Sodium Ion Pump Research Articles

Energy conservation in the decarboxylation of dicarboxylic acids by fermenting bacteria.

Decarboxylation of dicarboxylic acids (oxalate, malonate, succinate, glutarate, and malate) can serve as the sole energy source for the growth of fermenting bacteria. Since the free energy change of a decarboxylation reaction is small (around -20 kJ per mol) and equivalent to only approximately one-third of the energy required for ATP synthesis from ADP and phosphate under physiological conditions, the decarboxylation energy cannot be conserved by substrate-level phosphorylation. It is either converted (in malonate, succinate, and glutarate fermentation) by membrane-bound primary decarboxylase sodium ion pumps into an electrochemical gradient of sodium ions across the membrane; or, alternatively, an electrochemical proton gradient can be established by the combined action of a soluble decarboxylase with a dicarboxylate/monocarboxylate antiporter (in oxalate and malate fermentation). The thus generated electrochemical Na+ or H+ gradients are then exploited for ATP synthesis by Na+- or H+-coupled F1F0 ATP synthases. This new type of energy conservation has been termed decarboxylation phosphorylation and is responsible entirely for ATP synthesis in several anaerobic bacteria.

Cloning, sequencing and expression of the genes encoding the sodium translocating N5-methyltetrahydromethanopterin:coenzyme M methyltransferase of the methylotrophic archaeon Methanosarcina mazei Gö1

The N 5-methyltetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina mazei Gö1 is a membrane-associated, corrinoid-containing protein that uses a transmethylation reaction to drive an energy-conserving sodium ion pump. The eight open reading frames encoding the eight different subunits of the methyltransferase were identified and sequenced. All of these subunits are shown to be heterologously expressed in minicells of the Escherichia coli mutant DK6. Sequence comparisons with the methyltransferases of thermophilic and hypothermophilic methanogenic archaea are presented. The participation of the gene product of mtrD in sodium ion translocation as well as a consensus sequence of a corrinoid binding motif in MtrA are discussed.

Sodium ion translocation by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanosarcina mazei Gö1 reconstituted in ether lipid liposomes.

The N5-methyltetrahydromethanopterin (H,MPT):coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyl-tetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei Gö1 by a two-step solubilization with n-octyl-beta-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 microM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Gö1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na-/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl-cobalamin could substitute for methyl-H,MPT.

Pathways of energy conservation in methanogenic archaea

Methanogenic archaea are strictly anaerobic organisms that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H2+CO2 and formate are converted to CH4 via the CO2-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. A limited number of methanogenic organisms utilize acetate by the aceticlastic pathway. Redox reactions involved in these processes are partly catalyzed by membrane-bound enzyme systems that generate or, in the case of endergonic reactions, use electrochemical ion gradients. The H2:heterodisulfide oxidoreductase, the F420H2:heterodisulfide oxidoreductase and the CO:heterodisulfide oxidoreductase, are novel systems that generate a proton motive force by redox-potential-driven H+ translocation. The methyltetrahydromethanopterin:coenzyme M methyltransferase is a unique, reversible sodium ion pump that couples methyl transfer with the transport of Na+ across the cytoplasmic membrane. Formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation, of methanofuran. In summary, the pathways are coupled to the generation of an electrochemical sodium ion gradient and an electrochemical proton gradient. Both ion gradients are used directly for ATP synthesis via membrane integral ATP synthases. The function of the above-mentioned systems and their components in the metabolism of methanogens are described in detail.

