RESUMEN
The Na,K-ATPase alpha 3 isoform of the catalytic subunit has been isolated from pig kidney microsomes. The procedure employs immunoaffinity chromatography on Sepharose 4B covalently coupled with monospecific antibodies a-II against the synthetic peptide including the putative alpha 3 N-terminus. The structural analysis provides unambiguous proof that the isolated protein corresponds to the third transcript for the alpha 3 isoform. The N-terminal amino acid sequence determined. Met-Gly-Asp-Lys-Lys-Asp-Asp, shows that unlike the alpha 1 and alpha 2 proteins, the mature Na,K-ATPase isoform lacks post-translational proteolytic processing.
Asunto(s)
Isoenzimas/aislamiento & purificación , Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos , Complejo Antígeno-Anticuerpo , Microsomas/enzimología , Datos de Secuencia Molecular , PorcinosRESUMEN
At least three types of mRNA of the catalytic subunit of Na,K-ATPase namely alpha-,alpha+- and alpha 3-isoforms are identified in different tissues. Only two of them alpha and alpha+ have well known structural and catalytic properties. Here we present immunochemical data indicating that the alpha 3 protein really exists in pig and human kidney, and human brain. Crude membrane fractions and purified membrane-bound Na,K-ATPases were immunoblotted with alpha 3-specific antibodies raised against the synthetic peptide corresponding to the unique sequence of this isoform. The mature alpha 3-subunit is shown to include the sequence GDKKDDKSSPK followed by the initiating methionine residue. Nephron collecting tubules are proposed to specifically contain Na,K-ATPase alpha 3-isoform.
Asunto(s)
Isoenzimas/metabolismo , Riñón/enzimología , Microsomas/metabolismo , Tejido Nervioso/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , PorcinosRESUMEN
Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio , Animales , Membrana Celular/enzimología , Médula Renal/enzimología , Sustancias Macromoleculares , Modelos Moleculares , Conformación Proteica , Espectrometría Raman/métodos , Porcinos , TripsinaRESUMEN
To study the topology of Na+,K+-ATPase monoclonal antibodies (MAbs) specific for membrane-bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra- or intracellular portions of the alpha-subunit. The extracellular location of peptide loop 804-841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween-20), was shown to bind to the hydrophilic loop 868-945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887-904 and 810-825, respectively. The C-terminus of the alpha-subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity-purified antibodies to the synthetic peptide corresponding to fragment 999-1008.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Membrana Celular/enzimología , Epítopos/análisis , Médula Renal/enzimología , Sustancias Macromoleculares , Modelos Moleculares , Conformación Proteica , ATPasa Intercambiadora de Sodio-Potasio/inmunología , PorcinosRESUMEN
Exposed regions of the alpha- and beta-subunits of membrane-bound Na+,K+-ATPase were in turn hydrolyzed with trypsin. Resistance of the beta-subunit to proteolysis was shown to be due mainly to the presence of disulfide bridge(s) in the molecule. A model for the spatial organisation of the enzyme in the membrane was proposed on the basis of detailed structural analysis of extramembrane regions of both subunits.