Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound [formula omitted

Abstract

Compound 4880 (4880), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 4880 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 4880 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 4880 was found to be an inhinitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionaed, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 μg/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 4880 constituents to calmodulin can not fully account for the observed data. Direct interaction of 4880 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.