Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.     

Remobilization of toxic heavy metals adsorbed to bacterial wall-clay composites

Significant quantities of Ag(I), Cu(II), and Cr(III) were bound to isolated Bacillus subtilis 168 walls, Escherichia coli K-12 envelopes, kaolinite and smectite clays, and the corresponding organic material-clay aggregates (1:1, wt/wt). These sorbed metals were leached with HNO3, Ca(NO3)2, EDTA, fulvic acid, and lysozyme at several concentrations over 48 h at room temperature. The remobilization of the sorbed metals depended on the physical properties of the organic and clay surfaces and on the character and concentration of the leaching agents. In general, the order of remobilization of metals was Cr much less than Ag less than Cu. Cr was very stable in the wall, clay, and composite systems; pH 3.0, 500 microM EDTA, 120-ppm [mg liter-1] fulvic acid, and 160-ppm Ca remobilized less than 32% (wt/wt) of sorbed Cr. Ag (45 to 87%) and Cu (up to 100%) were readily removed by these agents. Although each leaching agent was effective at mobilizing certain metals, elevated Ca or acidic pH produced the greatest overall mobility. The organic chelators were less effective. Lysozyme digestion of Bacillus walls remobilized Cu from walls and Cu-wall-kaolinite composites, but Ag, Cr, and smectite partially inhibited enzyme activity, and the metals remained insoluble. The extent of metal remobilization was not always dependent on increasing concentrations of leaching agents; for example, Ag mobility decreased with some clays and some composites treated with high fulvic acid, EDTA, and lysozyme concentrations. Sometimes the organic material-clay composites reacted in a manner distinctly different from that of their individual counterparts; e.g., 25% less Cu was remobilized from wall- and envelope-smectite composites than from walls, envelopes, or smectite individually in 500 microM EDTA. Alternatively, treatment with 160-ppm Ca removed 1.5 to 10 times more Ag from envelope-kaolinite composites than from the individual components. The particle size of the deposited metal may account for some of the stability changes; those metals that formed large, compact aggregates (Cr and Ag) as seen by transmission electron microscopy were less likely to be remobilized. In summary, it is apparent that remobilization of toxic heavy metals in sediments, soils, and the vadose zone is a complicated issue. Predictions based on single inorganic or organic component systems are too simplistic.     

Functional regulation of reconstituted Na, K-ATPase by protein kinase A phosphorylation

Reconstituted Na⁺,K⁺-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P_i/mole -subunit in the pig kidney enzyme and 0.2 mol P_i/mol -subunit in the shark enzyme. In shark Na⁺,K⁺-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na⁺ activation and extracellular K⁺ activation without affecting the apparent K_m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na⁺,K⁺-ATPase.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

Anencephaly in trisomy 18 associated with elevated alpha-1-fetoprotein in amniotic fluid

Summary Elevated levels of alpha-1-fetoprotein (AFP) were found in the amniotic fluid of a 36-year-old woman in the 15th week of gestation. Because of this and the results of repeated ultrasonography, abortion was induced. An anencephalic fetus with trisomy 18 was delivered. The possible correlation among neural-tube defects, chromosomal abnormalities, and level of AFP is discussed.