Expression of the sodium ion pump methylmalonyl-coenzyme A-decarboxylase from Veillonella parvula and of mutated enzyme specimens in Escherichia coli

The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70). A DNA region upstream of mmdA apparently served as promoter in E. coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS(+)-derived expression plasmid. To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter. The derived plasmid, pT7mmd, was transformed into E. coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter. The synthesized proteins showed the typical properties of methylmalonyl-CoA-decarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin. In methylmalonyl-CoA-decarboxylase expressed in E. coli from pT7mmd, the gamma subunit was only partially biotinylated and the alpha subunit was present in substoichiometric amounts, resulting in a low catalytic activity. This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed alpha subunit. After these treatments methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose. To analyze the function of the delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd. E. coli cells transformed with pJHdelta2, which lacks mmdE and the 3' -terminal part of mmdD, showed no methylmalonyl-CoA-decarboxylase activity. In addition, a contrast, catalytically active methylmalonyl-CoA-decarboxylase was expressed in E. coli from plasmid pJHdelta1, which contained a deletion of the mmdE gene only. The mutant enzyme could be isolated, reconstituted into proteolipsomes, and shown to function in the transport of Na+ ions coupled to methylmalonyl-CoA decarboxylation. The small epsilon subunit therefore has no catalytic function within the methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex.

N5‐Methyltetrahydromethanopterin:Coenzyme M Methyltransferase from Methanobacterium thermoautotrophicum

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane associated, corrinoid-containing enzyme complex which uses a methyl-transfer reaction to drive an energy-conserving sodium ion pump. The purified methyltransferase from Methanobacterium thermoautotrophicum (strain Marburg) exhibited a rhombic EPR signal indicative of a base-on cob(II)amide. In this form, the enzyme was almost completely inactive. Upon addition of Ti(III)citrate, which is a one-electron reductant known to reduce corrinoids to the cob(I)amide form, the EPR signal was completely quenched. In the reduced form, the enzyme was active. When the purified complex was incubated in the presence of both Ti(III) and N5-methyltetrahydromethanopterin (CH3-H4MPT), enzyme-bound Co-methyl-5'-hydroxybenzimidazolyl cob(III)amide was formed. Upon incubation of the methylated enzyme with either tetrahydromethanopterin or coenzyme M, the enzyme was demethylated with the concomitant formation of CH3-H4MPT and methylcoenzyme M, respectively. Enzyme demethylation, in contrast to enzyme methylation, was not dependent on the presence of Ti(III). Methyl transfer from the methylated enzyme to coenzyme M was essentially irreversible. These results are interpreted to that the purified enzyme complex is active only when the enzyme-bound corrinoid is in the reduced cob(I)amide form, and that methyl transfer from CH3-H4MPT to coenzyme M proceeds via nucleophilic attack of the cobalt(I) on the N5-methyl substituent of CH3-H4MPT, forming an enzyme-bound CH3-corrinoid as intermediate. Methyl-coenzyme M formation from CH3-H4MPT and coenzyme M, as catalyzed by the purified methyltransferase, was stimulated by sodium ions, half-maximal activity being obtained at approximately 50 microM Na+. We therefore infer that the methyltransferase, as isolated, is capable of vectorial sodium ion translocation.

The energetics and sodium-ion dependence of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase studied with cob(I)alamin as methyl acceptor and methylcob(III)alamin as methyl donor.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane-associated enzyme complex that uses a methyl-transfer reaction to drive an energy-conserving sodium-ion pump. Methyl transfer occurs in two steps, first from N5-methyltetrahydromethanopterin (CH3-H4MPT) to an enzyme-bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H-S-CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH3-H4MPT and cob(I)alamin (reaction a), and methyl-coenzyme M formation from methylcob(III)alamin and H-S-CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH3-H4MPT to H-S-CoM. The free energy changes (delta G0') associated with reactions a and b were both between -10 kJ/mol and -20 kJ/mol, consistent with a free energy change of approximately -30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium-ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme-bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H-S-CoM is a site of coupling with sodium-ion translocation.

Sequence of the sodium ion pump methylmalonyl-CoA decarboxylase from Veillonella parvula.

