Studies of fragmented sarcoplasmic reticulum from human skeletal muscle

Sarcoplasmic reticulum (SR) vesicles were isolated from muscle biopsies of 4 normal volunteers, a patient with McArdle disease (before and during contracture), and a patient with normokalemic periodic paralysis. Fractions were analyzed for purity by electron microscopy and biochemical analysis of specific marker enzymes. Adenosine triphosphate (ATP)-dependent calcium ion uptake was measured kinetically by the absorbence changes of murexide, a metallochromic indicator of ionized Ca++ concentrations, in the absence of oxalate or other calcium-complexing anions. In these experiments, time resolution of the Ca++ transport rate was limited by the manual mixing of reagents, which occurred in 1 to 3 seconds. In 1 case the "true" initial velocity of Ca++ uptake was measured by rapid mixing of ATP in a stopped-flow apparatus and by following the change in absorbence of murexide in a storage oscilloscope. In SR from normal human muscle the ATP-dependent Ca++ uptake was 3.5 nmoles per second per milligram of protein for the first 5 seconds after ATP mixing. Fast kinetic experiments showed that Ca++ uptake proceeded linearly for the first 500 msec at a rate of 9 nmoles per second per milligram of protein (at 25 degrees C) and then progressively declined to reach steady-state levels in 40 to 50 seconds. No abnormality of Ca++ transport was found in SR vesicles from the patient with McArdle disease and the patient with normokalemic periodic paralysis.