Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells.
The Ca2+-ATPase accounts for the majority of Ca2+ removed from the cytoplasm during cardiac muscle relaxation. The Ca2+-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca2+-ATPase activity by decreasing the apparent affinity of the ATPase for Ca2+. To study the physical mechanism of Ca2+-ATPase regulation by phospholamban using spectroscopic and kinetic experiments, large amounts of both proteins are required. Therefore, we developed a Ca2+-ATPase and phospholamban preparation based on the baculovirus-insect cell expression system using High-Five insect cells to produce large amounts of microsomal vesicles that contain either Ca2+-ATPase expressed alone or Ca2+-ATPase co-expressed with phospholamban. The expressed proteins were characterized using immunofluorescence spectroscopy, Ca2+ -ATPase activity assays, Ca2+ uptake and efflux assays, and Western blotting. Our purification method yields 140 mg of microsomal protein per liter of infection (1.7 x 10(9)cells), and the Ca2+-ATPase and phospholamban account for 16 and 1.4%, respectively, of the total microsomal protein by weight, yielding a phospholamban:Ca2+-ATPase ratio of 1.6:1, similar to that observed in native cardiac SR vesicles. The enzymatic properties of the expressed Ca2+-ATPase are also similar to those observed in native cardiac SR vesicles, and when co-expressed with phospholamban, the Ca2+-ATPase is functionally coupled to phospholamban similar to that observed in cardiac SR vesicles.
Waggoner
Jason R
JR
Department of Biochemistry and Molecular Pharmacology, West Virginia University School of Medicine, Morgantown, WV 26506-9142, USA.
Huffman
Jamie
J
Griffith
Brian N
BN
Jones
Larry R
LR
Mahaney
James E
JE
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000235:genetics*; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D015202:Protein Engineering / Q000379:methods; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012717:Sesquiterpenes / Q000494:pharmacology; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004533:Egtazic Acid; D007783:Lactones; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012717:Sesquiterpenes; D000099211:Phospholamban; C046319:thapsigargicin