A. Purification of the Na,K-pump from mammalian kidney
Methods developed in 1969-1973 for purification of the Na,K-pump in membrane bound form from the basolateral membranes of thick ascending limbs of Henles loop of mammalian kidney (rabbit and pig). The purified preparation consists of tightly packed ab-units in disc-shaped lipid bilayer with all enzyme units in the same orientation in the membrane. This preparation has been predominant in all types of studies of the structure, kinetics and function of Na,K-ATPase.
B. Crystallization of Na,K-ATPase in the membrane
Methods developed in 1981 for crystallization of the purified Na,K-ATPase in the membrane by incubation in Mg2+-vanadate solution. This also led to crystallization of Ca-ATPase from sarcoplasmic reticulum and of H,K-ATPase, the proton pump in gastric mucosa. We found that a protomer ab-unit is the minimum asymmetric unit Na,K-ATPase in the crystals.
C. Soluble Na,K-ATPase
Operational definition in 1983 of a protomer ab-unit as the minimum functional unit of soluble Na,K-ATPase. We found that the cavity for occlusion of K+ or Na+-ions is formed within the structure of the ab-unit, as an alternative to earlier models of dimeric and oligomeric mechanisms. The soluble ab-unit is unstable in the absence of K+ or Na+-ions.
D. Protein Conformations of Na,K-ATPase
Demonstration of Na+ and K+ induced changes in conformation with proteolysis as a tool. Analysis of structural changes in the Na,K-pump protein related to transduction of the energy in ATP to transport of Na+ and K+ 1975-82. We also established methods for selective modification of membrane proteins in the study of molecular mechanisms for active transport.
E. Regulation and function of Na,K-pump in kidney
In addition to hormonal regulation, we found in 1968 that the amount of Na,K-ATPase in kidney tissue also depends on the electrolyte status and that changes of [Na+] activity alters the rate of biosynthesis of Na,K-pump protein in tubule cells. In 1975-76 we demonstrated that NaCl transport in thick ascending limbs of Henles loop is driven by the Na,K-pump and that Cl- transport is secondary active. This formed the basis for a unifying model in 1980 for primary Na,K-pumping and secondary active transport of solutes in all segments of kidney tubules (Physiological Reviews 60: 864-917, 1980).
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