Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding alpha 3-subunits of rat brain Na+,K+-ATPase

The cDNAs encoding alpha 3-subunits of rat brain Na+,K+-ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co-transfection method. Stably transformed cells were selected with 300 micrograms/ml of neomycin (G-418) for 6 weeks. Northern blot analysis using the 3'-non-translated region of the cDNA as a probe revealed that the alpha 3 mRNA appeared in transfected cells. Na+,K+-ATPase activity of the transfected cells was twice that of wild-type cells. Regarding ouabain sensitivity, the Na+,K+-ATPase showed two Ki values for ouabain (8 x 10(-8) and 4.5 x 10(-5) M) in transfected cells while wild-type cells displayed only the higher value. Ouabain sensitivity of Rb+ uptake also demonstrated two Ki values in the transfected cells (8 x 10(-8) and 4 x 10(-5) M) and a Ki in wild-type cells of 4 x 10(-5) M. It is concluded that alpha 3 is a highly ouabain-sensitive catalytic subunit of Na+,K+-ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the alpha-subunit rather than the beta-subunit. This is the first report on the catalytic characteristics of the alpha 3 isoform of Na+,K+-ATPase.     

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Stable structure of thermophilic proton ATPase beta subunit

F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli alpha and gamma subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria. The following results were obtained. Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits. Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. Codon usage: The codon usage of the TF1 beta gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.     

Changes in synthetic activity of cell walls during the cell cycle in a synchronous culture of Catharanthus roseus

The incorporation rates of [¹⁴C] glucose into various fractions of the cell walls and into the sugar constituent of each fraction were investigated in a synchronous culture of Catharanthus roseus (L.) G. Don in order to elucidate the synthetic aspects of the cell walls during the cell cycle. Changes in the incorporation of radioactivity were closely correlated with changes in the amount of each cell wall fraction as well as with those in sugar composition as reported previously (S. Amino et al. Physiol. Plant. 60: 326–332, 1984). The specific activity of galactose was higher than that of other sugars throughout the cell cycle, and a temporary increase in the incorporation of radioactivity into all cell wall fractions except cellulose was observed just before the increase in cell numbers. The synthetic activities may play key roles in the regulation of cell wall polysaccharide dynamics during the cell cycle.     

Reactivation of Cytoplasmic Streaming in a Tonoplast-Permeabilized Cell Model of Acetabularia

To find whether cytoplasmic streaming in Acetabularia is controlledby Ca²⁺, a tonoplast-permeabilized cell model was prepared usinga vacuolar perfusion technique. The cytoplasmic streaming remainedalmost normal after perfusion with EGTA medium (10 mM EGTA,40 mM PIPES, 5mM MgCl₂ and 800 mM sorbitol, pH 6.9), but stoppedwithin 10 min when saponin medium (EGTA medium plus 50 µg/mlsaponin, 50 µg/ml hexokinase and 5 mM glucose) was perfused.This model system was reactivated with a solution containing0.5 mM ATP and different concentrations of Ca²⁺ (reactivationmedium). With the reactivation medium at pCa 6–5, theresumed streaming lasted for about 10 min before the cytoplasmaggregated. At pCa 4–3, the streaming was observed onlyfor a few minutes because the cytoplasm aggregated quickly.At pCa 7, no reactivated movement was observed. Reactivationwas not induced in an ATP- or Mg²⁺-deficient medium even inthe presence of an adequate concentration of Ca²⁺, and was inhibitedby 50 µg/ml cytochalasin B or 1 mM N-ethylmaleimide. We concluded from these observations that the cytoplasmic streamingin Acetabularia is very likely to be driven by the actomyosinsystem in the presence of Mg-ATP and Ca²⁺ at pCa 6–5. (Received October 31, 1984; Accepted April 1, 1985)     

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.