Hasson E P, West C A
Division of Biochemistry, Department of Chemistry, University of California, Los Angeles, California 90024.
Plant Physiol. 1976 Oct;58(4):479-84. doi: 10.1104/pp.58.4.479.
Cytochrome P-450 and cytochrome b(5) at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b(5) reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b(5) and DPNH-cytochrome b(5) reductase activity were largely separated from one another and from an overlapping mixture of TPNH-cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination.The possible participation of these components in the mixed function oxidation of ent-kaur-16-ene and a number of its oxidized derivatives catalyzed by these microsomes is discussed in relation to the model which has been developed to explain the function of analogous components in mixed function oxidase reactions in mammalian liver microsomes.
通过光谱测量，在从未成熟的大果马拉种子胚乳组织制备的微粒体中，检测到细胞色素P - 450和细胞色素b(5)的含量分别约为每毫克微粒体蛋白0.10和0.60纳摩尔。这些微粒体中还存在TPNH - 细胞色素c还原酶、DPNH - 细胞色素c还原酶和DPNH - 细胞色素b(5)还原酶活性，其含量分别约为每毫克微粒体蛋白0.060、0.22和0.52单位。（一个还原酶单位是指每分钟催化还原1微摩尔电子受体的酶量。）用胰脂肪酶或胰蛋白酶处理微粒体，均无法有效地使这些电子传递成分以可检测的形式溶解。然而，用1%脱氧胆酸钠处理25%甘油中的微粒体悬浮液，导致约60%的蛋白质以及上述每种血红蛋白和电子传递活性释放到在105,000g下离心2小时后未沉淀的部分。去除去污剂后，在溶解部分可检测到一些贝壳杉-16-烯氧化酶活性。当脱氧胆酸钠溶解部分在DEAE - 纤维素柱上进行色谱分析时，细胞色素b(5)和DPNH - 细胞色素b(5)还原酶活性在很大程度上彼此分离，并与TPNH - 细胞色素c还原酶和DPNH - 细胞色素c还原酶的重叠混合物分离。在柱级分中未检测到细胞色素P - 450或细胞色素P - 420，并且当单独或组合测试柱级分时，未检测到贝壳杉-16-烯氧化酶活性。本文结合已建立的用于解释哺乳动物肝脏微粒体混合功能氧化酶反应中类似成分功能的模型，讨论了这些成分可能参与这些微粒体催化的贝壳杉-16-烯及其多种氧化衍生物的混合功能氧化反应。
