Sugumaran M, Nellaiappan K
Department of Biology, University of Massachusetts, Boston 02125.
Arch Insect Biochem Physiol. 1990;15(3):165-81. doi: 10.1002/arch.940150305.
The phenoloxidase system responsible for the sclerotization of cockroach ootheca is found to be present as an inactive form in the left colleterial gland of Periplaneta americana. The supernatant fraction obtained by centrifugation of the milky white secretions contained the inactive phenoloxidase which required both sodium dodecyl sulfate (SDS) and the insoluble sediment for exhibiting enzyme activity. Bovine serum albumin could replace the sediment in the activation process. Proteins separated from the supernatant fraction by molecular sieve chromatography on Sephadex G-25 did not require either albumin or the sediment, but required SDS for exhibiting the phenoloxidase activity. Among the detergents tested, SDS (anionic) and cetylpyridinium chloride (cationic) activated the phenoloxidase, but CHAPS (zwitterionic) or nonionic detergents failed to activate the enzyme. The activation caused by SDS occurred well below the critical micellar concentration of SDS indicating that SDS is causing the activation by binding to the protein and altering its conformation. Chloroform-methanol extracts of vestibulum or right gland could replace SDS confirming the presence of endogenous activator(s) of phenoloxidase system. A variety of exogenously added lipids could activate the latent enzyme, among which linoleate, oleate, laurate, linolenate, phosphatidylethanolamine, and phosphatidylglycerol proved to be the effective activators of the latent phenoloxidase. Partially purified phenoloxidase was found to be extremely labile and lost its activity on a) freezing and thawing, b) dialysis, and c) heating for 10 min at 55 degrees C. It exhibited a pH optimum of 7 and was inhibited drastically by phenylthiourea and diethyldithiocarbamate. It readily oxidized a number of o-diphenols such as 3,4-dihydroxybenzylalcohol, 3,4-dihydroxyphenethyl alcohol, catechol, N-acetyldopamine, N-acetylnorepinephrine, dopa, dopamine, etc., but failed to oxidize both 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde. It neither converted the typical laccase substrate syringaldazine to its quinone methide product, nor oxidized the p-diphenols, hydroquinone and methylhydroquinone. Therefore, the enzyme participating in the quinone tanning of cockroach ootheca appears to be a typical o-diphenol oxidase and not a laccase as previously thought.
负责蟑螂卵鞘硬化的酚氧化酶系统，在美国蟑螂的左粘液腺中以无活性形式存在。通过离心乳白色分泌物获得的上清液部分含有无活性的酚氧化酶，该酶需要十二烷基硫酸钠（SDS）和不溶性沉淀物才能表现出酶活性。牛血清白蛋白可以在激活过程中替代沉淀物。通过Sephadex G-25分子筛色谱从上清液部分分离的蛋白质，既不需要白蛋白也不需要沉淀物，但需要SDS来表现酚氧化酶活性。在所测试的去污剂中，SDS（阴离子型）和十六烷基氯化吡啶（阳离子型）激活了酚氧化酶，但CHAPS（两性离子型）或非离子型去污剂未能激活该酶。SDS引起的激活发生在SDS的临界胶束浓度以下，这表明SDS通过与蛋白质结合并改变其构象来引起激活。前庭或右腺的氯仿-甲醇提取物可以替代SDS，证实了酚氧化酶系统内源性激活剂的存在。多种外源添加的脂质可以激活潜在的酶，其中亚油酸、油酸、月桂酸、亚麻酸、磷脂酰乙醇胺和磷脂酰甘油被证明是潜在酚氧化酶的有效激活剂。部分纯化的酚氧化酶被发现极其不稳定，在以下情况下会失去活性：a）冷冻和解冻，b）透析，以及c）在55℃加热10分钟。它的最适pH值为7，并且被苯硫脲和二乙基二硫代氨基甲酸盐强烈抑制。它很容易氧化多种邻二酚，如3,4-二羟基苄醇、3,4-二羟基苯乙醇、儿茶酚、N-乙酰多巴胺、N-去甲肾上腺素、多巴、多巴胺等，但不能氧化3,4-二羟基苯甲酸和3,4-二羟基苯甲醛。它既不能将典型的漆酶底物丁香醛连氮转化为其醌甲基化物产物，也不能氧化对二酚、对苯二酚和甲基对苯二酚。因此，参与蟑螂卵鞘醌鞣制的酶似乎是一种典型的邻二酚氧化酶，而不是先前认为的漆酶。
