucose, but to lack this activity when grown anaerobically with nitrate. Also in this organism the absence of active GDH was due to lack of PQQ synthesis under these conditions, since GDH activity could be reconstituted by addition of PQQ to cell-free extracts.
Similar observations were made with cultures of Pseudomonas acidovorans and Rhodopseudomonas sphaeroides , indicating that control of GDH activity by PQQ synthesis maybe widespread among bacteria.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

UDP-sugar hydrolase isozymes in Salmonella enterica and Escherichia coli: Silent alleles of ushA in related strains of Group I Salmonella isolates, and of ushB in wild-type and K12 strains of E. coli, indicate recent and early silencing events, respectively

Abstract Escherichia coli contains a single periplasmic UDP-glucose hydrolase (5'-nucleotidase) encoded by ushA. Salmonella enterica , serotype Typhimurium, also contains a single UDP-glucose hydrolase but, in contrast to E. coli , it is membrane-bound and is encoded by the non-homologous ushB gene; Salmonella enterica (Typhimurium) also contains a silent allele of the ushA gene ( ushA⁰ ). In this report, we show that nearly all natural isolates of Salmonella contain both UDP-sugar hydrolases, i.e. they are UshA⁺ UshB⁺. The only exceptions are all from sub-group I ( S. gallinarum, S. pullorum , and most Typhimurium strains), are UshA⁻ UshB⁺, and several have been shown to contain an ushA⁰ allele. These data, together with the fact that these latter strains are closely related genetically, strongly suggests a recent silencing mutation(s). We also report the presence in E. coli K-12, and in natural isolates of E. coli , of a DNA sequence which is homologous to the ushB gene of Salmonella ; since E. coli does not contain UshB activity, we tentatively refer to this sequence as ushB⁰ . Since all E. coli strains investigated are UshB⁻, we conclude that the silencing mutation(s) occured relatively eary following the divergence of Escherichia coli and Salmonella from a common ancestor that was ushA⁺ ushB⁺ .     

The influences of calcium and magnesium ions on the exchange of monovalent cations in Neurospora crassa

Low-K⁺, high-Na⁺ cells of strain RL21a of Neurospora crassa , in steady state with 25 m M Na⁺, were used to study K⁺/Na⁺ exchanges in the presence or absence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺ and Mg²⁺, a low concentration of K⁺ (0.3 m M ) triggered a rapid exchange, but in the absence of the divalents, a high K⁺ concentration (30 m M ) was required to initiate the exchange at a rapid rate. In the absence of Ca²⁺ and Mg²⁺, K⁺ uptake did not occur at low K⁺ concentration, internal K⁺ did not regulate Na⁺ influx in the presence of external K⁺, and the efflux of Na⁺ proceeded at maximum activity at very low-K⁺ contents.     

Calcium and plant organelles

Abstract. The role of intracellular organelles in the regulation of cytosolic Ca²⁺ levels and whether changes in these levels affect organelle metabolism is considered. We have assessed the biochemical properties of the Ca²⁺ transporting systems in mitochondrial, chloroplast and microsomal fractions. It is proposed that although all of these organelles can transport Ca²⁺ to varying extents it would appear that in some tissues at least mitochondria do not play a significant role in the maintenance of cytosolic Ca²⁺. The most important Ca²⁺ transporting systems are probably the ATP dependent Ca²⁺ extrusion across the plasma membrane and Ca²⁺ uptake by endoplasmic reticulum, as well as light driven Ca²⁺ uptake by chloroplasts. Changes in cytoplasmic [Ca²⁺] do appear to regulate the activity of NAD kinase in chloroplasts, the mitochondrial external NADH dehydrogenase and intra-mitochondrial glutamate dehydrogenase, all of which play a key role in plant cell metabolism. Since some of these enzymes are affected by primary stimuli such as light or hormones, it is concluded that Ca²⁺ may act as a second messenger mediating some of the primary responses.     

Early Signals in Serum-Induced Increases in Ouabain-Sensitive Na+-K+ Pump Activity and in Glucose Transport in Rat Skeletal Muscle Are Amiloride-Sensitive

Abstract: The acute effects of serum on sodium-potassium (Na⁺-K⁺) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo-tubes in phosphate-buffered saline caused Na⁺-K⁺ pump activity (as measured by changes in the ouabain-sensitive component of both membrane potential and ⁸⁶Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage-dependent Na⁺ channels. On transfer of myotubes to Na⁺-free, choline buffer, resting Na⁺-K⁺ pump activity decreased to about 10% of that in phosphate-buffered saline. Addition of regular serum, but not Na⁺-free serum, caused Na⁺-K⁺ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na⁺ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na⁺-H⁺ exchange for serum-induced increases in Na⁺-K⁺ pump activity and glucose transport. The lack of complete blockade of serum-induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

The role of PQQ and quinoproteins in methylotrophic bacteria

Abstract Quinoprotein dehydrogenases play a non-exclusive role in the dissimilation of C₁ compounds. Methanol and methylamine oxidation occur by covalent catalysis while the reduction equivalents are transferred to the respiratory chain in one-electron steps. Cytochrome c _L is an excellent electron acceptor for methanol dehydrogenase at pH 7.0 and a bad one at pH 9.0. Efficient methanol oxidation (with NH₃ as activator) occurs at pH 9.0, but (due to the failure of NH₃) not at pH 7.0. Since stimulation occurred at the latter condition with a compound prepared from Hyphomicrobium X, most probably methanol oxidation in vivo requires the presence of a natural activator. The finding of pro-PQQ in methylamine dehydrogenase implicates that certain quinoproteins may have a modified tyrosine as cofactor. This type of quinoprotein is involved in assimilation routes which also occur in methylotrophs. l -Tyrosine and l -glutamate are the precursors of PQQ biosynthesis. Free intermediates in the route of biosynthesis have not been found. Most probably the whole process occurs on a protein matrix. In view of the significant amounts found in their culture fluid, methylotrophic bacteria seem particularly well suited for the fermentative production of PQQ.     

