The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase.

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.

Cellular and subcellular specification of Na,K-ATPase alpha and beta isoforms in the postnatal development of mouse retina.

The Na,K-ATPase is a dominant factor in retinal energy metabolism, and unique combinations of isoforms of its alpha and beta subunits are expressed in different cell types and determine its functional properties. We used isoform-specific antibodies and fluorescence confocal microscopy to determine the expression of Na,K-ATPase alpha and beta subunits in the mouse and rat retina. In the adult retina, alpha1 was found in Müller and horizontal cells, alpha2 in some Müller glia, and alpha3 in photoreceptors and all retinal neurons. beta1 was largely restricted to horizontal, amacrine, and ganglion cells; beta2 was largely restricted to photoreceptors, bipolar cells, and Müller glia; and beta3 was largely restricted to photoreceptors. Photoreceptor inner segments have the highest concentration of Na,K-ATPase in adult retinas. Isoform distribution exhibited marked changes during postnatal development. alpha3 and beta2 were in undifferentiated photoreceptor somas at birth but only later were targeted to inner segments and synaptic terminals. beta3, in contrast, was expressed late in photoreceptor differentiation and was immediately targeted to inner segments. A high level of beta1 expression in horizontal cells preceded migration, whereas increases in beta2 expression in bipolar cells occurred very late, coinciding with synaptogenesis in the inner plexiform layer. Most of the spatial specification of Na,K-ATPase isoform expression was completed before eye opening and the onset of electroretinographic responses on postnatal day 13 (P13), but quantitative increase continued until P22 in parallel with synaptogenesis.

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase.

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane-bound (Na+ + K+)-stimulated adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6--8-fold purification of (Na+ + K+)-ATPase is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350--400 mumol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the (Na+ + K+)-ATPase by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.

Anomalies in the electrophoretic resolution of Na+/K(+)-ATPase catalytic subunit isoforms reveal unusual protein--detergent interactions.

Three different isozymes of the Na+/K(+)-ATPase have slightly different different electrophoretic mobilities in sodium dodecyl sulfate (SDS). Certain procedures (reduction and alkylation, heating, and the use of sodium tetradecyl sulfate) have been reported either to improve the electrophoretic separation of isoforms or to reveal the presence of new isoforms. The variables affecting gel electrophoretic mobility were investigated here. Reduction and alkylation decreased the mobility of all three isozymes, and slightly improved the separation of alpha 1 from alpha 2 and alpha 3 without causing a qualitative change in the alpha isoforms detected. Heating the enzyme in SDS caused splitting into two bands. Both bands were intact polypeptides but migrated differently in 5% and 15% polyacrylamide, disclosing an anomalous conformation in detergent. The use of sodium tetradecyl or decyl sulfate instead of dodecyl sulfate altered the relative mobilities of the isozymes, revealing differences in detergent affinity, but no new isoforms were found. In conclusion, Na+/K(+)-ATPase alpha-subunit mobility reflects complex detergent-protein interaction that can be affected by experimental conditions. The existence of more than one band on gels may reflect different conformations in detergent, but should not be accepted alone as evidence for subunit structural heterogeneity.

Multiple digitalis receptors A molecular perspective.

The Na,K-ATPase is the only established receptor for cardiac glycosides like digoxin or ouabain. There are now known to be three different isoforms of its principal subunit. These isoforms can differ from one another in their intrinsic affinity for cardiac glycosides. Recent work examines the molecular structure of the binding site. The relative level of expression of the isoforms in cardiac tissue is modified in several developmental, hormonal, and pathological states, contributing to alterations in the digitalis sensitivity of the tissue.

Epitope and mimotope for an antibody to the Na, K-ATPase.

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.

Immunocytochemical localization of NaK-ATPase isoforms in the rat and mouse ocular ciliary epithelium.

