8-Azido-adenosine 5'-triphosphate as a photoaffinity label for bacterial F1 ATPase.

1. 8-Azido-adenosine 5'-triphosphate (n83ATP) is a suitable photoaffinity label for F1 ATPase from Micrococcus luteus. The nucleotide is a substrate in the presence of bivalent cations and inhibits the enzyme irreversibly upon irradiation with ultraviolet light above 300 nm. 2. More than 80% of the label is covalently bound to the beta subunits in the presence of bivalent cations. Labeling and inactivation is decreased by protection with ADP, ATP or adenyl-5'-yl imidodiphosphate. To a much smaller degree the alpha subunits also become labeled. 3. n83AMP does not specifically bind to the beta subunits upon irradiation. Like n83ATP and n83ADP, it also labels the alpha subunits to a small extent. 4. The F1 ATPase is inactivated after a single beta subunit per F1 complex has become labeled. A cooperativity of the beta subunits carrying nucleotide binding sites is suggested.     

Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling

The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)].oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits. With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed. The alpha subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.     

The effect of reducing agents on the structure of mammalian beta-adrenergic receptors

Under reducing conditions (5% beta-mercaptoethanol) the mammalian beta-adrenergic receptor binding site from both beta 1 (porcine heart membranes) and beta 2 receptors (hamster lung and rat erythrocyte membranes) appears to reside on peptides of Mr 62,000-65,000 as determined by photoaffinity labeling with p-azido-m-[125I]iodobenzylcarazolol and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When similar experiments are performed in these same systems under a variety of non-reducing conditions, there are minimal changes in the apparent molecular weight of both the beta 1- and beta 2-adrenergic receptor binding subunits and no specifically labeled higher molecular weight proteins are observed suggesting that there are no disulfide linked subunits in mammalian beta-adrenergic receptors.     

Identification of a novel 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide in calcium channel preparations

A 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide has been identified by photoaffinity labeling of purified rabbit and guinea pig skeletal muscle calcium channel preparations. The arylazide ligands (-)-[3H]azidopine and (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile [( N-methyl-3H]LU 49888) were used to label 1,4-dihydropyridine- and phenylalkylamine-binding sites, respectively. A single, 155 to 170-kDa polypeptide was specifically labeled by both ligands in rabbit and guinea pig preparations provided that the skeletal muscle membranes used for purification were derived from fresh and not previously frozen and thawed tissue. The photoaffinity labeled polypeptide (termed here alpha 1) is different from the previously described alpha subunit in that it has the identical electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels irrespective of pretreatment either with N-ethylmaleimide or with dithiothreitol. The use of transverse tubular membranes isolated from previously frozen and thawed skeletal muscle results in a purified calcium channel preparation devoid of the alpha 1 subunit. In these preparations proteolytic degradation products of alpha 1 are labeled with both (-)-[3H]azidopine and [N-methyl-3H]LU 49888. Another large molecular weight polypeptide (termed here alpha 2) was also present in every purified calcium channel preparation studied. alpha 2 is distinct from alpha 1 in that reduction with dithiothreitol changes its apparent mass from 160-190 to 130-150 kDa. The alpha 2 subunit is not photoaffinity labeled either with (-)-[3H]azidopine or [N-methyl-3H]LU 49888. These data suggest that two distinct high molecular weight polypeptides (termed alpha 1 and alpha 2) are putative subunits of skeletal muscle calcium channels. Only the alpha 1 subunit contains both 1,4-dihydropyridine and phenylalkylamine receptors. alpha 2 is the same as the previously described alpha subunit (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118), but is neither a 1,4-dihydropyridine- nor a phenylalkylamine-binding protein.     

