Endogenous, Ca(2+)-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle.
The association of an endogenous, Ca(2+)-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and beta-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.1 microM and maximal activity at 10 microM. Mg2+ is also an activator of this enzyme (CI50 = 22 microM). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356-1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383-1400. The RyR cleavage by the Ca(2+)-dependent thiol-protease is prevented in the presence of ATP (1-5 mM) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. A possible involvement of this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity is discussed.
Shoshan-Barmatz
V
V
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Weil
S
S
Meyer
H
H
Varsanyi
M
M
Heilmeyer
L M
LM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D008627:Mercuric Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D015220:Calcium Channels; D007976:Leupeptins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012965:Sodium Chloride; D008627:Mercuric Chloride; D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D002154:Calpain; D008274:Magnesium; C032854:leupeptin; D003545:Cysteine; D002118:Calcium; D004229:Dithiothreitol; D007460:Iodoacetamide