RESUMEN
Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.
Asunto(s)
Encéfalo/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Bioquímica/métodos , Tronco Encefálico/citología , Tronco Encefálico/enzimología , Dominio Catalítico , Bovinos , Inhibidores Enzimáticos/farmacología , Glicosilación , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Riñón/enzimología , Cinética , Microsomas/enzimología , Datos de Secuencia Molecular , Neuroglía/citología , Neuroglía/enzimología , Neuronas/enzimología , Ouabaína/farmacología , Homología de Secuencia de Aminoácido , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , Solubilidad , Especificidad por Sustrato , Tubulina (Proteína)/metabolismoRESUMEN
Active preparations of Na+,K(+)-ATPase containing three types of catalytic isoforms were isolated from the bovine brain to study the structure and function of the sodium pump. Na+,K(+)-ATPase from the brain grey matter was found to have a biphasic kinetics with respect to ouabain inhibition and to consist of a set of isozymes with subunit composition of alpha 1 beta 1, alpha 2 beta m and alpha 3 beta m (where m = 1 and/or 2). The alpha 1 beta 1 form clearly dominated. For the first time, glycosylation of the beta 1-subunit of the alpha 1 beta 1-type isozymes isolated from the kidney and brain was shown to be different. Na+,K(+)-ATPase from the brain stem and axolemma consisted mainly of a mixture of alpha 2 beta 1 and alpha 3 beta 1 isozymes having identical ouabain inhibition constants. In epithelial and arterial smooth muscle cells, where the plasma membrane is divided into functionally and biochemically distinct domains, the polarized distribution of Na+,K(+)-ATPase is maintained through interactions with the membrane cytoskeleton proteins ankyrin and spectrin (Nelson and Hammerton, 1989; Lee et al., 1996). We were the first to show the presence of the cytoskeleton protein tubulin (beta 5-isoform) and glyceraldehyde-3-phosphate dehydrogenase in a high-molecular-weight complex with Na+,K(+)-ATPase in brain stem neuron cells containing alpha 2 beta 1 and alpha 3 beta 1 isozymes. Consequently, the influence of not only subunit composition, but also of glycan and cytoskeleton structures and other plasma membrane-associated proteins on the functional properties of Na+,K(+)-ATPase isozymes is evident.
Asunto(s)
Tronco Encefálico/enzimología , Encéfalo/enzimología , Isoenzimas/análisis , ATPasa Intercambiadora de Sodio-Potasio/análisis , Animales , Bovinos , Células Epiteliales/enzimología , Riñón/enzimología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Neuronas/enzimología , Especificidad de ÓrganosRESUMEN
A hybrid protein, Il-Ox-K, was obtained from cells of E. coli TG1/pTOTEilox strain. The N-terminal sequence of this protein (63 amino acid residues) is a fragment of human interleukin-3, and the C-terminal sequence represents the full amino acid sequence of oxytocin flanked by a lysine residue. The modified oxytocinoyl-Lys containing S-sulfocysteine residues was isolated after tryptic digestion of S-sulfoderivative of the hybrid protein. The modified peptide was converted into the cyclic form containing the disulfide bonds [formula: see text]. Obtaining the oxytocinoyl-Lys proves the possibility of preparing short peptides using the microbiological synthesis.
Asunto(s)
Oxitocina/análogos & derivados , Oxitocina/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Interleucina-3/genética , Datos de Secuencia Molecular , Oxitocina/aislamiento & purificación , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Trypsin and cyanogen bromide were used for cleavage of the OSCP preparations. The peptide mixtures thus formed were separated into individual components by a combination of various chromatographic procedures: gel filtration, ion exchange and paper chromatography, as well as reversed-phase HPLC. As a result, 31 tryptic peptides and 9 out of 10 possible cyanogen bromide peptides were isolated. Determination of the amino acid sequences of these peptide allowed the alignment of cyanogen bromide fragments in the polypeptide chain that shed light on the "architecture" of the protein molecule as a whole. It also afforded the overlappings for tryptic peptides, 16 in the N-terminal and 8 in the C-terminal portions of the molecule.