Does the intestinal microflora synthesize pyrroloquinoline quinone?

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and glucose dehydrogenase. When chemically-defined diets without PQQ are fed to animals, lathyritic changes are observed. In previous studies, it was assumed that PQQ was produced by the intestinal microflora; consequently, antibiotics were routinely added to diets. In the present study this assumption is tested further in mice by: (i) examining the effects of dietary antibiotics on fecal PQQ excretion, (ii) isolating the intestinal flora to identify bacteria known to synthesize PQQ and (iii) determining in vitro if the intestinal microflora synthesizes PQQ from radio-chemically labeled precursors. The results of these experiments indicate that little if any PQQ is synthesized by the intestinal microflora. Rather, when PQQ is present in the intestine, the diet is a more obvious source.     

Synthesis of pyrroloquinoline quinone in vivo and in vitro and detection of an intermediate in the biosynthetic pathway.

In Klebsiella pneumoniae, six genes, constituting the pqqABCDEF operon, which are required for the synthesis of the cofactor pyrroloquinoline quinone (PQQ) have been identified. The role of each of these K. pneumoniae Pqq proteins was examined by expression of the cloned pqq genes in Escherichia coli, which cannot synthesize PQQ. All six pqq genes were required for PQQ biosynthesis and excretion into the medium in sufficient amounts to allow growth of E. coli on glucose via the PQQ-dependent glucose dehydrogenase. Mutants lacking the PqqB or PqqF protein synthesized small amounts of PQQ, however. PQQ synthesis was also studied in cell extracts. Extracts made from cells containing all Pqq proteins contained PQQ. Lack of each of the Pqq proteins except PqqB resulted in the absence of PQQ. Extracts lacking PqqB synthesized PQQ slowly. Complementation studies with extracts containing different Pqq proteins showed that an extract lacking PqqC synthesized an intermediate which was also detected in the culture medium of pqqC mutants. It is proposed that PqqC catalyzes the last step in PQQ biosynthesis. Studies with cells lacking PqqB suggest that the same intermediate might be accumulated in these mutants. By using pqq-lacZ protein fusions, it was shown that the expression of the putative precursor of PQQ, the small PqqA polypeptide, was much higher than that of the other Pqq proteins. Synthesis of PQQ most likely requires molecular oxygen, since PQQ was not synthesized under anaerobic conditions, although the pqq genes were expressed.     

Induction of the PhoE porin by NaCI as the basis for salt-induced acid sensitivity in Escherichia coli

Z. LAZIM, T.J. HUMPHREY AND R.J. ROWBURY. 1996. Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol 1⁻¹ added NaCl. Salt-induced acid sensitivity only occurs in relA⁺ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP. The finding that sensitization did not occur in a phoE strain but did occur in a phoE⁺ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions. In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac⁻ strain carrying pJP102 ( phoE-lacZ ) produced low levels of β-galactosidase but growth with added NaCl led to rapid and appreciable induction. Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl. Increased β-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA⁺ strains.
Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.     

Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.     

The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418

No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.Abbreviations PQQ 2, 7, 9-tricarboxy-1H-pyrrolo-(2,3-f)quinoline-4,5-dione - WB Wurster's Blue (1,4-bis-(dimethylamino)-benzene perchlorate)     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Glucose 6-Phosphate and Fructose 1,6-Bisphosphate Can Be Used as ATP-Regenerating Systems by Cerebellum Ca2+-Transport ATPase

Abstract : In this work, it is shown that the Ca²⁺-transport ATPase found in the microsomal fraction of the cerebellum can use both glucose 6-phosphate/hexokinase and fructose 1,6-bisphosphate/phosphofructokinase as ATP-regenerating systems. The vesicles derived from the cerebellum were able to accumulate Ca²⁺ in a medium containing ADP when either glucose 6-phosphate and hexokinase or fructose 1,6-bisphosphate and phosphofructokinase were added to the medium. There was no Ca²⁺ uptake if one of these components was omitted from the medium. The transport of Ca²⁺ was associated with the cleavage of sugar phosphate. The maximal amount of Ca²⁺ accumulated by the vesicles with the fructose 1,6-bisphosphate system was larger than that measured either with glucose 6-phosphate or with a low ATP concentration and phosphoenolpyruvate/pyruvate kinase. The Ca²⁺ uptake supported by glucose 6-phosphate was inhibited by glucose, but not by fructose 6-phosphate. In contrast, the Ca²⁺ uptake supported by fructose 1,6-bisphosphate was inhibited by fructose 6-phosphate, but not by glucose. Thapsigargin, a specific SERCA inhibitor, impaired the transport of Ca²⁺ sustained by either glucose 6-phosphate or fructose 1,6-bisphosphate. It is proposed that the use of glucose 6-phosphate and fructose 1,6-bisphosphate as an ATP-regenerating system by the cerebellum Ca²⁺-ATPase may represent a salvage route used at early stages of ischemia ; this could be used to energize the Ca²⁺ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.