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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.
Insights
Sarcoplasmic reticulum (SR) calcium uptake is normal in McArdle disease and periodic paralysis. This study measured ATP-dependent calcium transport in muscle SR vesicles, finding no abnormalities in patients with these conditions.
Area of Science:
- Muscle physiology
- Biochemistry
- Cellular transport
Background:
- Sarcoplasmic reticulum (SR) plays a crucial role in muscle contraction by regulating intracellular calcium levels.
- Disruptions in SR calcium handling are implicated in various muscle diseases.
- McArdle disease and normokalemic periodic paralysis are genetic disorders affecting muscle function.
Purpose of the Study:
- To investigate the adenosine triphosphate (ATP)-dependent calcium ion (Ca++) uptake in sarcoplasmic reticulum (SR) vesicles.
- To determine if abnormalities in SR Ca++ transport are present in patients with McArdle disease and normokalemic periodic paralysis.
Main Methods:
- Isolation of SR vesicles from muscle biopsies of normal volunteers and patients.
- Analysis of SR vesicle purity using electron microscopy and enzyme assays.
- Kinetic measurement of ATP-dependent Ca++ uptake using the metallochromic indicator murexide.
- Utilized stopped-flow apparatus for high-time-resolution kinetic measurements.
Main Results:
- Normal human SR exhibited an initial ATP-dependent Ca++ uptake rate of 3.5 nmoles/sec/mg protein.
- High-time-resolution studies revealed an initial linear Ca++ uptake rate of 9 nmoles/sec/mg protein (at 25°C) for the first 500 msec.
- No significant abnormalities in Ca++ transport kinetics were observed in SR vesicles from patients with McArdle disease or normokalemic periodic paralysis.
Conclusions:
- The ATP-dependent calcium transport function of sarcoplasmic reticulum is not impaired in McArdle disease or normokalemic periodic paralysis.
- These findings suggest that the underlying defects in these conditions do not directly involve the SR Ca++ pump mechanism.