Cysteine-scanning mutagenesis study of the sixth transmembrane segment of the Na,K-ATPase alpha subunit

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,K-ATPase alpha subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites.     

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.     

Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance.

The cardiac glycoside ouabain inhibits Na,K-ATPase by binding to the alpha subunit. In a highly ouabain resistant clone from the MDCK cell line, we have found two alleles of the alpha subunit in which the cysteine, present in the wild-type first transmembrane segment, is replaced by a tyrosine (Y) or a phenylalanine (F). We have studied the kinetics of ouabain inhibition by measuring the current generated by the Na,K-pump in Xenopus oocytes injected with wild-type and mutated alpha 1 and wild-type beta 1 subunit cRNAs. When these mutations, alpha 1C113Y and alpha 1C113F [according to the published sequence [Verrey et al. (1989) Am. J. Physiol., 256, F1034] were introduced in the alpha 1 subunit of the Na,K-ATPase from Xenopus laevis, the inhibition constant (Ki) of ouabain increased greater than 1000-fold compared with wild-type. A more conservative mutation, serine alpha 1C113S did not change the Ki. We observed that the decreased affinity for ouabain was mainly due to a faster dissociation, but probably also to a slower association. Thus we propose that an amino acid residue of the first transmembrane segment located deep in the plasma membrane participates in the structure and the function of the ouabain binding site.     

Structure of the 5th transmembrane segment of the Na,K-ATPase alpha subunit: a cysteine-scanning mutagenesis study

To study the structure of the pathway of cations across the Na, K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the alpha subunit of the Na,K-ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A(796)) was accessible from the extracellular side in the native Na,K-pump. After treatment with palytoxin, six other positions (Y(778), L(780), S(782), P(785), E(786) and L(791)), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an alpha-helical model but the pattern of reactivity would rather suggest a beta-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin-induced channel and indicate which amino acid positions are exposed to the pore of this channel.     

Functional role of cysteine residues in the (Na,K)-ATPase alpha subunit

The structural-functional roles of 23 cysteines present in the sheep (Na,K)-ATPase alpha1 subunit were studied using site directed mutagenesis, expression, and kinetics analysis. Twenty of these cysteines were individually substituted by alanine or serine. Cys452, Cys455 and Cys456 were simultaneously replaced by serine. These substitutions were introduced into an ouabain resistant alpha1 sheep isoform and expressed in HeLa cells under ouabain selective pressure. HeLa cells transfected with a cDNA encoding for replacements of Cys242 did not survive ouabain selective pressure. Single substitutions of the remaining cysteines yielded functional enzymes, although some had reduced turnover rates. Only minor variations were observed in the enzyme Na(+) and K(+) dependence as a result of these replacements. Some substitutions apparently affect the E1<-->E2 equilibrium as suggested by changes in the K(m) of ATP acting at its low affinity binding site. These results indicate that individual cysteines, with the exception of Cys242, are not essential for enzyme function. Furthermore, this suggests that the presence of putative disulfide bridges is not required for alpha1 subunit folding and subsequent activity. A (Na,K)-ATPase lacking cysteine residues in the transmembrane region was constructed (Cys104, 138, 336, 802, 911, 930, 964, 983Xxx). No alteration in the K(1/2) of Na(+) or K(+) for (Na,K)-ATPase activation was observed in the resulting enzyme, although it showed a 50% reduction in turnover rate. ATP binding at the high affinity site was not affected. However, a displacement in the E1<-->E2 equilibrium toward the E1 form was indicated by a small decrease in the K(m) of ATP at the low affinity site accompanied by an increase in IC(50) for vanadate inhibition. Thus, the transmembrane cysteine-deficient (Na,K)-ATPase appears functional with no critical alteration in its interactions with physiological ligands.     

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.     

Alanine scanning mutagenesis of oxygen-containing amino acids in the transmembrane region of the Na,K-ATPase.

Oxygen-containing amino acids in the transmembrane region of the Na, K-ATPase alpha subunit were studied to identify residues involved in Na+ and/or K+ coordination by the enzyme. Conserved residues located in the polar face of transmembrane helices were selected using helical wheel and topological models of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha1 isoform and expressed in HeLa cells. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of heterologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, Ser140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu818, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those carrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were introduced into the wild-type alpha1 sheep isoform and expressed in mouse NIH3T3 cells where [3H]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. Expression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at the plasma membrane was significantly lower than that of the wild-type isoform. Thus, these substitutions appear to impair the maturation process or targeting of the enzyme to the plasma membrane, but not cation-enzyme interactions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-containing residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme function. Consequently, other chemical groups, aromatic rings or backbone carbonyls, should be considered in models of cation-binding sites.     