Specific derivatization of the vesicle monoamine transporter with novel carrier-free radioiodinated reserpine and tetrabenazine photoaffinity labels

Two iodophenylazide derivatives of reserpine and one iodophenylazide derivative of tetrabenazine have been synthesized and characterized as photoaffinity labels of the vesicle monoamine transporter (VMAT2). These compounds are 18-O-[3-(3'-iodo-4'-azidophenyl)-propionyl]methyl reserpate (AIPPMER), 18-O-[N-(3'-iodo-4'-azidophenethyl)glycyl]methyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3H]dopamine uptake into purified chromaffin granule ghosts showed IC50 values of approximately 37 nM for reserpine, 83 nM for AIPPMER, 200 nM for IAPEGlyMER, and 2.1 microM for TBZ-AIPP. Carrier-free radioiodinated [125I]IAPEGlyMER and [125I]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [125I]IAPEGlyMER labeling of a polypeptide that migrated as a broad band (approximately 55-90 kDa), with the majority of the label located between 70 and 80 kDa. The labeling by [125I]IAPEGlyMER was blocked by 100 nM reserpine, 10 microM tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [125I]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 microM reserpine and 10 microM tetrabenazine. Incubation of [125I]TBZ-AIPP-photolabeled chromaffin granule membranes in the presence of the glycosidase N-glycanase shifted the apparent molecular weight of VMAT2 to approximately 51 kDa. These data indicate that [125I]IAPEGlyMER and [125I]TBZ-AIPP are effective photoaffinity labels for VMAT2.     

Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate

A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.     

The cardiac beta-adrenergic receptor. Structural similarities of beta 1 and beta 2 receptor subtypes demonstrated by photoaffinity labeling

The beta-adrenergic receptor photoaffinity ligand p-azido-m-[125I]iodobenzylcarazolol has been used to covalently label the beta 1 and beta 2 adrenergic receptor binding subunits present in left ventricular myocardial membranes derived from mammalian (including human) and nonmammalian species. Covalent incorporation of the photoaffinity ligand into membrane proteins was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the case of the human, canine, porcine, rabbit, and rat left ventricle, all of which contain predominantly or exclusively beta 1-adrenergic receptors, two peptides of Mr approximately equal to 62,000 (major component) and Mr approximately equal to 55,000 (minor component) were specifically labeled and visualized by autoradiography. Photoincorporation into these two bands could be blocked with the appropriate drugs to display a beta 1-adrenergic receptor pharmacological specificity. Simultaneous sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples from each species revealed that all of the Mr = 62,000 peptides co-migrated suggesting similarity in the beta 1-adrenergic receptor binding subunit peptides in all of these species. The minor component Mr approximately equal to 55,000 appears to be a proteolytic degradation product of the Mr = to 62,000 peptide. Its formation could be decreased by proteinase inhibitors. This suggests that the heterogeneity of the labeling pattern observed in mammalian tissues in this and previous studies may be the result of proteolytic degradation of the receptor subunit which occurs during membrane preparation. Photoaffinity labeling of frog ventricular membranes which contain predominantly beta 2-adrenergic receptors also revealed two peptides of Mr approximately equal to 62,000 (major component) and 55,000 (minor component) with the pharmacological selectivity of a beta 2-adrenergic receptor. These data suggest marked similarities in the beta 1- and beta 2-adrenergic receptor binding subunits of different species and suggest that the pharmacological subtype might be determined by the detailed structure, i.e. amino acid sequence, at the ligand binding sites of the receptor peptide.     

Structural and biosynthetic studies on the two molecular forms of the (Na+ + K+)-activated adenosine triphosphatase large subunit in Artemia salina Nauplii

The large subunit of (Na+ + K+)-activated ATPase from brine shrimp, Artemia salina, migrates as two bands in sodium dodecyl sulfate-polyacrylamide gels. The slower migrating band, as observed in neutral or alkaline gel systems, is designated alpha 1 and the faster, alpha 2. Structural and biosynthetic studies have been performed to determine if these two bands represent independent molecular forms or precursor products. Peptide mapping of partial proteolytic digests of alpha 1 and alpha 2 showed no distinguishable difference between them whereas this technique produced very distinct differences in the large subunit derived from three different species. The two large subunit bands also behaved identically when cross linked with cupric phenanthroline either in the presence or absence of digitonin, whereas other proteins in these preparations were unaffected. The peptide mapping and cross-linking experiments demonstrate that alpha 1 and alpha 2 have identical or nearly identical primary and probably higher order structure. Their different mobilities may be due to post-translational modification leading, for example, to different oligosaccharide composition. During development of the brine shrimp nauplius, alpha 1 increases in relative abundance while alpha 2 decreases. NaH14CO3 incorporation and pulse-chase experiments indicate that alpha 1 and alpha 2, as well as the small subunit of the brine shrimp (Na+ + K+)-activated ATPase, are synthesized at the same time during development and that all changes in the rates of synthesis of these subunits occur at the same time. The apparent rates of degradation of the subunits are also similar. These results are inconsistent with a precursor-product relationship between alpha 1 and alpha 2.     