The genes encoding methylmalonyl-CoA decarboxylase from Veillonella parvula were cloned on plasmids using oligonucleotides derived from N-terminal amino acid sequences as specific probes. The entire DNA sequence of the methylmalonyl-CoA decarboxylase genes together with upstream and downstream regions was determined. The genes encoding subunits alpha (mmdA), delta (mmdD), epsilon (mmdE), gamma (mmdC), and beta (mmdB) of the decarboxylase were clustered on the chromosome in the given order. The previously unnoted epsilon-chain (M(r) 5,888) was clearly shown to be a subunit of the decarboxylase by correspondence of the N-terminal amino acid sequence with that deduced from the DNA sequence of mmdE. The alpha-subunit was 60% identical with the carboxyltransferase domain of rat liver propionyl-CoA carboxylase, the beta-subunit showed 61% sequence identity with the beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae, and the biotin-containing gamma-subunit was 29-39% identical with biotin-domains of other biotin enzymes. The delta-subunit of methylmalonyl-CoA decarboxylase and the gamma-subunit of oxaloacetate decarboxylase did not show significant sequence homology. The gross structure of both proteins, however, was similar, consisting of a hydrophobic membrane anchor near the N terminus, a proline/alanine linker, and a remarkable accumulation of charged amino acids in the C-terminal part. The sequence of the small epsilon-subunit could be aligned to the C-terminal region of the delta-subunit downstream of the proline/alanine linker, where the two subunits were 47% identical. Of considerable interest for the mechanism of Na+ transport are the long stretches of complete sequence identity between the hydrophobic beta-subunits of methylmalonyl-CoA decarboxylase and oxaloacetate decarboxylase and the presence of two conserved aspartic acid residues within putative membrane-spanning helices.

Acetogenesis and ATP synthesis inAcetobacterium itwoodiiare coupled via a transmembrane primary sodium ion gradient

In cell suspensions of Acetobacterium woodii the acetyl-CoA pathway is coupled to net ATP formation. Acetate formation as well as ATP synthesis and the generation of a transmembrane sodium ion gradient are not inhibited by protonophores but by sodium ionophores. Acetogenesis from CO or formaldehyde + CO as catalyzed by inverted vesicles is coupled to sodium ion uptake. Both processes are not inhibited by protonophores but by sodium ionophores. These experiments are in accordance with the presence of a primary sodium ion pump connected to the acetyl-CoA pathway which enables the cells to synthesize net ATP by means of a ΔμNa+ in concert with a Na+-translocating ATPase.

Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate.

Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anaerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m = 170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).

The sodium ion pumping oxaloacetate decarboxylase of Klebsiella pneumoniae Metal ion content, inhibitors and proteolytic degradation studies

Oxaloacetate decarboxylase of Klebsiella pneumoniae was shown to contain between 0.6 and 1.0 mol zinc per mol enzyme in different preparations. The decarboxylase activity was completely abolished after 15 min incubation with 1 mM Hg(NO 3) 2 in phosphate buffer, while the activity decreased only 20% if the incubation was performed in MES/Trix buffer. Treatment of the isolated subunits with Hg(NO 3) 2 indicated that the binding site for Hg 2+ ions is on the α subunit. Other inhibitors of the decarboxylase are KSCN and diethylstilbestrol. Inactivation of the enzyme with 2% 1-butanol was significantly reduced by 100 mM NaCl. Sodium ions also protected the isolated β+γ subunits from a digestion with trypsin.

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5 M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na+ (Km = 3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent Km for Na+ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, alpha (m = 65 kDa), beta (33 kDa), gamma (19 kDa), and delta (16 kDa) with the functions of a carboxy transferase (alpha), a carboxy lyase (beta and probably delta) and a biotin carrier (gamma). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the alpha- and the biotin containing subunits of the three decarboxylases and that from Peptostreptococcus asaccharolyticus could be detected on Western blots.

