Pituitary adenylate cyclase-activating peptide (PACAP) and PAC1 receptor in the testis of cartilaginous fish Torpedo marmorata: A molecular and phylogenetic study.

The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one. Finally, we demonstrated that in T. marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis.

Design, synthesis, and characterization of a 39 amino acid peptide mimic of the main immunogenic region of the Torpedo acetylcholine receptor.

We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers: This 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10(-10)M, which is smaller than (7.13±1.20)×10(-10)M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10(-10)M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.

Expression of VIP and its receptors in the testis of the spotted ray Torpedo marmorata (Risso 1880).

The aim of this work was to study, by immunoprecipitation, in situ hybridization and immunohistochemistry, and the expression of the vasoactive intestinal peptide (VIP) and of its receptors (VPAC(1)R and VPAC(2)R) in the testis of a nonmammalian vertebrate, the cartilaginous fish Torpedo marmorata. We demonstrated that, differently from mammals, VIP and VPAC(2)R were widely distributed in the testicular cells while the VPAC(1)R had a limited distribution. In details, we showed that VIP and VPAC(2)R were present in mitotic and differentiating germ cells as well as in the cells involved in the steroidogenesis, i.e., Leydig, Sertoli cells, and prespermatogonia and spermatogonia. The possibility that VIP is involved in the spermatogenesis and particularly in the steroidogenesis of T. marmorata is discussed.

Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17ß-estradiol.

In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17ß-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed.

Expression of functional human α6ß2ß3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.

α6ß2ß3* acetylcholine receptors (AChRs) on dopaminergic neurons are important targets for drugs to treat nicotine addiction and Parkinson's disease. However, it has not been possible to efficiently express functional α6ß2ß3* AChRs in oocytes or transfected cells. α6/α3 subunit chimeras permit expression of functional AChRs and reveal that parts of the α6 M1 transmembrane domain and large cytoplasmic domain impair assembly. Concatameric subunits permit assembly of functional α6ß2ß3* AChRs with defined subunit compositions and subunit orders. Assembly of accessory subunits is limiting in formation of mature AChRs. A single linker between the ß3 accessory subunit and an α4 or α6 subunit is sufficient to permit assembly of complex ß3-(α4ß2)(α6ß2) or ß3-(α6ß2)(α4ß2) AChRs. Concatameric pentamers such as ß3-α6-ß2-α4-ß2 have been functionally characterized. α6ß2ß3* AChRs are sensitive to activation by drugs used for smoking cessation therapy (nicotine, varenicline, and cytisine) and by sazetidine. All these are partial agonists. (α6ß2)(α4ß2)ß3 AChRs are most sensitive to agonists. (α6ß2)2ß3 AChRs have the greatest Ca²+ permeability. (α4ß2)(α6ß2)ß3 AChRs are most efficiently transported to the cell surface, whereas (α6ß2)2ß3 AChRs are the least efficiently transported. Dopaminergic neurons may have special chaperones for assembling accessory subunits with α6 subunits and for transporting (α6ß2)2ß3 AChRs to the cell surface. Concatameric pentamers and pentamers formed from combinations of trimers, dimers, and monomers exhibit similar properties, indicating that the linkers between subunits do not alter their functional properties. For the first time, these concatamers allow analysis of functional properties of α6ß2ß3* AChRs. These concatamers should enable selection of drugs specific for α6ß2ß3* AChRs.

Pore structure of the Cys-loop ligand-gated ion channels.

The Cys-loop receptor family of ligand-gated ion channels (LGICs) play a key role in synaptic transmission in the central nervous system of animals. Recent advances have led to the elucidation of two crystal structures of related prokaryotic LGICs and the electron micrograph derived structure of the acetylcholine receptor from Torpedo marmorata. Here, we review the structural and biochemical data that form our understanding of the structure of the channel pore. We introduce original data from the glycine receptor using the substituted-cysteine accessibility technique and show that while the helical structure of the segment that surrounds the channel pore is generally agreed, the location of the channel gate, the pore diameter and the structure that forms the entry to the channel pore are likely to differ between receptors. The fundamental structural differences between anion and cation selective receptors and how these differences are related to the pore structure are also considered.

Probing protein packing surrounding the residues in and flanking the nicotinic acetylcholine receptor M2M3 loop.

