Ogata K, Dai X, Volini M
Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu 96822.
J Biol Chem. 1989 Feb 15;264(5):2718-25.
The mitochondrial sulfurtransferase, rhodanese, has been analyzed for phosphate content. Significant amounts of protein-bound phosphate (30-40%) were measured in the six rhodanese preparations examined. Chromatographic experiments followed by phosphate analyses done on two of the preparations indicated that rhodanese A and rhodanese B, two enzyme forms that were previously resolved on DEAE-Sephadex by Blumenthal and Heinrikson (Blumenthal, K., and Heinrikson, R. L. (1971) J. Biol. Chem. 240, 2430-2437), correspond to dephospho- and phosphorhodanese, respectively. The phosphorylation of rhodanese by [gamma-32P]ATP is catalyzed by cAMP-dependent protein kinase. The stoichiometry of 32P incorporation based on the amount of dephosphorhodanese in the enzyme preparation approaches 1.0. The phosphorylation site is accessible in rhodanese that is free of substrate sulfur but not in the covalent enzyme-sulfur intermediate which is formed as an obligatory step during the course of catalysis. Because the cellular localization of cAMP-dependent protein kinase makes it unlikely as the physiologic modulator of rhodanese activity, liver extracts have been tested for a rhodanese kinase that does not require cAMP. Rhodanese kinase activity which is independent of cAMP is observed in extract fractions resolved by Affi-Gel Blue chromatography and freed from endogenous rhodanese by chromatography on Sephadex G-100. These results together with previous findings from this and other laboratories have led to a working model of a bicyclic cascade system that can modulate the rate of mitochondrial respiration. The essence of the model is a transduction and amplification of cellular signals into the altered covalent phosphorylation of rhodanese. Rhodanese, in turn, serves as a converter enzyme which directly alters the rate of the respiratory chain and, thus, ATP production by the reversible sulfuration of key iron-sulfur centers. The model, when expanded to include signal pathways initiated by hormones or neurotransmitters, represents a mechanism by which mitochondria can recognize and meet changing energy demands.
已对线粒体硫转移酶——硫代硫酸硫转移酶进行了磷含量分析。在所检测的六种硫代硫酸硫转移酶制剂中，测定出了大量与蛋白质结合的磷（30 - 40%）。对其中两种制剂进行色谱实验并随后进行磷分析表明，硫代硫酸硫转移酶A和硫代硫酸硫转移酶B这两种酶形式，先前由布卢门撒尔和海因里克森在DEAE - 葡聚糖凝胶上分离得到（布卢门撒尔，K.，和海因里克森，R. L.（1971年）《生物化学杂志》240，2430 - 2437），分别对应脱磷酸硫代硫酸硫转移酶和磷酸化硫代硫酸硫转移酶。[γ - 32P]ATP催化硫代硫酸硫转移酶的磷酸化反应由环磷酸腺苷（cAMP）依赖性蛋白激酶催化。基于酶制剂中脱磷酸硫代硫酸硫转移酶的量，32P掺入的化学计量比接近1.0。磷酸化位点在没有底物硫的硫代硫酸硫转移酶中是可及的，但在催化过程中作为必要步骤形成的共价酶 - 硫中间体中则不可及。由于cAMP依赖性蛋白激酶的细胞定位使其不太可能作为硫代硫酸硫转移酶活性的生理调节剂，因此已对肝脏提取物进行检测以寻找一种不需要cAMP的硫代硫酸硫转移酶激酶。在通过Affi - Gel Blue色谱分离并通过Sephadex G - 100色谱去除内源性硫代硫酸硫转移酶的提取物组分中，观察到了与cAMP无关的硫代硫酸硫转移酶激酶活性。这些结果与本实验室和其他实验室先前的研究结果共同形成了一个双环级联系统的工作模型，该模型可以调节线粒体呼吸速率。该模型的核心是将细胞信号转导并放大为硫代硫酸硫转移酶共价磷酸化的改变。反过来，硫代硫酸硫转移酶作为一种转换酶，通过关键铁硫中心可逆的硫化作用直接改变呼吸链的速率，从而改变ATP的产生。当该模型扩展到包括由激素或神经递质引发的信号通路时，它代表了一种线粒体能够识别并满足不断变化的能量需求的机制。