PURPOSE: Ion gradients established by NaK-adenosine triphosphatase (ATPase) in the ocular ciliary epithelium (CE) contribute to the production of aqueous humor. Modulation of NaK-ATPase activity in the CE may alter aqueous inflow, aqueous turnover, and intraocular pressure. To understand the role of NaK-ATPase, it is necessary to examine the distribution of NaK-ATPase subunit isoforms within the epithelium. METHODS: Isoform-specific antibodies and scanning laser confocal microscopy were used to localize NaK-ATPase subunit isoforms in the CE of the mouse and rat. RESULTS: The nonpigmented epithelium (NPE) expressed alpha2 and beta3 at very high levels on its basolateral surface, and alpha1 and beta2 at much lower levels. The pigmented epithelium (PE) expressed alpha1 and beta1 subunits on its basolateral surface along its entire length, whereas alpha3 was expressed in the pars plana only. The distribution and apparent expression levels of isoforms were similar for mouse and rat, with only minor discrepancies, most likely caused by antibody sensitivity. CONCLUSIONS: The results indicate that sodium pumps in the NPE are primarily composed of alpha2 and beta3, whereas those in the PE are alpha1 and beta1. This specialization in isoform expression implies that NaK-ATPase has distinct physiological functions in the two epithelia and that its activity is likely to be regulated by different mechanisms.

Carbachol and nitric oxide inhibition of Na,K-ATPase activity in bovine ciliary processes.

PURPOSE: Nitric oxide (NO) donors and cholinergic agents decrease intraocular pressure, in part because they induce a decrease in aqueous humor production. Because Na,K-adenosine triphosphatase (ATPase) is involved in aqueous humor formation, this study was conducted to investigate the hypothesis that NO and cholinomimetics regulate its activity in bovine ciliary processes. METHODS: Bovine tissue slices were incubated with agonists and antagonists in a physiological buffer in vitro. Na,K-ATPase activity was determined by assaying hydrolysis of adenosine triphosphate (ATP) in suspended permeabilized tissue slices. RESULTS: Carbachol-induced inhibition of Na,K-ATPase activity correlated with increases in cGMP. This inhibition was abolished by the muscarinic blocker atropine, the NO inhibitor N(w)-nitro-L-arginine (L-NAME) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Sodium nitroprusside (SNP) mimicked the actions of carbachol. The SNP-induced decrease in Na,K-ATPase activity correlated with an increase in cGMP and was also abolished by ODQ. Both 8-bromo (Br)-cGMP and okadaic acid also inhibited Na,K-ATPase activity. CONCLUSIONS: Carbachol-induced inhibition of Na,K-ATPase activity involves muscarinic receptor activation. That SNP mimics and L-NAME reverses carbachol's effect on Na,K-ATPase activity suggests that the actions of carbachol are mediated by NO. Carbachol's and SNP's effects on Na,K-ATPase activity involved soluble guanylate cyclase and cGMP. Inhibition of Na,K-ATPase activity by 8-Br-cGMP and okadaic acid indicates that protein phosphorylation events may mediate SNP-induced inhibition of Na,K-ATPase activity.

Constraints on models for the folding of the Na,K-ATPase.

We have attempted to bring together in graphic fashion the available evidence on the structure of the Na,K-ATPase and the H,K-ATPase. There appears to be much room for modification of the existing models for transmembrane folding. More sites on each side of the membrane need to be identified. Whether these will be antibody epitopes, sites of covalent modification, or tags inserted by mutagenesis is less important than that there be many of them and that each be verified by alternative approaches. If any single principle has emerged from the study of the topography of membrane proteins, it is that it is easy to reach conclusions too soon.

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting.

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).

Overlapping and diverse distribution of Na-K ATPase isozymes in neurons and glia.

The Na-K ATPase is the plasma membrane enzyme that catalyzes the active uptake of K+ and extrusion of Na+, thereby establishing ion concentration gradients between the inside and outside of the cell. It consumes a large fraction of the energy used in the brain. The enzyme is present in both neurons and glia. Studies of ion flux and of the properties of membrane-associated ATPase activity have suggested that there is more than one functional type of Na-K ATPase in the central nervous system. Molecular cloning has demonstrated that there are three different genes encoding catalytic (alpha) subunits and at least two genes encoding glycoprotein (beta) subunits; all are expressed in the brain. This brief review summarizes the current understanding of Na-K ATPase isozyme distribution and properties. Both neurons and glia can express different isoforms in a cell-specific manner.

Post-translational modification and evoked release of two large surface proteins of sympathetic neurons.