A hybrid adenosine triphosphatase composed of F1 of Escherichia coli and F0 of Propionigenium modestum is a functional sodium ion pump

Analyses on immunoblots indicated strong binding of the alpha- and beta-subunits of the ATPase of Propionigenium modestum to antibodies raised against the corresponding subunits of the F1F0 ATPase of Escherichia coli. Cross-reactivities of antibodies against the other ATPase subunits were not observed. The use of Na+ or H+ as alternate coupling ions, observed previously for the P. modestum ATPase [Laubinger, W., & Dimroth, P. (1989) Biochemistry 28, 7194-7198], is not found for the F1F0 ATPase of E. coli, which is specific for protons. However, a hybrid consisting of the F1 moiety of the E. coli ATPase and F0 of that from P. modestum performed Na+ or H+ transport in a reconstituted system. As with the homologous ATPase of P. modestum, H+ pumping of the hybrid was abolished at Na+ concentrations of greater than 1 mM. The F0 sector and not F1, therefore, determines the cation specificity of these F1F0 ATPases.

Photosynthesis, Respiration, and Salt Gland Activity of Distichlis spicata in Relation to Soil Salinity

Photosynthesis in Distichlis spicata was negatively correlated with soil salinity in both field and green-house plants. This relationship was stronger and statistically more significant in the greenhouse population where salinity and other environmental factors were more tightly controlled. In contrast, leaf respiration showed no significant correlation with soil salinity in either group of plants. Sodium ion pumping rates by leaf salt glands had a statistically significant positive correlation with soil salinity. When these rates were compared to leaf respiration, the relationship was also positive and statistically significant. We postulate that both reduced photosynthesis and increased leaf respiration contribute to the reduced stem growth found with increasing soil salinity.

Isolation and characterization of oxaloacetate decarboxylase of Salmonella typhimurium, a sodium ion pump

Anaerobic growth of Salmonella typhimurium on citrate is Na+-dependent and requires induction of the necessary enzymes during a 20-40 h lag phase. The citrate fermentation pathway involves citrate lyase and oxaloacetate decarboxylase. The decarboxylase is a membrane-bound, Na+-activated, biotin-containing enzyme that functions as a Na+ pump. Oxaloacetate decarboxylase was isolated by affinity chromatography of a Triton X-100 extract of the bacterial membranes on avidin-Sepharose. The enzyme consists of three subunits alpha, beta, gamma, with apparent molecular weights of 63,800, 34,500 and 10,600. The alpha-chain contains a covalently attached biotin group and binds to antibodies raised against the alpha-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae. The Na+ transport function was reconstituted by incorporation of the purified enzyme into proteoliposomes.

Reconstitution of the purified (Na + + Mg 2+)-ATPase from Acholeplasma laidlawii B membranes into lipid vesicles and a characterization of the resulting proteoliposomes

The purified (Na + + Mg 2+)-ATPase from Acholeplasma laidlawii B membranes was reconstituted with dimyristoylphosphatidylcholine using a cholate solubilization and dialysis procedure. The incorporation of this enzyme into the phospholipid bilayer is accompanied by an enhancement of its specific activity and by a restoration of its lipid phase state-dependent properties which were lost during solubilization and purification from native membranes. Moreover, reconstitution of this ATPase with phospholipid also stabilizes it against cold inactivation at low temperatures ( ⋍ 0° C ), oxidative degradation at room temperature, and thermal denaturation at elevated temperatures ( ⋍ 55° C ). The elution profile from a Sepharose 4B-CL column indicates that all of the ATPase protein is associated with the phospholipid vesicles and that the Stoke's radius of the proteoliposomes formed is smaller than that of the lipid vesicles formed in the absence of any protein. The latter conclusion is supported by sedimentation velocity measurements and by an electron microscopic examination of negatively stained preparations. The electron microscopic studies demonstrate that sealed vesicles are formed only at low protein-to-lipid ratios. These observations indicate that the Acholeplasma laidlawii B (Na + + Mg 2+)-ATPase has been structurally and functionally reconstituted into lipid vesicles and that the proteoliposomes formed are amenable to studies aimed at the clarification of its proposed role as a sodium ion pump.

Origin of 5-hydroxytryptamine-induced hyperpolarization of the rat superior cervical ganglion and vagus nerve.