Nicotinic acetylcholine receptors (nAChR) are cation-selective, ligand-gated ion channels of the cysteine (Cys)-loop gene superfamily. The recent crystal structure of a bacterial homolog from Erwinia chrysanthemi (ELIC) agrees with previous structures of the N-terminal domain of AChBP (acetylcholine-binding protein) and of the electron-microscopy-derived Torpedo nAChR structure. However, the ELIC transmembrane domain is significantly more tightly packed than the corresponding region of the Torpedo nAChR. We investigated the tightness of protein packing surrounding the extracellular end of the M2 transmembrane segment and around the loop connecting the M2 and M3 segments using the substituted cysteine accessibility method. The M2 20' to 27' residues were highly water accessible and the variation in reaction rates were consistent with this region being alpha-helical. At all positions tested, the presence of ACh changed methanethiosulfonate ethylammonium (MTSEA) modification rates by <10-fold. In the presence of ACh, reaction rates for residues in the last extracellular alpha-helical turn of M2 and in the M2M3 loop increased, whereas rates in the penultimate alpha-helical turn of M2 decreased. Only three of eight M2M3 loop residues were accessible to MTSEA in both the presence and absence of ACh. We infer that the protein packing around the M2M3 loop is tight, consistent with its location at the interdomain interface where it is involved in the transduction of ligand binding in the extracellular domain to gating in the transmembrane domain. Our data indicate that the Torpedo nAChR transmembrane domain structure is a better model than the ELIC structure for eukaryotic Cys-loop receptors.

Effect of 17beta-estradiol and progesterone on vitellogenesis in the spotted ray Torpedo marmorata Risso 1810 (Elasmobranchii: Torpediniformes): studies on females and on estrogen-treated males.

The influence of 17beta-estradiol (E(2)) on vertebrate vitellogenesis is well ascertained. The aim of the present paper is to study the involvement of E(2) and progesterone (P) in the induction and regulation of vitellogenesis in females and experimental E(2)-treated males of Torpedo marmorata. We analyzed females in various stages of the reproductive cycle and E(2) experimentally treated males. The presence of vitellogenin was investigated in the plasma and in the liver by western blot and immunohistochemistry; its site of synthesis was investigated by in situ hybridization. The steroid levels in the plasma were measured by Enzyme Immunoassay. In treated males, E(2) induces in the liver the synthesis of VTG which is then secreted into the bloodstream as a 205-kDa polypeptide, the same that is found in the plasma of non-pregnant vitellogenic females. In females, E(2) is naturally present in the plasma and its level is correlated with VTG synthesis in the liver and with the female reproductive cycle. Indeed, large amounts of E(2) are only found in mature vitellogenic females, whose liver is involved in VTG synthesis and secretion. By contrast, small amounts of E(2) are evident in juveniles whose ovaries are lacking in vitellogenic follicles and in females preparing for ovulation. Low titers are also found in gravid females, whose liver is not engaged in VTG synthesis. We show that P, which is absent in untreated males and juvenile females, is evident in the blood serum of E(2)-treated males and sexually mature females. Interestingly, in treated males P appears in the plasma just 24h after the first injection of E(2) and its titer increases; a week after the last injections, the P level is similar to that recorded in non-gravid vitellogenic females. Finally, it is noteworthy that the highest titer of P was recorded in pregnant females. We demonstrate that in Torpedo vitellogenin synthesis, as in other vertebrates, is under the control of E(2) but also that this synthesis is probably under the control of progesterone.

Mechanisms of cholinesterase inhibition by inorganic mercury.

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Distribution of PACAP in the brain of the cartilaginous fish torpedo marmorata.

In this article, we investigated the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNA by immunohistochemistry, in situ hybridization, and RT-PCR techniques, in the central nervous system of the elasmobranch Torpedo marmorata. RT-PCR analysis showed that the CNS of T. marmorata expresses a messenger encoding PACAP. The immunohistochemistry and in situ hybridization patterns were partly overlapping, with a major expression in the hypothalamo-pituitary region and, surprisingly, in the saccus vasculosus. Our results show that, in T. marmorata, PACAP is synthesized and widely distributed in the CNS, suggesting an as yet unidentified role for this peptide in elasmobranch brain physiology.

