Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

College of Graduate Studies

First Advisor

George E. Lindenmayer

Second Advisor

Ervin E. Bagwell

Abstract

The cardiac sarcolemmal Na+/Ca[superscript 2+] exchanger promotes the coupled movement of sodium and calcium across the cell membrane in a reversible, electrogenic manner. The goal of this study was to identify the protein(s) responsible for Na+/Ca[superscript 2+] exchange across cardiac sarcolemma. Purification of the Na+/Ca[superscript 2+] exchanger from canine ventricle was accomplished using the following sequence: 1) isolation of sarcolemma (SL); 2)alkaline extraction of membranes; 3) solubilization with CHAPS; 4) DEAE chromatography; 5) gel filtration HPLC; 6) addition of cholate, salt and phospholipids; 7) wheat germ agglutinin chromatography followed by 8)reconstitution into proteoliposomes by detergent dilution. Specific activity was increased from 5.2 (SL) to 3766 nmol/mg/sec with 100/0 recovery of activity and 0.02% recovery of protein. SDS-PAGE of the purified Na+/Ca[superscript 2+] exchanger preparation under reducing conditions revealed prominent proteins of 75, 120 and 140 kDa. One major protein with a molecular mass of 140 kDa was detected under nonreducing conditions. Polyclonal antibodies against the reconstituted, purified Na+/Ca[superscript 2+] exchanger preparation recognized the three proteins and immunoprecipitated 97.4%+1.3% (n=4) of the Na+/[Ca[superscript 2+] exchange activity from detergent-solubilized sarcolemma. Subsequently, antibodies against the 75, 120and 140 kDa proteins were antigen-purified and found to immunoprecipitate 92%, 91 % and 83%, respectively, of the Na +/Ca[superscript 2+] exchange activity from detergent-solubilized sarcolemma. Furthermore, the antigen-purified antibodies cross-reacted with each of the other two proteins on immunoblots of sarcolemmal protein. Immunoblots with samples prepared from isolated canine ventricular myocytes revealed predominantly the 140 kDa protein and trace amounts of the 75 kDa protein. The 120 kDa protein was not detected. The polyclonal antibodies were subsequently tested for the ability to affect Na +/Ca[superscript 2+] exchange activity manifested by sarcolemmal vesicles. IgG from immune serum stimulated exchange activity 3.5-fold in a concentration-dependent manner (ED[subscript 50] = 0.5 .µg IgG/µg sarcolemmal protein) while IgG from preimmune serum had little or no effect. Curiously, IgG from a rabbit immunized against total sarcolemmal protein stimulated activity nearly as much, but was unable to immunoprecipitate exchange activity. Stimulation appeared to be due to a small increase in V max and a larger decrease in the K[subscript 0.5] for extra vesicular calcium. Molecular cloning of the canine cardiac Na+/ Ca[superscript 2+] exchanger has recently been reported (Nicoll et al., 1990). RNA synthesized from the cDNA clone induced expression of Na +/Ca[superscript 2+] exchange activity when injected into Xenopus oocytes. The cDNA codes for a protein of 970 amino acids (~108 kDa). Antibodies developed against a synthetic peptide based on the deduced amino acid sequence reacted with sarcolemmal proteins of 70, 120 and 160 kDa. Antibodies generated against a partially purified canine cardiac Na+/Ca[superscript 2+] exchanger preparation also recognized proteins of 70, 120 and 160 kDa (Philipson et al., 1988). These antibodies (provided by K. D. Philipson) recognized proteins of 75, 120 and 140 kDa on immunoblots of sarcolemmal protein and the purified Na+/Ca[superscript 2+] exchanger preparation in this laboratory (Ambesi et al., 1991c). These data support the hypothesis that the 70-75, 120 and 140-160 kDa proteins are involved with Na+/Ca[superscript 2+] exchange across cardiac sarcolemma and that the 70-75 and 120 kDa proteins are fragments of the 140-160 kDa protein formed during isolation of the sarcolemmal preparation.

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