John R. Sachs

αβ protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: Formation of higher oligomers does not modify molecular activity

The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.

The L Antibody and Potassium Fluxes in LK Red Cells of Sheep and Goats

Populations of sheep and goats are dimorphic with respect to the cation composition of their red blood cells. HK individuals have high K and low Na concentrations while LK individuals have the converse concentrations. This difference in cation composition, which is controlled by a single genetic locus with two alleles (Evans and King, 1955), can be explained in sheep by the higher pump fluxes and lower passive fluxes of K in HK than in LK cells (Tosteson and Hoffman, 1960). The difference in pump fluxes is due in part to a greater number of Na-K pump sites per cell on HK cells (Dunham and Hoffman, 1971a). In sheep the locus controlling the M-L antigen system is closely linked to (or identical with) the locus which controls cation composition, such that LK cells always have the L antigen and HK cells always have M (Rasmusen and Hall, 1966). Recent evidence indicates that the HK-LK system in goats has a similar basis (Ellory and Tucker, 1970b). Goats also have the M-L system; the genetic control in goats is not as well understood as in sheep, but if there is linkage to the locus controlling transport it is not as close as in sheep (Ellory and Tucker, 1970b). An important aspect of the relationship between the antigen and the Na-K pump is the demonstration that anti-L antiserum produced in an HK sheep against LK sheep cells stimulates the K pump in LK cells from both sheep and goats (Ellory and Tucker, 1969; 1970b). The mechanisms of action of the antibody in stimulating the pump may involve an increase in the number of pump sites and/or an alteration of the affinities of the pump sites for Na and K. This report presents our recent results on several specific aspects of the effect of anti-L on K fluxes in LK cells.