Tuinstra Robbyn L, Burgner John W, Miziorko Henry M
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Arch Biochem Biophys. 2002 Dec 15;408(2):286-94. doi: 10.1016/s0003-9861(02)00584-2.
Unprocessed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, retaining the mitochondrial signal sequence, has been proposed to correspond to a peroxisomal isoform. Using a modified expression plasmid and purification protocol, it is now possible to isolate substantial amounts (>10mg) of highly purified peroxisomal HMG-CoA lyase. These improvements facilitate more detailed protein chemistry approaches for characterization of the enzyme, which exhibits substantial (eightfold) dithiothreitol (DTT) stimulation of activity. The C323S mutant shows little DTT activation. Superose gel filtration chromatography data have prompted other investigators to hypothesize that the peroxisomal isoform is a monomer. This study confirms the elution properties presented in that earlier report, but also demonstrates anomalous elution up on Superose chromatography. Elution properties observed using a polyacrylamide resin (Bio-Gel P100) suggest a dimeric, rather than monomeric, enzyme. This observation has been further tested by protein chemistry experiments. The peroxisomal enzyme forms a covalently linked dimeric species upon crosslinking with dibromopropanone or o-phenylenedimaleimide or upon disulfide formation as a result of incubation with diamide. Cysteine-323 is required for intersubunit covalent crosslinking. Crosslinking efficiency is not dependent on HMG-CoA lyase protein concentration nor is it influenced by the presence of varying concentrations of an unrelated protein, such as ovalbumin. Sedimentation equilibrium analyses do not indicate a monomeric form of either human mitochondrial or human peroxisomal HMG-CoA lyase; the results suggest that these proteins are predominantly dimers. The retention of the basic N-terminal mitochondrial signal sequence in the peroxisomal HMG-CoA lyase isoform may influence elution from Superose gel filtration media but does not alter the oligomeric status of the enzyme.
未加工的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）裂解酶保留了线粒体信号序列，有人提出它对应于一种过氧化物酶体异构体。使用改良的表达质粒和纯化方案，现在可以分离出大量（>10mg）高度纯化的过氧化物酶体HMG-CoA裂解酶。这些改进有助于采用更详细的蛋白质化学方法来表征该酶，该酶表现出显著的（八倍）二硫苏糖醇（DTT）对活性的刺激作用。C323S突变体几乎没有DTT激活作用。Superose凝胶过滤色谱数据促使其他研究人员推测过氧化物酶体异构体是单体。本研究证实了早期报告中呈现的洗脱特性，但也证明了在Superose色谱上的异常洗脱。使用聚丙烯酰胺树脂（Bio-Gel P100）观察到的洗脱特性表明该酶是二聚体而非单体。这一观察结果已通过蛋白质化学实验进一步验证。过氧化物酶体酶在与二溴丙酮或邻苯二甲酰亚胺交联时或与二酰胺孵育形成二硫键时会形成共价连接的二聚体物种。亚基间共价交联需要半胱氨酸-323。交联效率不依赖于HMG-CoA裂解酶蛋白浓度，也不受不同浓度无关蛋白（如卵清蛋白）存在的影响。沉降平衡分析未表明人线粒体或人过氧化物酶体HMG-CoA裂解酶的单体形式；结果表明这些蛋白质主要是二聚体。过氧化物酶体HMG-CoA裂解酶异构体中基本的N端线粒体信号序列的保留可能会影响从Superose凝胶过滤介质中的洗脱，但不会改变该酶的寡聚状态。
