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Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle
Abstract:
From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35--45% sucrose and a small protein fraction at 45--55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35--45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35--45%. The NADH-oxidase activity was highest in the 35% fraction and the 5'-nucleotidase activity in the 40,000 X g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [3H]-ouabain and the incorporation of [3H]-leucine was most pronounced in the 35% fraction. In a K+-free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35--45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.
Insights
Researchers isolated rabbit colon muscle subcellular fractions using differential centrifugation. The 35% sucrose fraction, rich in endoplasmic reticulum and plasma membrane markers, showed significant ATP hydrolysis and protein synthesis.
Area of Science:
- Cell Biology
- Biochemistry
- Muscle Physiology
Background:
- Understanding the specific functions of subcellular components in muscle tissue is crucial for comprehending muscle physiology and disease.
- Rabbit colon muscle serves as a model system for investigating smooth muscle cell structures and their biochemical properties.
Purpose of the Study:
- To isolate and characterize subcellular fractions from rabbit colon muscle.
- To investigate the biochemical properties and morphological features of these fractions.
- To identify the cellular origins of different sucrose density gradient fractions.
Main Methods:
- Differential centrifugation was employed to isolate crude microsomal fractions from rabbit colon muscle homogenates.
- Sucrose density gradient centrifugation was used to separate microsomes into subfractions.
- Biochemical assays were performed to determine the distribution of lipids, RNA, and enzyme activities (e.g., cytochrome c oxidase, NADH-oxidase, 5'-nucleotidase, ATPase, adenylate cyclase, phosphodiesterase).
- Morphological characterization and analysis of specific molecule binding ([3H]-ouabain, [3H]-leucine) and ATP hydrolysis were conducted.
Main Results:
- Fractions were obtained at 35% sucrose (vesicular microsomes), 35-45% sucrose (sarcolemma, mitochondria, microsomes), and 45-55% sucrose (protein).
- The 35% sucrose fraction showed high NADH-oxidase activity, RNA localization, and stimulated ATP hydrolysis in the presence of Ca2+, indicating endoplasmic reticulum and plasma membrane components.
- The 35-45% sucrose fraction contained sarcolemma, mitochondrial fragments, and microsomal vesicles, with significant cytochrome c oxidase contamination and distributed cholesterol and phospholipids.
Conclusions:
- The 35% sucrose fraction is primarily derived from the endoplasmic reticulum and plasma membrane.
- The 35-45% sucrose fraction originates from the plasma membrane, mitochondria, and to a lesser extent, the endoplasmic reticulum.
- These distinct fractions provide a basis for further studies into the specific roles of these subcellular components in rabbit colon muscle function.