Two high molecular weight glycoproteins, exposed on the surface of sympathetic neurons, are modified after they have been translated, glycosylated, and inserted in the plasma membrane. B1 (apparent Mr = 230,000 by electrophoresis in sodium dodecyl sulfate) and B3 (Mr approximately 200,000) are each modified to give proteins of lower apparent molecular weight: B2 (Mr approximately 215,000) and B4 (Mr approximately 185,000). B1 and B3 are derived from two precursors, P1 (Mr approximately 210,000) and P3 (Mr approximately 185,000) which are nonsialylated, mannose-rich proteins not exposed on the cell surface. In unstimulated cells, B1 and B3 are converted to B2 and B4 with a half-life of 4 to 6 hr. In cells which have been treated chemically to evoke the release of neurotransmitter, the modification appears to be accelerated, and B2 and B4 are shed into the medium in soluble form (S2 and S4). This evoked release of protein is calcium dependent and is detected only in conditions which favor the rapid release of neurotransmitter. In the absence of exogenous calcium, however, transmitter release can be evoked without the accompanying release of protein. Thus the release of protein is not an essential step of transmitter release, but may follow it. B1, its precursor, and derivatives are immunologically related to the NILE (nerve growth factor-inducible, large external) glycoprotein of pheochromocytoma PC12 cells (McGuire, J. C., L. A. Greene, and A. V. Furano (1978) Cell 15: 357-365). B3 does not cross-react with B1 or NILE antigenically, but otherwise is synthesized, processed, and released in a similar manner.

Environmentally regulated expression of soluble extracellular proteins of sympathetic neurons.

Sympathetic neurons in dissociated cell culture release 16-18 soluble proteins into the medium, which appear to be distinct from the cell membrane glycoproteins. The released proteins are major cellular products, comprising 2-3% of the newly synthesized protein. They turn over with a half-life of approximately 9 h. Their release is spontaneous and does not correlate with the release of neurotransmitter. Release occurs from distal axon regions and possibly from cell bodies and dendrites as well. Sympathetic neurons are adrenergic in culture, but become cholinergic if grown in medium conditioned by certain types of non-neuronal cells but not others (Patterson, P. H. (1978) Annu. Rev. Neurosci. 1, 1-17). Those conditioned media which induce cholinergic development also dramatically alter the expression of 4 of the 18 released proteins, suggesting a correlation between the expression of these secreted proteins and neurotransmitter choice. Such extracellular proteins may play a part in intercellular communication during the development of the nervous system.

Size, shape, and solubility of a class of releasable cell surface proteins of sympathetic neurons.

Exposed on the cell surface of sympathetic neurons in culture is a family of high molecular weight glycoproteins (B1, B2, B3, and B4, related to the NILE protein) which undergoes post-translational modification (Sweadner, K. J. (1983) J. Neurosci. 3: 2504-2517). B1 and B3 are converted to B2 and B4 by what might be limited proteolysis. These proteins normally require detergents to release them from the cells. When neurotransmitter release is evoked chemically, however, derivatives of the proteins (S2 and S4) are released into the medium. A hydrodynamic analysis of the structure of the released proteins and their membrane-associated precursors was undertaken to determine whether the proteins are released as membrane fragments, aggregates, or monomers in solution, and to give information on the structure and disposition of the proteins on the cell surface. Measurements of the Stokes radii, sedimentation coefficients, partial specific volumes, and frictional coefficients of the proteins indicate that they are released into the medium as soluble monomers. The hydrodynamic analysis also indicates that they are nonglobular (probably fibrous) in shape, both before and after post-translational modification and release. Their true molecular weights are calculated to be approximately 130,000 to 170,000. Although B1, B2, B3, and B4 are probably intrinsic membrane proteins, their releasability suggests that most of their mass is exposed to the aqueous extracellular medium.

Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin.

The brain contains two distinct molecular forms of the (Na,K)-ATPase (sodium and potassium ion-stimulated adenosine triphosphatase). They can be resolved by gel electrophoresis in sodium dodecyl sulfate, and can be identified by sodium-dependent, potassium-sensitive phosphorylation by [gamma-32P]ATP. They are present in the brain of every animal species examined, while only one molecular form is detected in the other organs examined. They are located in different kinds of cells within the brain, and can be physically separated while fully active by gentle tissue fractionation procedures. One is the only (Na,K)-ATPase of brain non-neuronal cells (astrocytes), while the other is the only (Na,K)-ATPase of axolemma (plasma membrane of myelinated axons). They differ in at least one kinetic parameter: the affinity for the specific inhibitor strophanthidin. They have similar one-dimensional peptide maps, but differ in their sensitivity to digestion by trypsin and in the number or reactivity of sulfhydryl groups. It is anticipated that they will be found to play functionally different roles in the complex ion transport mechanisms of the brain.