Electrogenicity of Na,K- and H,K-ATPase activity and presence of a positively charged amino acid in the fifth transmembrane segment

The transport activity of the Na,K-ATPase (a 3 Na+ for 2 K+ ion exchange) is electrogenic, whereas the closely related gastric and non-gastric H,K-ATPases perform electroneutral cation exchange. We have studied the role of a highly conserved serine residue in the fifth transmembrane segment of the Na,K-ATPase, which is replaced with a lysine in all known H,K-ATPases. Ouabain-sensitive 86Rb uptake and K+-activated currents were measured in Xenopus oocytes expressing the Bufo bladder H,K-ATPase or the Bufo Na,K-ATPase in which these residues, Lys800 and Ser782, respectively, were mutated. Mutants K800A and K800E of the H,K-ATPase showed K+-stimulated and ouabain-sensitive electrogenic transport. In contrast, when the positive charge was conserved (K800R), no K+-induced outward current could be measured, even though rubidium transport activity was present. Conversely, the S782R mutant of the Na,K-ATPase had non-electrogenic transport activity, whereas the S782A mutant was electrogenic. The K800S mutant of the H,K-ATPase had a more complex behavior, with electrogenic transport only in the absence of extracellular Na+. Thus, a single positively charged residue in the fifth transmembrane segment of the alpha-subunit can determine the electrogenicity and therefore the stoichiometry of cation transport by these ATPases.     

Structure of the alpha 1 subunit of horse Na,K-ATPase gene

Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264. The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.     

Functional role of conserved transmembrane segment 1 residues in human sodium-dependent vitamin C transporters

Sodium-dependent vitamin C transporters, SVCT1 and SVCT2, are the only two known proteins for the uptake of ascorbate, the active form of vitamin C. Little structural information is available for SVCTs, although a transport activity increase from pH 5.5 to 7.5 suggests a functional role of one or more conserved histidines (p K a approximately 6.5). Confocal fluorescence microscopy and uptake kinetic analyses were used here to characterize cells transfected with mutants of EGFP-tagged hSVCTs. Mutating any of the four conserved histidine residues (His51, 147, 210, or 354) in hSVCT1 to alanine did not affect the apical membrane localization in polarized MDCK cells. His51Ala (in putative transmembrane segment 1, TM1) was the only mutation that resulted in a significant loss of ascorbate transport and an increase in apparent Km with no significant effect on Vmax. The corresponding mutation in hSVCT2, His109Ala, also led to a loss of transport activity. Among eight other mutations of His51 in hSVCT1, significant sodium-dependent ascorbate transport activity was only observed with asparagine or tyrosine replacement. Thus, our results suggest that uncharged His51, directly or indirectly, contributes to substrate binding through the hydrogen bond. His51 cannot account for the observed pH dependence as neutral amino acid substitutions failed to abolish the pH-dependent activity increase. The importance of TM1 is further strengthened by the comparable loss of sodium-dependent ascorbate transport activity upon the mutation of adjacent conserved Gln50 and the apparent change in substrate specificity in the hSVCT1-His51Gln mutation, which showed a specific increase in sodium-independent dehydroascorbate transport.     

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.     

The transmembrane segment of the human Na,K-ATPase beta-subunit acts as the membrane incorporation signal

The human Na,K-ATPase beta-subunit is anchored to the membrane by a single stretch of 28 hydrophobic amino acids; the hydrophilic amino terminus faces the cytoplasm and the carboxyl terminus is exoplasmic. Glycosylation and insertion of the Na,K-ATPase beta-subunit into the endoplasmic reticulum membrane are shown to be co-translational and SRP-dependent. The hydrophilic amino terminus is not required for the membrane insertion. The membrane-anchor domain is necessary for membrane insertion, and a 16 amino acid stretch has been identified as an element sufficient for the insertion.     

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.     

Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance.

We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.     

Accessibility of tryptophan residues in Na,K-ATPase

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit

The gamma subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E(1) <--> E(2) conformational equilibrium and (ii) an increase in K(+) antagonism of cytoplasmic Na(+) activation. Antibodies against the C terminus common to both variants (anti-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Expression and functional role of the gamma subunit of the Na, K-ATPase in mammalian cells

The functional role of the gamma subunit of the Na,K-ATPase was studied using rat gamma cDNA-transfected HEK-293 cells and an antiserum (gammaC33) specific for gamma. Although the sequence for gamma was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a gammaC33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of gammaC33-treated kidney pumps shows that gamma regulates the apparent affinity for ATP. Thus, gamma-transfected cells have a decreased K'ATP as shown in measurements of (i) K'ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 microM ATP. Consistent with the behavior of gamma-transfected cells, gammaC33 pretreatment increases K'ATP of the kidney enzyme and K+ inhibition (1 microM ATP) of both kidney and gamma-transfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma subunit stabilizes the E2(K) form of the enzyme (Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634). Overall, our data demonstrate that gamma is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum.