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Membrane-bound redox proteins of the murine Friend virus-induced erythroleukemia cell
Abstract:
We have obtained and studied a 105,000-g pellet from T-3-Cl-2 cells, a cloned line of Friend virus-induced erythroleukemia cells. By difference spectrophotometry, the pellet was shown to contain cytochrome b5 and cytochrome P-450, hemeproteins that have been shown to participate in electron-transport reactions of endoplasmic reticulum and other membranous fractions of various tissues. The pellet also possesses NADH-cytochrome c reductase activity which is inhibited by anti-cytochrome b5 gamma-globulin, indicating the presence of cytochrome b5 reductase. This is the first demonstration of membrane-bound forms of these redox proteins in erythroid cells. Dimethyl sulfoxide-treated T-3-Cl-2 cells were also shown to possess membrane-bound cytochrome b5 and NADH-cytochrome c reductase activity. We failed to detect soluble cytochrome b5 in the 105,000-g supernatant fraction from homogenates of untreated or dimethyl sulfoxide-treated T-3-Cl-2 cells. In contrast, erythrocytes obtained from mouse blood were shown to possess soluble cytochrome b5 but no membrane-bound form of this protein. These findings are supportive of our hypothesis that soluble cytochrome b5 of erythrocytes is derived from endoplasmic reticulum or some other membrane structure of immature erythroid cells during cell maturation.
Insights
Researchers identified membrane-bound cytochrome b5 and cytochrome P-450 in erythroid cells, suggesting their role in electron transport. Soluble cytochrome b5 in mature erythrocytes may originate from immature erythroid cell membranes.
Area of Science:
- Biochemistry
- Cell Biology
- Hematology
Background:
- Cytochrome b5 and cytochrome P-450 are hemeproteins involved in endoplasmic reticulum electron transport.
- Erythroid cells, particularly during maturation, undergo significant biochemical and structural changes.
Purpose of the Study:
- To investigate the presence and localization of cytochrome b5 and cytochrome P-450 in erythroid cells.
- To explore the potential origin of soluble cytochrome b5 in mature erythrocytes.
Main Methods:
- Differential centrifugation to isolate cellular fractions from T-3-Cl-2 erythroleukemia cells.
- Difference spectrophotometry to identify hemeproteins.
- Enzyme activity assays (NADH-cytochrome c reductase) and inhibition studies using specific antibodies.
- Comparison with fractions from mouse erythrocytes.
Main Results:
- Identified membrane-bound cytochrome b5 and cytochrome P-450 in a 105,000-g pellet from T-3-Cl-2 cells.
- Demonstrated NADH-cytochrome c reductase activity, indicating the presence of cytochrome b5 reductase, in the erythroid cell pellet.
- Showed that dimethyl sulfoxide treatment of T-3-Cl-2 cells also resulted in membrane-bound cytochrome b5 and reductase activity.
- Found soluble cytochrome b5 in mouse erythrocytes but not in the supernatant of T-3-Cl-2 cells, while membrane-bound forms were present in the latter.
Conclusions:
- This study provides the first evidence of membrane-bound cytochrome b5 and P-450 in erythroid cells.
- The findings support the hypothesis that soluble cytochrome b5 in mature erythrocytes is derived from membrane structures of immature erythroid cells during maturation.