Abstract
Reactive disulfide reagents (RDSs) with a biotin moiety have been synthesized and found to cause Ca2+ release from sarcoplasmic reticulum (SR) vesicles. The RDSs oxidize SH sites on SR proteins via a thiol-disulfide exchange, with the formation of mixed disulfide bonds between SR proteins and biotin. Biotinylated RDSs identified a 106-kDa protein which was purified by biotin-avidin chromatography. Disulfide reducing agents, like dithiothreitol, reverse the effect of RDSs and thus promoted active re-uptake of Ca2+ and dissociated biotin from the labeled protein indicating that biotin was covalently linked to the 106-kDa protein via a disulfide bond. Several lines of evidence indicate that this protein is not Ca2+, Mg2+-ATPase and is not a proteolytic fragment or a subunit of the 400-kDa Ca2+-ryanodine receptor complex (RRC). Monoclonal antibodies against the ATPase did not cross-react with the 106-kDa protein, and polyclonal antibodies against the 106-kDa did not cross-react with either the ATPase or the 400-kDa RRC. RDSs did not label the 400-kDa RRC with biotin. Linear sucrose gradients used to purify the RRC show that the 106-kDa protein migrated throughout 5-20% linear sucrose gradients, including the high sucrose density protein fractions containing 400-kDa RRC. Protease inhibitors diisopropylfluorophosphate used to prevent proteolysis of 400-kDa proteins did not alter the migration of 106-kDa in sucrose gradients nor the patterns of biotin labeling of the 106-kDa protein. Incorporation of highly purified 106-kDa protein (free of RRC) in planar bilayers revealed cationic channels with large Na+ (gNa+ = 375 +/- 15 pS) and Ca2+ (gCa2+ = 107.7 +/- 12 pS) conductances which were activated by micromolar [Ca2+]free or millimolar [ATP] and blocked by micromolar ruthenium red or millimolar [Mg2+]. Thus, the SR contains a sulfhydryl-activated 106-kDa Ca2+ channel with apparently similar characteristics to the 400-kDa "feet" proteins.
Highlights
RDSs oxidize SH sitesonSRproteinsviaathioldisulfide exchange,with the formationof mixed disul- In the previous article, we have described a class of “reacfide bonds between sarcoplasmic reticulum (SR) proteins and biotin
The present study shows that RDSs can be used to crosslink biotin to an SRprotein containing the critical sulfhydryl site involved in the opening and closing of a Ca2+channel pathway
The reduction of the newly formed disulfide bond betweetnhe SR protein and the biotin resulted in active re-uptake of Ca2+
Summary
SPDP and SPDP-BiotCinonjugates Induce SRCa2+Release kDaantibody (1:250 dilutionin blockingbuffer), (ii)preimmune uiu a n SH Oxidation Reaction-To test the effect of SPDP serum (1:250 dilution inblocking buffer), or (iii) monoclonal antibody and its bioticnonjugates on SRCa2+ releaset,he vesicles were directedagainst Ca2+,Mg2'-ATPase (1:lOOO dilutionin blocking buffer) for 4, 4, and 1 h, respectively. Of sulfhydryl reagent that causes rapid Ca2r+elease from the This protein represented approximately 0.3% of total SR protein, a polypeptide with an apparent molecular mass of -106 kDa as determined by SDS-PAGE. After the completion of Caz+efflux, DTT was added to reduce the disulfidebond linking biotin to SR proteins which resulted in active re-uptake of Ca2+by the vesicles. Higher concentration of these reagents increased the rates and extent of SR Caz+. The measurement shows that PDP-biotin hydrazide oxidized a maximum of 1.7 nmol of SH/mg SR protein From such a measurement, it cannot be determined how many of these SH sites aredirectly associated with Ca2+-releasechannelproteins and thenumber of oxidized SH/Ca2+release channel is not known. The avidin beads were washed with buffer to elutenonbiotinylated proteins, washed with DTT to dissociate the proteins linked to biotin via a disulfide bond and tocollect
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