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Effect of digitonin on rat myometrium subcellular membrane fractions
Abstract:
Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.
Insights
Digitonin treatment shifted plasma membrane markers to higher densities in rat myometrium microsomes. This indicates that ATP-dependent calcium uptake is a function of the plasma membrane, not other organelles.
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Biology
Background:
- Rat myometrium plays a crucial role in reproduction.
- Understanding calcium (Ca) uptake mechanisms in myometrium is vital for reproductive health.
- Plasma membrane (PM) and endoplasmic reticulum (ER) are key organelles involved in cellular Ca regulation.
Purpose of the Study:
- To investigate the localization of Ca uptake in rat myometrium.
- To determine whether ATP-dependent Ca uptake is associated with plasma membranes or other cellular compartments.
- To characterize the effects of digitonin on membrane fractions.
Main Methods:
- Isopycnic centrifugation using sucrose density gradients was employed.
- Microsomal and purified membrane fractions were analyzed.
- Distribution of specific membrane markers (5'-nucleotidase, NADPH-dependent cytochrome c reductase, cytochrome c oxidase) was assessed.
- ATP-dependent Ca uptake assays were performed in the presence and absence of digitonin.
Main Results:
- Digitonin treatment shifted the distribution of the PM marker 5'-nucleotidase to higher densities.
- Similar, less pronounced shifts were observed for ER and mitochondrial markers.
- Digitonin inhibited ATP-dependent Ca uptake in a concentration-dependent manner, particularly in fractions N1 and N2.
- ATP-dependent, azide-insensitive Ca uptake was inhibited by digitonin in all fractions.
Conclusions:
- Fractions N1 and N2 are confirmed as subfractions of the plasma membrane.
- ATP-dependent, azide-insensitive Ca uptake in rat myometrium is primarily a function of the plasma membrane.