During a lactoperoxidase-catalysed iodination study of the topographical distribution of proteins in isolated rat liver nuclei and nuclear envelope, it was observed that the controls without exogenous peroxidase also incorporated lz5I into the proteins, which could be prevented reversibly by 5 m~-2-mercaptoethanol, 5 mM-NaN3, 10mM-KCN and irreversibly by 0.2m~-3-amino-l,2,4-triazole, in the presence of 40p~-H,O,. Endogenous peroxidase activity has been demonstrated cytochemically by Strum & Karnovsky (1970), who showed a wide subcellular distribution of theenzyme in thyroid follicular cells. We here report on the distribution and activity of a peroxidase found in subcellular fractions from rat liver. Adult male Wistar rats (approx. 200-25Og body weight) were used for the preparation of nuclei and nuclear envelope by the method of Harris & Milne (1974), with the addition of O.OOOl% butylated hydroxytoluene to the homogenization medium as recommended by Welton & Aust (1972). Mitochondria were prepared from the post-nuclear supernatant by centrifuging at 3000ga,. for 10min. The pellet was discarded and the supernatant recentrifuged at 8000ga,. for 10min. The pelleted mitochondria were washed three times in homogenization medium at 8000ga,. for lOmin and finally resuspended in homogenization medium. The post-mitochondria1 supernatant was centrifuged at 100000g,,, for 60min to pellet the total microsomal fraction (rough plus smooth), which was resuspended in homogenization medium by gentle homogenization. The clear post-microsomal supernatant was carefully withdrawn from below the floating fatty layer and constituted the soluble fraction. Plasma membranes were isolated by the procedure described by Touster et al. (1970). All operations were performed at W I T . The subcellular fractions were assayed by two methods: method 1, spectrophotometrically, using 2,2’-azino-di-(3-ethylbenzothiazoline-6-sulphonate) (diammonium salt) and HzOz as substrates (Boehringer, 1975) with the addition of Triton X-100 (Sigma (London) Chemical Co., Kingston upon Thames, U.K.) to a final concentration of 0.1 %; method 2, by lZ5I incorporation; typically this was a 75Opl suspension of membrane in homogenization medium (nuclear envelope was in 10mM-Tris/HC1, pH7.4), 1OOpCi carrier-free Nalz5I (The Radiochemical Centre, Amersham, U.K.) followed by five additions of 3,d of 1 . 2 5 ~ 10-4~-Hz02 at 5min intervals, at 4°C. Incorporated radioactivity was measured by trichloroacetic acid precipitation using the ‘disc-batch’ method in the presence of 50m~-K’~’ I (Hubbard & Cohn, 1975). The cellulose acetate discs (Millipore AA, 0.8pm) were counted for radioactivity in a Nuclear Enterprises model NE 1600 y-counter. Controls were carried out in the absence of H202. Protein determinations were performed by using the method of Lowry et al. (1951). Table 1 compares the results of the iodine incorporation assay with those of the spectrophotometric assay. Plasma membrane, released chromatin, mitochondria and the soluble fraction showed no detectable activity. Both assays show very close agreement although the microsomal specific activities were variable, but low enough to be considered as trace contamination by nuclear envelope. This point was investigated further by subjecting the tissue to increasing homogenization, with samples of the total homogenate taken at intervals of 1, 2, 5, 7.5 and 10min. The total microsomal fraction was prepared from each sample, and assayed by the spectrophotometric procedure. Table 2 shows that increased amounts of peroxidase activity were associ-
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Feb 1, 1983
S H Kaufmann + 2
