Continuous monitoring of Ca2+ uptake in membrane vesicles with fura-2.
The Ca2+-selective, fluorescent dye, fura-2, was used to monitor ATP-dependent Ca2+ uptake by membrane vesicles isolated from rabbit skeletal muscle. Micromolar fura-2 concentrations, added outside the vesicles, served as a high-affinity, low-capacity Ca2+ buffer. Changes in fura-2 fluorescence resulted from the decline in free Ca2+ concentration [( Ca2+]free) owing to active Ca2+ accumulation by the vesicles. Ca2+ uptake (delta[Ca2+]total) was calculated from changes in [Ca2+]free and from the Kd value for the fura-2-Ca2+ complex. The velocity of Ca2+ uptake determined in this manner had an apparent [Ca2+]0.5 of approximately 200 nM. The Hill coefficient for dependence of uptake velocity on [Ca2+]free was congruent to 2. Changes in [Ca2+]free and Ca2+ uptake expected for Ca2+ transport by skeletal muscle sarcoplasmic reticulum were determined theoretically from known kinetic parameters and found to be similar to experimental values. This method of directly monitoring Ca2+ uptake can be used to determine the kinetic parameters for Ca2+ transport with small amounts of vesicles and with greater precision than possible with radiometric techniques.
Kargacin
M E
ME
Department of Physiology, University of Massachusetts Medical School, Worcester 01655.
Scheid
C R
CR
Honeyman
T W
TW
D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D016257:Fura-2; D066298:In Vitro Techniques; D008991:Monitoring, Physiologic / Q000379:methods*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013997:Time Factors
D001572:Benzofurans; D002118:Calcium; D016257:Fura-2