Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates.
Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.
Ji
Y
Y
Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0542, USA.
Loukianov
E
E
Periasamy
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005615:Freezing; D066298:In Vitro Techniques; D017136:Ion Transport; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D008851:Micropore Filters; D008861:Microsomes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium