Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum.
The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.
Lalonde
R J
RJ
Department of Biology, University of Waterloo, Ontario, Canada.
Lepock
J R
JR
Kruuv
J
J
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002234:Carbodiimides; D003451:Cryoprotective Agents; D016650:Fluorescein-5-isothiocyanate; D005615:Freezing; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000596:Amino Acids; D002234:Carbodiimides; D003451:Cryoprotective Agents; D014364:Tryptophan; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium