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Studies on the heterogeneity of sarcoplasmic reticulum vesicles
Abstract:
Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.
Insights
Rabbit white muscle sarcoplasmic reticulum vesicles were separated into light and heavy fractions. Both fractions originate from sarcoplasmic reticulum membranes, with the light fraction identified as "inside-out" vesicles.
Area of Science:
- Muscle physiology
- Cellular biology
- Biochemistry
Background:
- Sarcoplasmic reticulum (SR) is crucial for muscle calcium regulation.
- SR vesicles are often studied using isolated fractions.
- Understanding SR vesicle orientation is key to interpreting functional data.
Purpose of the Study:
- To characterize and differentiate light and heavy sarcoplasmic reticulum vesicle fractions from rabbit white muscle.
- To determine the orientation of vesicles within these fractions.
- To investigate the implications of fraction composition on SR function.
Main Methods:
- Density gradient centrifugation to isolate sarcoplasmic reticulum (SR) vesicle fractions.
- Analysis of protein patterns, Ca2+-binding proteins, and Ca2+-dependent ATPase activity.
- Immunological cross-reactivity assays.
- Assessment of vesicle permeability and trypsin digestion.
- Lipid distribution analysis.
Main Results:
- Both light and heavy fractions derived from SR membranes, showing similar protein patterns but different acidic, Ca2+-binding protein ratios.
- The 105,000 dalton protein in the light fraction cross-reacted with the Ca2+-dependent ATPase in the heavy fraction.
- Ca2+-dependent ATPase activity was present in both fractions, reaching equal specific activity after permeabilization.
- Light fraction vesicles exhibited characteristics of "inside-out" orientation, including low Ca2+ accumulation, altered trypsin digestion, and reversed aminophospholipid distribution compared to heavy fraction vesicles.
- Light SR fraction had a higher phospholipid to protein ratio.
Conclusions:
- Both light and heavy fractions are derived from sarcoplasmic reticulum membranes.
- The light fraction consists of "inside-out" vesicles, explaining functional differences observed.
- Fraction composition and vesicle orientation suggest derivation from distinct regions of the sarcoplasmic reticulum system.