METHODS FOR DETERMINING CARDIAC SARCOPLASMIC-RETICULUM CA2+ PUMP KINETICS FROM FURA 2 MEASUREMENTS

We explored the use of four methods for analyzing real and simulated f ura 2 measurements of Ca2+ uptake by membrane vesicles derived from th e sarcoplasmic reticulum (SR) of cardiac muscle. Uptake velocity was c alculated 1) directly from the raw data, 2) after segmenting the raw d ata and averaging the data points in each segment, 3) after smoothing of the raw data by moving-window averaging, and 4) by Savitsky-Golay c onvolution. Methods 2, 3, and 4 could be used to determine maximum upt ake velocity, the Hill coefficient, and the Ca2+ concentration at half -maximal pump velocity from Ca2+ concentration vs. time and velocity c urves that were too noisy to analyze directly. Data analysis using the se methods should have general applicability to biological experiments , especially those in which large numbers of measurements are made. Th e fluorometric method we describe for measuring Ca2+ uptake by cardiac SR vesicles opens up the possibility of studying SR function from ver y small starting tissue samples.