1 5-Hydroxytryptamine (5-HT)-induced membrane potential changes were recorded extracellularly from rat superior cervical ganglia (SCG) and cervical vagus nerves in vitro. 2 On the SCG, low concentrations of 5-HT (1 X 10(-8)-3 X 10(-7) M) induced concentration-related hyperpolarization responses. Higher concentrations of 5-HT (1 X 10(-6) 1 X 10(-4) M) induced complex responses which typically consisted of an initial hyperpolarization, followed by a depolarization and subsequent after-hyperpolarization. The depolarization, but not the initial hyperpolarization, was blocked by metoclopramide (3 X 10(-5) M), quipazine (1 X 10(-6) M) or MDL 72222 (1 X 10(-5) M). 3 5-HT-induced hyperpolarization of the SCG was potentiated when the amount of calcium chloride added to the superfusion medium was reduced from 2.5 to 0.15 mmol l-1. Hyperpolarization responses recorded from SCG preparations superfused with this low-calcium medium were unaffected by the substitution of lithium chloride for sodium chloride and were potentiated by the omission of potassium ions. Ouabain (1 X 10(-3) M) abolished both the hyperpolarization and the depolarization induced by 5-HT. 4 On the vagus nerve, 5-HT (1 X 10(-7) - 3 X 10(-5)M) did not induce initial hyperpolarization in either normal or low-calcium Krebs-Henseleit medium. However, in the latter solution only, depolarization responses induced by 5-HT at concentrations of 1 X 10(-6)M or greater were followed by hyperpolarization. Both the depolarization and the post-5-HT hyperpolarization were blocked by metoclopramide (3 X 10(-5)M) but were unaffected by spiperone (1 X 10(-7)M). 5 On the vagus nerve, post-5-HT hyperpolarization responses were selectively and reversibly inhibited by ouabain, and by superfusion with Krebs-Henseleit medium that was either potassium-free or contained lithium chloride in place of sodium chloride. 7 These results demonstrate the generation in the rat SCG of a 5-HT-induced hyperpolarization response that is not mediated through 5-HT3 receptors and is unlikely to be a consequence of depolarization. In contrast, on the rat vagus nerve, the post-5-HT hyperpolarization observed in the present study had the characteristics expected of depolarization-dependent activation of a sodium ion pump.

Sodium and potassium ion pump in kidney tubules.

Sodium and potassium ion pump in kidney tubules.

Application of a cation exchange membrane to a sodium ion pump.

Application of a cation exchange membrane to a sodium ion pump.

The number of sodium ion pumping sites in skeletal muscle and its modification by insulin.

1. [3H]ouabain binding by frog sartorius muscles shows at least two components: one linked to inhibition of the pump and another not related to transport inhibition. This is suggested by the finding that [3H]ouabain uptake continued to increase when (a) the glycoside concentration was increased beyond that causing maximum transport inhibition, and (b) exposure times longer than those required to produce full inhibition were used. 2. A number of 1600 pumping sites per mum2 of membrane was estimated considering only the cylindrical surface of the muscle. 3. Insulin stimulated the ouabain-sensitive components of 22Na efflux and 134Cs influx. It also increased [3H]ouabain binding to a level of 1-7 times the total resting value. The increases in [3H]ouabain binding and in 22Na efflux followed a similar relationship with respect to insulin concentration. 4. Insulin stimulated the Na pump in muscles whose pumping sites had been inhibited by ouabain and then transferred to a glycoside-free solution. This stimulation was observed before detecting any recovery of the initial pumping activity. 5. When both the resting and the insulin-stimulated 22Na efflux had been blocked by ouabain, an additional dose of insulin, in a ouabain-free solution, had no further effects on 22Na efflux. 6. The effects of insulin were unaffected by cycloheximide or by high concentrations of butyryl derivatives of cyclic AMP and cyclic GMP. 7. We conclude that there are two pools of Na pumping sites in muscle cells: one active and another inactive. Insulin unmasks the inactive pumping sites by a mechanism that is independent of protein synthesis, increases in intracellular [Na] or decreases in intracellular [K].