Anexperiment eliminating therotating carrier mechanism forthe active transport ofCaioninsarcoplasmic reticulum membranes

Anexperiment hasbeencarried outtotest the rotating carrier mechanism ofthetranslocation event inmem- brane transport. TotheCa-ATPase inintact sarcoplasmic re- ticulum membranes, 2,4{f3H)dinitrophenyl-cadaverine hasbeen covalently attached bytheaction oftheenzyme, transglutam- inase. Thebinding ofanti-2,4-dinitrophenyl antibodies tothe 2,4-dinitrophenyl-modified membranes wasfound tohaveno effect oneither theCa-ATPase activity ortheCaiontransport rate ofthemembranes. These results rule outtherotating carrier mechanism inthis system. Adifferent scheme forthetranslo- cation process, theaggregate rearrangement mechanism, is discussed. Thetransport ofstnall hydrophilic molecules andions (ligands) through biological membranes isgenerally mediated byspecific proteins that areintegral tothemembrane. Themechanisms proposed forthetranslocation oftheligand fromoneside ofthe membrane totheother can, for ourpurposes, beplaced into two classes (1): (a)thetransport protein rotates across ordiffuses through themembrane, carrying theligand bound ataspecific site ontheprotein fromonemembrane surface totheother (rotating carrier mechanisms) or(b) thetransport protein forms afixed channel that spans thethickness ofthemembrane, through which theligand somehow passes (fixed pore mecha- nisms). Thermodynamic considerations (2, 3)that ledtothe formulation ofthefluid mosaic model ofmembrane structure (4) suggested tousthat arotating carrier mechanism wasmost unlikely (4, 5). Ithasbeenimpossible fromstudies ofthekinetics oftransport todiscriminate between these twoclasses of translocation mechanisms. Oneexperimental approach tosuch discrimination, however, isasfollows. Ifalarge protein mole- cule (antibody, lectin, etc.) canbespecifically andstoichio- metrically bound toatransport protein inanintact membrane, andifsuch binding wereshown tohavenosignificant effect onthekinetics ofthetransport process, then arotating carrier mechanism would beruled out(see Discussion). Whenwe initiated theexperiment reported here, only onesuch experi- ment,carried outinthis laboratory byKyte(6), hadbeen published. Inaddition, since that timetwosimilar experiments have beenreported (7, 8). Theexperiments wecarried outweredesigned toovercome certain ambiguities encountered inthis type ofexperiment (see Discussion). Tothis end, wechose tostudy theCaionactive transport system inmuscle sarcoplasmic reticulum (s.r.) membranes. Toavoid anyquestions about antibody specificity,