j Mol Cell Cardiol 19 (Supplement IV) (1987)

109 POLYCLONAL ANTIBODIES AGAINST THE PURIFIED SUBUNITS OF THE VOLTAGE SENSITIVE CALCIUM CHANNEL. Q. Yi and B.S. Tuaua. Departme,lt of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Canada, M5S lAg.

The voltage sensitive calcium channel from rabbit skeletal muscle was purified by lectin affinity chromatography. Reversible labelling of the associated calcium antagonist receptor with various calcium antagonists was used to identify and estimate the purity of the calcium channel. Polypeptides of 145, I00, 56 and 32 kDa were found to be enriched in the purified receptor fraction. The polypeptides wer~ separated in SDS-PAGE and antibodies were raised against each protein in guinea-pigs. Antibodies against the 145 kDa polypeptide pp145 Ab recognized the 145 and 50 kDa polypeptides in western blots of the purified receptor fraction. Similarly antibodies against the 50 kDa protein and the 32 kDa protein reacted with the 145 and 56 kDa polypeptides of the purified receptor. In native transverse tubule membranes the pp145 AD reacted against a fragment of 28 kDa. A fragment of 28 and 5b kDa was also recognized by pp145 Ab in sarcolemmal membranes from cardiac and vascular smooth muscle as well as brain plasma membranes. The pp145 Ab and pp56 Ab specifically inhibited the binding of dihydropyridiues to the Ca 2+ channel. These results suggest that the lower molecular weight proteins may be proteolytic products of a larger precursor. In addition, the data de:nonstrate that the polypeptide(s) comprising the Ca 2+ channel from different tissues share coml~1on epitopes. (Supported by MRC.)

110 COVALENT MODIFICATION OF CALCIUM ANTAGONIST RECEPTORS IN HEART SARCOLEMMA. B.J. Murphy and B.S. Tuana. Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, CANADA, M5S fag.

The binding of the dihydropyridine-type calcium antagonist [3H]PN20<)-I[0 (p) was studied in rabbit heart muscle. P binding sites were found to be enriched in the sarcolemma[ (SL) membrane fraction. Due to the proteinaneous nature of the dihydropyridine binding site we examined the possible role of disulfide (-SS-), sulfhydryl (SH) and phosphate (PO4) groups in the specific biuding of P in cardiac sarcolemma. DTT and reduced glutathione were employed to assess the role of disulfide groups in P binding. DTT had no significant effect on P bincling over a wide concentration range (I0 -I[ - 10-2M). Reduced glutathione however was a potent inhibitor of P binding (IC50 = 0.5 urn). NEM, PMC and iodoacetamide were employed to investigate the effect of alkylation of free sulfhydryl groups on P bindb~g. PMCS completely inhibited P binding (IC50 = 20 uM). NSM ollly partially inhibited P binding, 40% at 100 mM, while iodoacetamide was without a~fect on P binding over the concentration range (I0 -I0 - IO-2M). cAHP-depeudent phosphorylation of SL membranes did not alter the characteristics of P binding. Ca2+/cahnodulin-dependent phosphorylation of cardiac (SL) and skeletal t-tubule membranes increased P binding by 15% without altering the K D. (Supported by OHSF.)

111 MONOCLONAL ANTIBODY THAT IMMUNOPRECIPITATES SKELETAL MUSCLE 1,4-DIHYDROPYRIDINE RECEPTORS, H. Smilowitz, R.J. Chang~ and C. Bowik ~. Dept. of Pharmacology, Univ. of Conn. Health Center, Farmington, CT 06032.

Rabbit skeletal muscle membranes (I0 pmole 1,4-dihydropyridine bi~ding sites/rag protein) were preklabeled with the dihydropyridine antagonist H PN200-110, extracted with digitonin and subjected to wheat get~ affinity chromatography (approximately I00-150 fold purification) followed by Sephacryl S-400 chromatography (approximately 2-fold purification). Generally 500 mg of starting membrane protein was used. When an eight protease inhibitor cocktail was used, about 0.5 to 1 mg of protein (40-50% 1,4-d~hydropyridine receptor) was obtained in about 20 hours. SDS PAGE (non-reducing conditions) revealed two major broad a bands upon coomassie staining at about 142-155 Kd and 155-170 Kd as well as less prominent bands at ~57 Kd and ~30 Kd. When only four protease inhibitors were used, the lower

was selectively lost followin~ Sephacryl S-400 chromatography. Under reducing conditions, most of the ~ materlal ran at 135-145 Kd; some protein stain remained in the 145-170 Kd region of the gel. Wheat germ purified material (about I000 pmole PN200-110 sites/mg protein) was used as immunogen. Mice were repeatedly injected until their serum gave a 1/2 maximum signal on ELISA at about a 1/5000 dilution. Spleen cells were fused with NSO myeloma cells. Screening of hybridoma supernatants was first by ELISA. Positives were screened again by immunoblot, Several hybridoma supernalants that ir~aunoblotted to a -170 Kd polypeptide were obtained and the cells were cloned. One such clone, antibody #78 (IgG1) , has been purified on Protein A agarose and investigated in greater detail, it has been found to: (a) inm~unoblot to a 150-170 Kd polypeptide under bo~h non-reducing and reducing conditions; (b) immunoprecipitate 20-30% of the H PN200-110 labeled material extracted from prelabeled skeletal membranes. We are currently testing the hypothesis that antibody #78 binds to the skeletal muscle 1,4-dihydropyridine sensitive calcium channel. Supported by NIH grant HL33026.

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