Anderson J V, Guy C L
Department of Environmental Horticulture, University of Florida, Gainesville 32611-0512, USA.
Planta. 1995;196(2):303-10. doi: 10.1007/BF00201389.
Spinach (Spinacia oleracea L.) leaf tissue 70-kilodalton heat-shock cognate was purified by ATP-agarose affinity and gel filtration. Gel filtration of the affinity-purified protein resolved it into three forms: monomer, dimer, and oligomer. In the absence of ATP, the majority of the heat-shock cognate existed as a monomeric form with lesser amounts of dimer and oligomer. Addition of 3 mM ATP to the purified protein, containing all three forms, converted the dimeric and monomeric forms to a high-molecular-weight complex. Removal of ATP from the complex by dialysis resulted in the reappearance of the dimeric and monomeric forms. Addition of ATP to the highly purified monomer had no effect on its gel-filtration migration. Neither purified monomeric or dimeric forms showed stable binding to denatured proteins; however, both forms of the purified heat-shock cognate were able to stabilize the enzymatic activity of bovine adrenal glucose-6-phosphate dehydrogenase over a 48-h period at 25 degrees C. In addition, the activity of glucose-6-phosphate dehydrogenase in the presence of purified heat-shock cognate dimer or monomer could be rapidly decreased in an ATP-dependent fashion depending on the order of the substrate addition to the reaction mixture. Circular-dichroism studies indicated that addition of ATP to the spinach 70-kDa heat-shock cognate caused a conformation change from alpha-helical to a greater beta-sheet content. How conformational character may influence the stabilizing activity of the heat-shock cognate in a mechanism which does not require stable peptide binding is discussed.
通过ATP-琼脂糖亲和层析和凝胶过滤法对菠菜（Spinacia oleracea L.）叶片组织中的70千道尔顿热休克同源蛋白进行了纯化。对亲和纯化后的蛋白进行凝胶过滤，结果显示它可分为三种形式：单体、二聚体和寡聚体。在没有ATP的情况下，大多数热休克同源蛋白以单体形式存在，二聚体和寡聚体的含量较少。向含有所有三种形式的纯化蛋白中添加3 mM ATP后，二聚体和单体形式转变为一种高分子量复合物。通过透析去除该复合物中的ATP后，二聚体和单体形式又重新出现。向高度纯化的单体中添加ATP对其凝胶过滤迁移没有影响。纯化后的单体或二聚体形式均未显示出与变性蛋白的稳定结合；然而，在25℃下，纯化后的热休克同源蛋白的这两种形式均能够在48小时内稳定牛肾上腺葡萄糖-6-磷酸脱氢酶的酶活性。此外，根据向反应混合物中添加底物的顺序，在纯化的热休克同源蛋白二聚体或单体存在的情况下，葡萄糖-6-磷酸脱氢酶的活性可能会以ATP依赖的方式迅速降低。圆二色性研究表明，向菠菜70 kDa热休克同源蛋白中添加ATP会导致其构象从α-螺旋转变为β-折叠含量更高的构象。本文讨论了构象特征如何在不需要稳定肽结合的机制中影响热休克同源蛋白的稳定活性。
