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Purification and characterization of intact cytochrome b5 from yeast microsomes
Abstract:
A method for purification of detergent-solubilized cytochrome b5 to gel electrophoretic homogeneity from yeast (Saccharomyces cerevisiae) microsomes is described. The purified preparation shows the same absorption spectra as the trypsin-solubilized cytochrome (Y. Yoshida, H. Kumaoka, and R. Sato J. Biochem. 75, 1211-1219 (1974)) in the visible and Soret regions. The detergent-solubilized cytochrome is an amphipathic protein having a monomeric molecular weight of about 18,000 and exists as a hexa- or heptameric aggregate (Mr 122,000) in aqueous media. In the presence of low concentrations of Triton X-100, it interacts effectively with the intact form of NADH-cytochrome b5 reductase purified either from yeast microsomes or from rabbit liver microsomes. Upon trypsin digestion, it is converted to a heme-containing, hydrophilic fragment (Mr 13,000) which retains the spectral characteristics of the original cytochrome, does not form aggregates, and interacts with the reductase only poorly. It is concluded that the preparation purified in this study represents the intact form of yeast cytochrome b5 consisting of a hydrophilic, heme-containing moiety (Mr 13,000) and a hydrophobic, membrane-binding tail (Mr 5000).
Insights
Researchers purified detergent-solubilized cytochrome b5 from yeast microsomes. This amphipathic protein exists as aggregates but can interact with NADH-cytochrome b5 reductase.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Cytochrome b5 is an important protein involved in various metabolic processes.
- Understanding its structure and function is crucial for cellular respiration research.
- Previous studies have characterized trypsin-solubilized cytochrome b5, but detergent-solubilized forms require further investigation.
Purpose of the Study:
- To develop a method for purifying detergent-solubilized cytochrome b5 from yeast microsomes.
- To characterize the properties of the purified yeast cytochrome b5.
- To investigate the interaction of purified cytochrome b5 with NADH-cytochrome b5 reductase.
Main Methods:
- Microsomal preparation from Saccharomyces cerevisiae (yeast).
- Detergent solubilization and purification of cytochrome b5 to gel electrophoretic homogeneity.
- Spectroscopic analysis (visible and Soret regions).
- Molecular weight determination (monomeric and aggregated forms).
- Enzyme interaction studies with NADH-cytochrome b5 reductase.
- Limited proteolysis using trypsin.
Main Results:
- A method was established for purifying detergent-solubilized cytochrome b5 from yeast.
- The purified cytochrome b5 exhibited spectral properties similar to previously described forms.
- The detergent-solubilized cytochrome b5 is an amphipathic protein (Mr 18,000 monomer) that forms aggregates (Mr 122,000).
- It effectively interacts with NADH-cytochrome b5 reductase in the presence of Triton X-100.
- Trypsin digestion yielded a hydrophilic heme-containing fragment (Mr 13,000) with poor reductase interaction.
Conclusions:
- The purified preparation represents the intact form of yeast cytochrome b5.
- Yeast cytochrome b5 consists of a hydrophilic heme-binding part and a hydrophobic membrane-binding tail.
- The intact form is essential for effective interaction with NADH-cytochrome b5 reductase.