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The Preparation of Microsomal Fractions of Rodent Respiratory Tract and Their Characterization¹

Department of Biochemistry, The University of Texas Health Science Center, Dallas, Texas 75235

A method for preparing rodent lung microsomes using normal differential centrifugation methods is described which allows the spectral characterization of these lung fractions. Further, procedures have been established to quantitate the microsomal cytochromes b₅ and P-450 in microsomes, which contain appreciable amounts of contaminating hemoglobin and methemoglobin. Physical biochemical characterizations of the rodent lung microsomal cytochromes include reduced-minus-oxidized difference spectra, low-temperature difference spectra, substrate-binding difference spectra, and enzymatic cytochrome P-450 reduction studies. In general, the lung microsomal cytochromes are similar to those found in the liver microsomes except that their specific content is 20- to 40-fold lower than in liver. The lung microsomal fraction appears to use reduced nicotinamide adenine dinucleotide more effectively as an electron donor to reduce cytochrome P-450 than do liver microsomal fractions.

Administration i.p. of 3-methylcholanthrene induces the rodent lung microsomal cytochrome P-450-dependent mixed-function oxidase system by increasing the specific content of cytochrome P-450 and shifts the difference-absorption maximum of the CO derivative of reduced cytochrome P-450 from 450 nm to 448 nm. Phenobarbital treatment was without effect on the content of cytochrome P-450. Although benzphetamine demethylase and biphenyl hydroxylase activities in lung microsomes from 3-methylcholanthrene-induced animals are as high as the activities of normal liver, the lung benzo(a)pyrene hydroxylase is 400-fold less active than the comparable liver hydroxylase of control animals and the induced lung benzo(a)pyrene hydroxylase is 100-fold less active than the hydroxylase of liver microsomes from carcinogen-treated rats.

¹ Supported by National Cancer Institute Contract NO1 CP 33362.

² Present address: The Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, Japan.

³ To whom correspondence should be directed.

Received 4/15/74. Accepted 5/10/74.

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