Site of synthesis of the alpha and beta subunits of the Na,K-ATPase in brine shrimp nauplii

Developing nauplii (embryos) of the brine shrimp Artemia salina are an excellent model system for studying the biogenesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase). The nauplii exhibit a burst of Na,K-ATPase synthesis between 6 and 32 h of development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We have now determined the sites of synthesis of the alpha and beta subunits of the Na,K-ATPase in developing A. salina nauplii. Membrane-bound and free polysomes were isolated from nauplii, and RNA was extracted from the polysomes. The polysomal RNA was translated in vitro in a rabbit reticulocyte lysate, and the translation products were immunoprecipitated by anti-subunit antisera. The immunoprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Our data show that the alpha subunit precursor is synthesized on membrane-bound polysomes and the beta subunit precursor is synthesized on free polysomes. In addition, the alpha subunit precursor appears as two separate peptides on sodium dodecyl sulfate-polyacrylamide gels, which suggests that the two alpha subunit forms seen in mature brine shrimp Na,K-ATPase are products of two distinct messenger RNAs. The beta subunit precursor appears as a single discrete band, unlike the mature beta subunit, which appears as a diffuse band.     

Inactivation of beef heart mitochondrial F1-ATPase by the 2',3'-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.     

Identifying the lipid-protein interface of the alpha4beta2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.     

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.     

8-N(3)-3'-biotinyl-ATP, a novel monofunctional reagent: differences in the F(1)- and V(1)-ATPases by means of the ATP analogue

A novel photoaffinity label, 8-N(3)-3'-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F(1)-ATPase from the thermophilic bacterium PS3 (TF(1)). UV irradiation of TF(1) in the presence of 8-N(3)-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic alpha and the catalytic beta subunits of TF(1), demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V(1)-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the gamma subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N(3)-3'-biotinyl-ATP.     

Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin

A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.     

Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium. Contribution of a nucleophilic amino acid residue of the alpha1 subunit.

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.     

Differences in phosphorylation of the two large subunits of brine shrimp Na,K-ATPase

Analysis of purified Na,K-ATPase from brine shrimp nauplii revealed two molecular forms of the alpha subunit separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The molecular form with lower mobility is designated alpha 1 and the one with higher mobility, alpha 2, in a neutral or alkaline gel system. Differences in Na+-dependent, K+-sensitive phosphorylation of these two molecular forms have been investigated by directly measuring the radioactivity present in each phosphoprotein after separation of the two forms by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Na+,Mg2+, and ATP, when the ATP concentration is above 1 microM, both alpha subunits are phosphorylated, although the phosphoprotein content of alpha 1 is considerably greater than that of alpha 2. Below 1 microM ATP, the phosphoprotein content of alpha 2 is even further reduced. These striking differences in phosphorylation at low ATP concentrations are not due to a greater instability of the alpha 2 phosphoprotein during the long electrophoresis times or during fixation, staining, and destaining. The proportion of total phosphoprotein content in alpha 2, as well as the relationship between phosphoprotein content and ATP concentration, is unchanged when the radioactive analysis is performed on frozen gels that have been electrophoresed for shorter times, even though the actual amount of phosphorylation is 15 times greater than with fixed gels. Since the concentration of alpha 1 and alpha 2 vary during development [G.L. Peterson, L. Churchill, J.A. Fisher, and L.E. Hokin (1982) J. Exp. Zool. 221:295], the differences in phosphorylation may be relevant to differences in Na,K-ATPase activity during different development stages.