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R M Philpot

Publications and source records attributed to R M Philpot.

143 records · Page 8Linked to original sources
PubMedDate not supplied

Cellular localization of flavin-containing monooxygenase in rabbit lung.

A specific form of flavin monooxygenase has been identified in the lungs of a number of species. Distribution of the pulmonary flavin-containing monooxygenase (FMOp) is of interest because it oxidatively metabolizes a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics, some of which form highly toxic reactive intermediates. We have identified the nonciliated bronchiolar epithelial (Clara) cell as the predominant location for this enzyme in rabbit lung. In addition, protein in ciliated, endothelial, type I, and type II cells and in tracheal lining layer reacted with antibodies to FMOp. In all these cell types antigen was found associated with cytoplasmic organelles, and in the Clara cell antigen was most concentrated in areas rich in smooth endoplasmic reticulum. Staining of ciliated surfaces was also observed at both the light and electron microscopy levels. Extracellular antigen was also apparent in tracheal lining layer smeared onto glass slides. We compared the location of the FMOp with that of two enzymes of the cytochrome P-450 monooxygenase system (studied here and elsewhere), cytochrome P450 IIB (P450 IIB), and NADPH cytochrome P450 reductase (reductase), and concluded that (1) FMOp is detected in all cells where P450 IIB and reductase are both present (Clara, type II, and ciliated); (2) FMOp and P450 IIB, but not reductase, are detected in endothelial cells; (3) P450 IIB alone is detected in the plasma membrane, cilia, and microvillae of ciliated cells and plasma membrane of endothelial cells; and (4) FMOp alone is detected in type I cells.

Animals↗
PubMedDate not supplied

Cytochrome P-450 content and mixed-function oxidase activity in microsomes isolated from mouse skin.

A microsomal fraction, prepared from mouse skin, catalyzed the hydroxylation of benzpyrene and aniline and the deethylation of 7-ethoxycoumarin. Contamination of the preparation by cytochrome oxidase and cytochrome P-420 was determined by spectral analysis. The enzyme activities studied in mouse skin (Swiss-Webster CD-1) did not respond to topical application of 3-MC. Twenty-four hours after topical application of TCDD to mice, microsomes from skin had 50% greater benzpyrene hydroxylase and 7-ethoxycoumarin deethylase activity, and 4- to 8-fold greater activity of these enzymes was seen after 72 hr. Increases in cytochrome P-450 content of skin microsomes could be demonstrated 24 and 72 hr after topical TCDD treatment of mice. Cholate treatment (solubilization) of skin microsomes, followed by centrifugation, removed the contaminating cytochrome oxidase. Quantitative and qualitative analyses of cytochrome P-450 difference spectra were made from the solubilized preparations.

Aniline Hydroxylase↗
PubMedDate not supplied

Tissue, species, and substrate concentration differences in the position-selective hydroxylation of N-nitrosodibutylamine. Relationship to the distribution of cytochrome P-450 isozymes 2 (IIB) and 5 (IVB).

The relative rates of 3- and 4-hydroxylation of N-nitrosodibutylamine in microsomal preparations depend upon species, tissue, and substrate concentration. With rabbits, at a substrate concentration of 200 microM, the two reactions differ by less than 30% in preparations from liver, lung, or intestine, whereas the ratio of 4- to 3-hydroxylation is 2.0 with bladder and only 0.4 with kidney. As the substrate concentration is lowered, this ratio increases to a maximum of about 2.0 in hepatic and pulmonary preparations. The sum of the rates of 3- and 4-hydroxylation of N-nitrosodibutylamine in microsomal preparations from untreated rabbits is highest in those from lung (about 2-fold greater than liver). Following treatment of rabbits with phenobarbital, however, the highest rate is with hepatic microsomes (about 2-fold greater than pulmonary microsomes). Antibodies to cytochrome P-450 isozymes 2 (IIB) and 5 (IVB) together inhibit greater than 90% of the microsomal 3- and 4-hydroxylation of N-nitrosodibutylamine. The ratio of 4- to 3-hydroxylation with antibodies to isozyme 2 present is the same (2.0) as that obtained with purified isozyme 5, and the ratio with antibodies to isozyme 5 present is the same (0.2) as that with purified isozyme 2. The Km with isozyme 5 is less than 10 microM, whereas the Km with isozyme 2 is 55 microM, a difference that explains why position selectivity in microsomal incubations is dependent upon substrate concentration. Also, differences in the relative and absolute rates of hydroxylation among various tissues reflect differences in the contents of isozymes 2 and 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Reconstitution of the rabbit pulmonary microsomal mixed-function oxidase system from solubilized components.

