Interaction of sodium and potassium ions with Na+, K+-ATPase. III. Cooperative effect of ATP and Na+ on complete release of K+ from E2K

The effects of Na+ and ATP on the K+ binding to Na+, K+-ATPase were investigated by the centrifugation method with radioactive K+ in the absence of Mg2+. In the presence of 10 microM 43KCl, 0.6 and 10 mM Na+ decreased the amount of bound K+ to one-half and zero, respectively. On the other hand, 10 microM and 10 mM ATP decreased the amount of K+ to 60 and 25-40%, respectively. When the combined effect of ATP and Na+ was tested, 10 microM ATP decreased the Na+ concentration giving half-maximal inhibition of the K+ binding to one-third, showing synergistic inhibition by both ligands, though increase in ATP concentration seemed to depress the inhibitory effect of Na+. The synergistic inhibition by ATP and Na+ suggests that the release of K+ from E2K is not completed by the binding of ATP alone but is completed by the binding of Na+ in addition to ATP during the cycle of Na+, K+-dependent ATP-hydrolysis as well as ion-transport.     

Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.     

Detection of H-2K mRNA in mouse 8-cell embryo by cDNA cloning

Mouse MHC class I gene expression in 8-cell embryo was examined by cDNA cloning. We constructed a cDNA library from 8-cell embryos of ICR mice and isolated a class I cDNA from 3.0 x 10(5) phage clones of the library. Sequencing analysis of this clone revealed it to include the cDNA fragment extending from the exon 6 of the cytoplasmic portion to 3' untranslated region 1 of H-2K gene. Qa, Tla or other embryonic class I cDNA have not been isolated in the library.     

Isolation of endo A cDNA from mouse 8-cell stage embryos

To analyse the species of genes expressed in a cleavage stage mouse embryo, we have constructed a cDNA library containing 3.0 x 10(5) independent clones from about 2 x 10(3) embryos at the 8-cell stage of development. Endo A cDNA prepared from parietal yolk sac endoderm like PYS-2 cells was used to screen the library. Southern blot analyses using the endo A sequence as a probe and restriction mapping analyses revealed that four independent recombinants had been inserted endo A sequence. Sequencing data of these clones showed that endo A mRNA present in the 8-cell stage embryo is identical to that of parietal yolk sac endoderm cells.     

A simple preparation method for apoaspartate aminotransferase from Escherichia coli B, and its application for the assay of pyridoxal and pyridoxamine 5'-phosphate

A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.     

Reconstruction of the pharyngoesophagus following pharyngoesophagectomy and irradiation therapy

During the 5-year period between 1978 and 1983, a total of 56 individuals have undergone pharyngoesophageal reconstruction at our hospitals. To restore the continuity of the upper enteric tract, conventional skin flaps with or without the underlying muscle were used in 27 and a segmental free intestinal graft by means of microvascular technique was used in 29. Two-thirds of both groups received irradiation therapy before surgery. Fistula formation was encountered in 8 individuals who received a skin flap. This incidence was found to increase with the use of preoperative irradiation. In contrast, no patient developed a fistula among the 29 patients who received the segmental free intestinal graft.     

Interpretation of the stokes radius of macromolecules determined by gel filtration chromatography

From a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6 M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration.     

Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.