Evolutionarily conserved allosteric network in the Cys loop family of ligand-gated ion channels revealed by statistical covariance analyses.

The Cys loop family of ligand-gated ion channels mediate fast synaptic transmission for communication between neurons. They are allosteric proteins, in which binding of a neurotransmitter to its binding site in the extracellular amino-terminal domain triggers structural changes in distant transmembrane domains to open a channel for ion flow. Although the locations of binding site and channel gating machinery are well defined, the structural basis of the activation pathway coupling binding and channel opening remains to be determined. In this paper, by analyzing amino acid covariance in a multiple sequence alignment, we have identified an energetically interconnected network in the Cys loop family of ligand-gated ion channels. Statistical coupling and correlated mutational analyses along with clustering revealed a highly coupled cluster. Mapping the positions in the cluster onto a three-dimensional structural model demonstrated that these highly coupled positions form an interconnected network linking experimentally identified binding domains through the coupling region to the gating machinery. In addition, these highly coupled positions are also condensed in the transmembrane domains, which are a recent focus for the sites of action of many allosteric modulators. Thus, our results revealed a genetically interconnected network that potentially plays an important role in the allosteric activation and modulation of the Cys loop family of ligand-gated ion channels.

Oxidation and reduction control of the inactivation gating of Torpedo ClC-0 chloride channels.

Oxidation and reduction (redox) are known to modulate the function of a variety of ion channels. Here, we report a redox regulation of the function of ClC-0, a chloride (Cl(-)) channel from the Torpedo electric organ. The study was motivated by the occasional observation of oocytes with hyperpolarization-activated Cl(-) current when these oocytes expressed ClC-0. We find that these atypical recording traces can be turned into typical ClC-0 current by incubating the oocyte in millimolar concentrations of reducing agents, suggesting that the channel function is regulated by oxidation and reduction. The redox control apparently results from an effect of oxidation on the slow (inactivation) gating: oxidation renders it more difficult for the channel to recover from the inactivated states. Introducing the point mutation C212S in ClC-0 suppresses the inactivation state, and this inactivation-suppressed mutant is no longer sensitive to the inhibition by oxidizing reagents. However, C212 is probably not the target for the redox reaction because the regulation of the inactivation gating by oxidation is still present in a pore mutant (K165C/K165 heterodimer) in which the C212S mutation is present. Taking advantage of the K165C/K165 heterodimer, we further explore the oxidation effect in ClC-0 by methane thiosulfonate (MTS) modifications. We found that trimethylethylammonium MTS modification of the introduced cysteine can induce current in the K165C/K165 heterodimer, an effect attributed to the recovery of the channel from the inactivation state. The current induction by MTS reagents is subjected to redox controls, and thus the extent of this current induction can serve as an indicator to report the oxidation state of the channel. These results together suggest that the inactivation gating of ClC-0 is affected by redox regulation. The finding also provides a convenient method to "cure" those atypical recording traces of ClC-0 expressed in Xenopus oocytes.

Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays.

Intact acetylcholine receptors have been purified on a novel affinity resin from three electric fish endemic to Australian waters. Their binding properties and morphology are compared with those of their northern hemisphere homolog, Torpedo marmorata. All four exhibit apparent dissociation constants, Kd, in the nanomolar range for the snake neurotoxin alpha-bungarotoxin and have a distinctive rosette-like appearance when viewed in negative stain under the electron microscope. Furthermore, these rosettes are paired, indicating that acetylcholine receptors from southern ocean electric fish exist as dimers, in the same fashion as their northern hemisphere counterparts. The cDNAs of the receptor's four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits, alpha and delta, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the delta subunit of each of the newly sequenced fish species is a cysteine residue. Its conservation suggests that the mechanism for the observed dimerization of acetylcholine receptors is disulfide bond formation between the delta subunit of adjacent receptors, analogous to acetylcholine receptor dimers observed in other electric fish. It appears that this mechanism for receptor clustering is unique to acetylcholine receptors packed and organized in the specialized organs of electric fish. Alignment of the deduced protein sequences with the equivalent sequences from Torpedo californica and humans reveals a high degree of homology.

Trimerization domain of the collagen tail of acetylcholinesterase.

