ASSOCIATION OF GLYOXYLATE AND BETA-OXIDATION ENZYMES WITH PEROXISOMES OF SACCHAROMYCES-CEREVISIAE
ASSOCIATION OF GLYOXYLATE AND BETA-OXIDATION ENZYMES WITH PEROXISOMES OF SACCHAROMYCES-CEREVISIAE
复制标题
DOI：
10.1128/jb.172.10.5816-5827.1990
复制
发表时间：
1990-10-01
影响因子：
3.2
通讯作者：
GOODMAN, JM
中科院分区：
文献类型：
--
作者：
MCCAMMON, MT;VEENHUIS, M;GOODMAN, JM
虽然在正常生长条件下很难在酿酒酵母中鉴定出过氧化物体，但当细胞在油酸上培养时，它们就会增殖。我们利用这一发现详细研究了这些细胞器的蛋白质组成。油酸培养细胞的过氧化物体在不连续的蔗糖梯度上纯化；它们迁移到46%至50%(wt/wt)的蔗糖界面。从形态和过氧化体β-氧化途径的所有酶的存在来鉴定过氧化体组分。这些细胞器还含有乙醛酸途径的两种酶--苹果酸合成酶和苹果酸脱氢酶-2的重要但很小的一部分。免疫电子显微镜证实了苹果酸合成酶在过氧化体中的定位。据推测，乙醛酸途径的酶在细胞裂解时很容易和优先地从过氧化体中释放出来，这是它们从分离的细胞器中不完全恢复的原因。用标记酶在蔗糖梯度上检测到来自甘油培养物的未诱导的小的过氧化物体。在这些条件下，过氧化氢酶、酰基辅酶A氧化酶和苹果酸合成酶在接近线粒体峰值的平衡状态下共分离，表明细胞器比生长在油酸上的细胞器更小、密度更低。采用浮力密度离心法对油酸培养物中的过氧化物膜进行纯化。观察到3种丰富的蛋白质，大小分别为24、31和32千道尔顿。
Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid. We used this finding to study the protein composition of these organelles in detail. Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface. The peroxisomal fraction was identified morphologically and by the presence of all the enzymes of the peroxisomal beta-oxidation pathway. These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2. The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy. It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles. Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes. Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid. Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation. Three abundant proteins of 24, 31, and 32 kilodaltons were observed.