Lung microsomal cytochrome P-450 was solubilized and purified 2-fold. NADPH-cytochrome c reductase (EC 1.6.2.3) was solubilized and purified 4-6 fold by three methods with use of sonication and detergent digestion followed by either DEAE-cellulose chromatography or ammonium sulfate fractionation. Benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities were reconstituted when NADPH-cytochrome c reductase and cytochrome P-450 fractions were combined. Reductase fractions prepared by sodium cholate digestion of microsomes were highly active in supporting the hydroxylation activity in the reconstituted systems, whereas those prepared with sodium deoxycholate were not. About twice as much NADPH-cytochrome c reductase was required for saturation of the 7-ethoxycoumarin deethylase activity as for saturation of the benzphetamine N-demethylase activity. A heat-stable lipid fraction was necessary for maximum hydroxylation activity. NADH did not support benzphetamine N-demethylation in the reconstituted system or increase the rate of reaction when added with NADPH.

Animals↗
PubMedDate not supplied

Cytochrome P-450 isozymes 2 and 5 in rabbit lung and liver. Comparisons of structure and inducibility.

Rabbit cytochrome P-450 isozymes 2 and 5 were purified from pulmonary and hepatic microsomal preparations. Purification of isozyme 5 was monitored by immunochemical methods so that contamination by isozymes 2, 4, and 6 could be avoided. Partial proteolysis of hepatic and pulmonary isozyme 5 showed minor differences in peptide formation when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by the silver staining method. In contrast, identical patterns were observed when the peptides were transferred to nitrocellulose paper and visualized immunochemically. The differences observed between the results obtained with the two methods was apparently caused by differences in small amounts of contaminants present in both preparations. HPLC profiles of peptides formed by treatment of pulmonary and hepatic isozyme 5 with trypsin appeared to be the same. In addition, it was found that the pulmonary and hepatic isozymes had identical sequences for the first 20 NH2-terminal amino acids. Three distinct fractions of hepatic cytochrome P-450 isozyme 2 were obtained when chromatography on DEAE-cellulose was used as the final step in the purification procedure. In contrast, only a single fraction of purified pulmonary isozyme 2 was isolated by the same method. Analysis of the pulmonary and three hepatic preparations of isozyme 2 by partial proteolysis and visualization of peptides by silver staining or immunoblotting showed no differences. Analysis of tryptic digests by HPLC also produced the same results for each of the four preparations. The first 24 NH2-terminal amino acids were identical for all four preparations of isozyme 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Metabolism of benzo[a]pyrene by the isolated perfused rabbit lung.

The metabolism of benzo[a]pyrene (BP) was studied in the isolated perfused rabbit lung and in rabbit pulmonary microsomes. Pretreatment of rabbits with 3-methylcholanthrene did not increase the metabolism of BP by microsomal preparations, and the pretreatment did not induce cytochrome P-448 in pulmonary microsomes. In the isolated perfused lung, BP was metabolized at a rate of about 6 nmol/min/g of lung. The intermediate arene oxides formed from BP in the isolated perfused lung were metabolized nonoxidatively by epoxide hydrase and glutathione S-transferases. The rates of the latter reactions were at least an order of magnitude less than the overall rate of metabolism. Pretreatment of the animals with 3-methylcholanthrene increased only the apparent rate of the epoxide hydrase reaction. In the isolated perfused lung, BP and some of its less polar metabolites (i.e., quinones and phenolic derivatives) were preferentially partitioned into lung tissue, precluding accurate measurement of metabolic rates by analysis of the perfusion medium alone. Covalent binding of BP-derived radioactivity to lung tissue occurred, but relatively high variability in this parameter in lungs from 3-methylcholanthrene-pretreated animals did not allow measurement of a significant difference from control lungs.

Animals↗
PubMedDate not supplied

Distribution of microsomal monooxygenases in the rabbit respiratory tract.

The distribution of microsomal cytochrome P-450 isozymes 2, 4, 5, and 6 and the pulmonary FAD-containing monooxygenase was determined in 10 different anatomical regions of the respiratory tract using immunoblot analysis and enzymatic assays. Cytochrome P-450 isozymes 2 and 5 and the FAD-containing monooxygenase were detected by immunoblotting in all of the pulmonary and nasal samples, although levels in nasal tissues were generally much lower than those levels found in the lung. Cytochrome P-450 isozyme 4, which is generally not present in extrahepatic tissues, was detected in nasal ethmoturbinates and mucosa. Different isozymes of cytochrome P-450 appear to be responsible for the N-demethylation of benzphetamine in lung as compared with nasal tissues. Isozyme 2 is responsible for the N-demethylation of benzphetamine in the lung, whereas another isozyme, possibly isozyme 3a is responsible for N-demethylation in nasal tissues. The presence of isozyme 3a in nasal samples was indicated by the presence of high rates of aniline hydroxylation.

Animals↗