A new simple preparation method for NaK-ATPase-rich membrane fragments

A method for the isolation of highly active membrane bound NaK-ATPase without detergents in quantity from the electric organ of the electric eel (Electrophorus electricus) is described. This method consists of the homogenation of electric organ with an isotonic solution containing sucrose, histidine, EDTA, and arginine, and of the separation of the higher active membrane fraction from the microsomal fraction by density gradient centrifugation. The enzyme has a specific activity of about 20 μmol Pi/min/mg at 37°C, and 13 μmol Pi/min/mg at 30°C. Although it is not as pure as the detergent-treated enzyme preparation based on the level of phosphorylated protein, ouabain binding, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its enzyme activity is comparable to that of the purified enzymes. This preparation is very stable and is able to change its medium by Sephadex chromatography without any loss of enzyme activity and protein content. This preparation is also expected to keep the original characteristics as well as the enzyme in the tissue.     

Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.

     

Phosphorylated intermediates of Na,K-ATPase proteoliposomes controlled by bilayer cholesterol. Interaction with cardiac steroid

Fragmental Na,K-ATPase from the electric eel forms three phosphorylated intermediates (EP) with MgATP and Na+: ADP-sensitive K+-insensitive EP (E1P), ADP- and K+-sensitive EP (E*P), and K+-sensitive ADP-insensitive EP (E2P). The EP composition varied with the Na+ concentration. In the reconstituted Na,K-ATPase proteoliposomes (PL), the EP composition of the inside-out form was controlled not only by the intravesicular (extracellular) Na+ concentration, but also by the temperature and the cholesterol content of the lipid bilayer. When the lipid bilayer of PL contained less than 30 mol % cholesterol, the E*P content did not change significantly while the E2P content increased with an elevation in temperature (3-20 degrees C). In contrast, when the lipid bilayer contains more than 35 mol % cholesterol, the E*P content increased while the E2P content stayed less than 10% under the same temperature change. These observations suggest that a high cholesterol content in the lipid bilayer interferes with the E*P to E2P conversion. This cholesterol effect was reversed by ionophores (monensin, nigericin, and A23187). Therefore, E1P-rich EP, E*P-rich EP, or E2P-rich EP could be obtained in the PL under a constant Na+ concentration by using various concentrations of cholesterol and ionophores. The reaction between the proteoliposomal EPs and digitoxigenin (lipid-soluble cardiac steroid) occurred in a single turnover, thereby avoiding unphysiologically high Na+ concentrations. The increase in the ADP- and K+-insensitive EP, which indicated formation of the digitoxigenin-Na,K-ATPase complex, was equivalent to the decrease in the E*P under six different sets of conditions, without any significant change in the E1P and E2P contents. This result indicated that E*P is the active intermediate of the Na,K-ATPase for cardiac steroid binding. Although the E2P has been thought to be the active form for binding, it cannot bind with the cardiac steroid in the presence of Na+ and in the absence of free Mg2+.     

Postglacial environmental change of the Pacific Ocean off the coasts of central Japan

Downcore changes in microfossil assemblages and oxygen isotope ratios in three piston cores recovered from the Northwestern Pacific, off central Japan, show that the subtropical Kuroshio front was located to the south of C-4 core site (Lat. 33° N) during the last glacial. The front then advanced northward, passing over the C-4 site and the C-6 site (34.6° N) at about 13 ka and 10 ka, respectively, and reached the C-1 core site (36° N) at about 7 ka. After 5.5 ka it retreated to the area between the C-1 and C-6 sites. A brief but significant cold event, the readvance of the cold Oyashio Current, is recognized between 11 and 10 ka in the two northern cores, but the current did not reach the southern C-4 site. A contemporaneous cold event is known in the North Atlantic, and the cooling was probably a global phenomenon likely to be associated with lowering of sea level. Contamination of isotopically light water is apparent between 14 and 11 ka in the marked change in isotopic composition of benthic foraminifers. Oxygen isotope ratios of planktonic foraminifers show that prior to the advance of the Kuroshio front, the surface water at these core sites was isotopically lighter than the Kuroshio water at that time.     

In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit.

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.