This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP–Fe2+, which directs the Fe2+ to the normal ATP–Mg2+ site. Fe2+- and ATP–Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane–water interface. Cu2+- or Cu2+-DPP-catalyzed cleavages are providing information on α/β subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.

A series of creep experiments were performed on Mt. Burnet Dunite, including stress relaxation and the incremental (stepped) variation of temperature and stress on a single sample. The steady-state flow behavior is given by the equation: εs = 1.9 · 1010[sinh(σ3.50)]3.00 ± 0.20exp(−QRT) where εs is the steady-state strain rate in sec−1, and σ is the uniaxial compressive stress in kbar. Q is 93.8 ± 2.8 kcal/mole for dunite deformed in a hydrous environment, and Q is about 126 kcal/mole for dunite deformed in an anhydrous environment. The other coefficients in the above equation appear to be insensitive to sample water content, based on limited data. Water thus seems to affect the strain rate primarily through its effect on the activation energy. The activation energy and stress exponent appear to be about the same in transient creep as in steady-state creep. Ductile faulting, characterized by narrow to wide shear zones and gradual stress drops, is prevalent in the samples. This phenomenon may be similar to that occurring in deep-focus earthquakes.

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.

1.1. The net transport of potassium and sodium across the human erythrocyte membrane were observed at 37° in cells prepared by cold storage and fortified with nucleoside. Passive transport was minimized by adjustment of sodium and potassium concentrations in the medium. Active transport was distinguished from passive transport by the use of strophanthin, which stops active transport specifically.2.2. The active transport of potassium inward and sodium outward occured only at a ratio which was constant over a wide range of rates and independent of extracellular and intracellular sodium and potassium concentrations. Two atoms of potassium wer e transported inward for every three atoms of sodium that were tramsported outward.3.3. Ammonium appeared to substitute directly for potassium adn required a concentration 3 to 7 times greater than potassium to produce a comparable effect.4.4. These findings indicate that active potassium and sodium transport across the human erythrocyte membrane are parts of a single tightly-linked system.

Journal Article Control of Boxelder Bugs Get access J. A. Munro, J. A. Munro North Dakota Agr. Expt. Station, Fargo Search for other works by this author on: Oxford Academic PubMed Google Scholar R. L. Post R. L. Post North Dakota Agr. Expt. Station, Fargo Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Economic Entomology, Volume 42, Issue 6, 1 December 1949, Page 994, https://doi.org/10.1093/jee/42.6.994 Published: 01 December 1949