Ade P, Banchelli Soldaini M G, Castelli M G, Chiesara E, Clementi F, Fanelli R, Funari E, Ignesti G, Marabini A, Orunesu M
Ecotoxicol Environ Saf. 1984 Oct;8(5):423-46. doi: 10.1016/0147-6513(84)90064-2.
Differential centrifugation methods already in use were applied to purify rat, quail, and trout liver microsomes and modified as necessary to purify microsomes from mussel digestive gland and whole water flea. All these microsomal preparations were comparatively characterized with respect to protein and RNA content, levels of markers of subcellular contaminants, ultrastructural morphology, differential spectra of cytochromes P-450 and b5, monoxygenase activity, and in vitro metabolism of p-dichlorobenzene. Yields of microsomal proteins of the tested organisms differed widely, with mussel showing the lowest yield. Very low levels of nuclear and mitochondrial contaminants were detected in all microsomal preparations, but cell membrane contaminants were clearly present in most preparations. Daphnia microsomes were significantly contaminated by plasma membranes, and hepatopancreas microsomes contained significant amounts of partially disrupted secretory granules and plasma membrane. From a qualitative standpoint differential spectra of cytochrome b5 were very similar for all the preparations, whereas cytochrome P-450 spectra were largely dependent on the microsomal preparation as well as on the assay method used. The content of cytochrome P-450 was highest for rat liver microsomes and very low or absent in Daphnia and mussel preparations; the range of cytochrome b5 contents was much narrower. Significant differences were observed among monoxygenase activities of the different preparations. In Daphnia and mussel microsomes, aniline hydroxylase was absent and benzo[a]pyrene hydroxylase activity was much lower than in rat and quail microsomes. Benzo[a]pyrene hydroxylase activity of trout liver microsomes was similar to that of rat and quail microsomes, whereas hydroxylation of substrates which in rat liver are preferentially metabolized by phenobarbital-inducible forms of cytochrome P-450 was much lower in trout microsomes.
采用已有的差速离心法来纯化大鼠、鹌鹑和鳟鱼的肝脏微粒体，并根据需要进行改进，以纯化贻贝消化腺和整个水蚤的微粒体。对所有这些微粒体制剂在蛋白质和RNA含量、亚细胞污染物标记物水平、超微结构形态、细胞色素P - 450和b5的差异光谱、单加氧酶活性以及对二氯苯的体外代谢方面进行了比较表征。受试生物微粒体蛋白的产量差异很大，贻贝的产量最低。在所有微粒体制剂中均检测到极低水平的核和线粒体污染物，但大多数制剂中明显存在细胞膜污染物。水蚤微粒体被质膜显著污染，肝胰腺微粒体含有大量部分破碎的分泌颗粒和质膜。从定性的角度来看，所有制剂中细胞色素b5的差异光谱非常相似，而细胞色素P - 450光谱在很大程度上取决于微粒体制剂以及所使用的测定方法。大鼠肝脏微粒体中细胞色素P - 450的含量最高，而水蚤和贻贝制剂中的含量非常低或不存在；细胞色素b5含量的范围要窄得多。不同制剂的单加氧酶活性存在显著差异。在水蚤和贻贝微粒体中，不存在苯胺羟化酶，苯并[a]芘羟化酶活性远低于大鼠和鹌鹑微粒体。鳟鱼肝脏微粒体的苯并[a]芘羟化酶活性与大鼠和鹌鹑微粒体相似，而在鳟鱼微粒体中，大鼠肝脏中优先由苯巴比妥诱导型细胞色素P - 450代谢的底物的羟化作用要低得多。
