Studies of fragmented sarcoplasmic reticulum from human skeletal muscle

Abstract

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.

Calcium accumulation by the sarcoplasmic reticulum in two populations of chemically skinned human muscle fibers. Effects of calcium and cyclic AMP.

“…On this basis the data of Table I yield 0.18 t.mol/mg protein for quadriceps SR in the absence of oxalate and 6 [tmol/mg protein with oxalate present (4 t,mol/mg for pectoralis; see Table 11) . These values for SR capacity in skinned fibers compare favorably with isolated-vesicle preparations from human muscles, where steady-state capacities were 0.08 [,mol/mg protein for loading without oxalate (Takagi, 1971 ;Scarpa et al, 1978) . With oxalate, the capacity of isolated vesicles increased to 2.0 f,mol/mg protein for gastrocnemius plus vastus lateralis (Samaha and Gergely, 1965) and 3.9 tmol/mg protein for pectoralis (Takagi, 1971) .…”