Fig. 1. HPLC procedures for extensive purification of NCX_IF.

NCX_IF was extracted and partially purified by solvent precipitation, gel-filtration, and ion-exchange procedures, as described in Materials and Methods. Partially purified NCX_IF was further loaded on the HPLC columns in the following order: A. Synergi-Polar (10 ml/min). B. Krumasil-Silica (5 ml/min). C. TSK-gel Amide-80 (5 ml/min). D. ZIC-HILIC (0.5 ml/min). E. Hypera (1.0 ml/min). F. Cyclobond (1 ml/min). All other chromatographic conditions are described in Materials and Methods. Aliquots were removed from collected fractions, lyophilized, and then assayed for inhibition of the Na⁺/Ca²⁺ exchanger by using the standard assay of Na_i-dependent ⁴⁵Ca-uptake in sarcolemma vesicles (see Materials and Methods). The active fractions obtained from 20–50 injections were pooled, lyophilized, and then the concentrated fractions were injected into the next column. Bars indicate the fractions containing the inhibitory activity of NCX_IF.