In the collagen-tailed forms of cholinesterases, each subunit of a specific triple helical collagen, ColQ, may be attached through a proline-rich domain (PRAD) situated in its N-terminal noncollagenous region, to tetramers of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). This heteromeric assembly ensures the functional anchoring of AChE in extracellulare matrices, for example, at the neuromuscular junction. In this study, we analyzed the influence of deletions in the noncollagenous C-terminal region of ColQ on its capacity to form a triple helix. We show that an 80-residue segment located downstream of the collagenous regions contains the trimerization domain, that it can form trimers without the collagenous regions, and that a pair of cysteines located at the N-boundary of this domain facilitates oligomerization, although it is not absolutely required. We further show that AChE subunits can associate with nonhelical collagen ColQ monomers, forming ColQ-associated tetramers (G4-Q), which are secreted or are anchored at the cell surface when the C-terminal domain of ColQ is replaced by a GPI-addition signal.

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase.

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.

Identification of a dynein molecular motor component in Torpedo electroplax; binding and phosphorylation of Tctex-1 by Fyn.

The microtubule protein Tctex-1 was cloned from Torpedo electroplax, a biochemical model of the neuromuscular junction, using the unique domain of Fyn in the yeast two hybrid system. Binding of Tctex-1 and Fyn also occurred in vitro. Torpedo Tctex-1 was contained within the molecular motor protein dynein. A Src class kinase was also complexed with dynein. Tctex-1 was enriched in electric organ vs. skeletal muscle, was present in the postsynaptic membrane, and coprecipitated with the acetylcholine receptor. The sequence of Tctex-1 contained a tyrosine phosphorylation motif and Tctex-1 could be phosphorylated by Fyn in vitro and in vivo. These data demonstrated that Tctex-1-containing dynein is a cytoskeletal element at the acetylcholine receptor-enriched postsynaptic membrane and suggested that Tctex-1 may be a substrate for Fyn.

Isolation and characterization of a skate retinal GABA transporter cDNA.

PURPOSE: The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is believed to play a crucial role in the processing of information within the vertebrate retina. Extracellular concentrations of GABA are thought to be tightly regulated by carrier-mediated transport proteins in neurons and glial cells. The purpose of this work was to isolate the gene that encodes one of these transport proteins in the skate retina. METHODS: cDNA clones were isolated from a skate retinal cDNA library using a mouse retinal GABA transporter (GAT1) cDNA as a probe. The PCR technique was used to fill sequence gaps, and 5' and 3' RACE were employed to amplify the 5' and 3' untranslated regions. The amplified fragments were subcloned into a T-vector. Blots containing RNA from 10 different tissues were probed to determine the size of the transcript and the tissue distribution. RESULTS: Sequence analysis revealed that the skate retinal GABA transporter cDNA shared 72% identity with the mouse GABA transporter-1 at the DNA level and 80% identity at the amino acid level. Multiple sequence alignments showed that our sequence is closest to the Torpedo GABA transporter-1. Two transcripts, 4.5 and 7 kb, were detected in retina and possibly brain by RNA blot analysis. Fourteen introns were detected in the skate GABA transporter gene. CONCLUSIONS: We successfully isolated a full length GABA transporter cDNA from the retina of the skate. The size of the full length sequence of the skate retinal GABA transporter is in agreement with the size of the smaller transcript detected on RNA blots. The larger transcript observed on the RNA blot may be the result of either alternative splicing or utilization of a downstream poly A signal.

Structure-function relationships of Na+/K(+)-pumps expressed in Xenopus oocytes.

During the last years we have examined structure-function relationships in the Na+/K(+)-ATPase with respect to interactions of the external cations with the pump molecule. We have analysed in voltage-clamp experiments the influence of extracellular Na+ and K+ on the current generated by Na+/K(+)-pumps expressed in Xenopus oocytes. Our results demonstrated that external Na+ and K+ have to pass an access channel in the electrical field of the membrane to reach their binding sites. This external access, therefore, is voltage-dependent and is affected by lysine residues within the cytoplasmic N-terminus, by glutamic acid residues in intramembraneous domains, the ouabain sensitivity and phosphorylation by protein kinases.

Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target.

BACKGROUND: Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. RESULTS: The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 A resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (approximately 2000 A2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. CONCLUSIONS: Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.

A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in Xenopus laevis oocytes.

We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto-oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co-precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc-tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits.