Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle.
In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.
Shoshan-Barmatz
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V
Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Pressley
T A
TA
Higham
S
S
Kraus-Friedmann
N
N
D000818:Animals; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D002478:Cells, Cultured; D003620:Dantrolene / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D015345:Oligonucleotide Probes; D051381:Rats; D011919:Rats, Inbred Strains; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012965:Sodium Chloride / Q000494:pharmacology
D015345:Oligonucleotide Probes; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D012965:Sodium Chloride; D004110:Diltiazem; D003620:Dantrolene