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Purification and characterization of two unique forms of cytochrome P-450 from rabbit nasal microsomes
1Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109.
Abstract:
Two forms of cytochrome P-450, designated P-450NMa and P-450NMb, were purified to electrophoretic homogeneity from rabbit nasal microsomes. The purified cytochromes, which contained 14-16 nmol of P-450/mg of protein, exhibited apparent monomeric molecular weights of 49,500 and 51,000, respectively. As indicated by several criteria, including the amino acid composition, absorption spectra, and peptide maps, the two nasal forms of P-450 are distinct from each other. Furthermore, as judged by the NH2-terminal amino acid sequences, they are distinct from all other P-450 cytochromes described to date. In the ferric form, P-450NMa is in the low-spin state, whereas P-450NMb is predominantly in the high-spin state. When reconstituted with NADPH-cytochrome P-450 reductase and phospholipid, P-450NMa is very active in the oxidation of ethanol as well as several nasal procarcinogens, including the N-deethylation of N-nitrosodiethylamine, the O-deethylation of phenacetin, and the N-demethylation of hexamethyl-phosphoramide. P-450NMb also metabolizes these substrates, but at lower rates. Both nasal forms are also active with testosterone, with P-450NMa oxidizing the substrate in the 17-position to give androstenedione and P-450NMb catalyzing hydroxylation in the 15 alpha-, 16 alpha-, and 19-positions. The two cytochromes represent the major portion of the total P-450 in nasal microsomes, but the corresponding forms could not be detected in hepatic microsomes.
Insights
Two distinct cytochrome P-450 forms, P-450NMa and P-450NMb, were purified from rabbit nasal microsomes. These enzymes metabolize ethanol, procarcinogens, and testosterone, showing unique activity profiles and spin states.
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Cytochrome P-450 enzymes are crucial for metabolizing xenobiotics and endogenous compounds.
- Nasal P-450 forms play a significant role in the metabolism of inhaled substances, including procarcinogens.
- Understanding specific P-450 isoforms is key to predicting drug efficacy and toxicity.
Purpose of the Study:
- To purify and characterize novel cytochrome P-450 forms from rabbit nasal microsomes.
- To investigate the substrate specificity and catalytic activities of these purified nasal P-450 enzymes.
- To compare the properties of these nasal P-450 forms with known hepatic P-450 enzymes.
Main Methods:
- Purification of cytochrome P-450 to electrophoretic homogeneity from rabbit nasal microsomes.
- Characterization using amino acid composition, absorption spectra, peptide mapping, and N-terminal sequencing.
- Reconstitution assays with NADPH-cytochrome P-450 reductase and phospholipid to determine enzymatic activity.
- Substrate metabolism studies using ethanol, procarcinogens (N-nitrosodiethylamine, phenacetin, hexamethylphosphoramide), and testosterone.
Main Results:
- Two distinct cytochrome P-450 forms, P-450NMa and P-450NMb, were isolated with molecular weights of 49,500 and 51,000 Da, respectively.
- These nasal forms exhibited unique amino acid compositions, spectral properties, and N-terminal sequences, differentiating them from each other and other known P-450s.
- P-450NMa showed high activity in ethanol oxidation and procarcinogen metabolism, while P-450NMb displayed lower rates for these substrates.
- Both forms metabolized testosterone differently: P-450NMa at the 17-position and P-450NMb at the 15α, 16α, and 19-positions.
- P-450NMa was low-spin and P-450NMb was predominantly high-spin in their ferric forms.
- These nasal P-450 forms constituted the major P-450 content in nasal microsomes but were not detected in hepatic microsomes.
Conclusions:
- Rabbit nasal microsomes contain at least two unique cytochrome P-450 forms, P-450NMa and P-450NMb.
- These enzymes possess distinct biochemical and catalytic properties, suggesting specialized roles in nasal xenobiotic metabolism.
- The findings highlight the tissue-specific expression of P-450 enzymes and their potential involvement in the activation or detoxification of inhaled compounds.