Ogasawara Y, Suzuki T, Ishii K, Tanabe S
Department of Hygienic Chemistry, Meiji College of Pharmacy, Tokyo, Japan.
Biochim Biophys Acta. 1997 Feb 11;1334(1):33-43. doi: 10.1016/s0304-4165(96)00072-4.
Liver cytosolic gamma-cystathionase catalyzes the generation of reduced sulfur species, referred to as "bound sulfur,' in the presence of cystine. Incubating a rat liver cytosol fraction in the presence of cystine or oxidized glutathione inactivated certain cytosolic enzyme activities. The activities of cytosolic phosphofructokinase (PFK) and pyruvate kinase rapidly decreased at pH 7.4 during incubation with a lower concentration of cystine than during incubation with oxidized glutathione. Hexokinase and 11 other enzymes in the system were affected minimally or not at all. Adding dithiothreitol to the system reactivated the modified enzymes. Inactivated PFK activity could also be recovered when reduced glutathione or NADPH was added to the cytosol fraction. In these reconstitution systems, purified rat liver PFK was directly inactivated with cystine trisulfide (one of the low molecular types of bound sulfur), but not by cystine (below 0.1 mM). Purified PFK was also inactivated by incubation with cystine plus gamma-cystathionase freshly prepared from cytosol. This was not observed, however, when gamma-cystathionase was pretreated with a specific inhibitor, D,L-propargylglycine. The cystine-dependent inactivation of PFK observed in liver cytosol is shown to be caused mainly by the reaction between bound sulfur and the enzyme, but not by the direct thiol/disulfide exchange. Thus, in vitro modification of the cytosolic enzymes by bound sulfur generated from cystine with gamma-cystathionase has high potency and relatively specific.
肝胞质γ-胱硫醚酶在胱氨酸存在的情况下催化还原型硫物种（称为“结合硫”）的生成。在胱氨酸或氧化型谷胱甘肽存在的情况下孵育大鼠肝脏胞质部分会使某些胞质酶活性失活。在pH 7.4条件下，与氧化型谷胱甘肽孵育相比，较低浓度的胱氨酸孵育期间，胞质磷酸果糖激酶（PFK）和丙酮酸激酶的活性迅速下降。该系统中的己糖激酶和其他11种酶受到的影响最小或根本没有受到影响。向系统中加入二硫苏糖醇可使修饰后的酶重新激活。当向胞质部分加入还原型谷胱甘肽或NADPH时，失活的PFK活性也可以恢复。在这些重构系统中，纯化的大鼠肝脏PFK直接被三硫化胱氨酸（结合硫的低分子类型之一）失活，但不受胱氨酸（低于0.1 mM）的影响。纯化的PFK与胱氨酸加新从胞质中制备的γ-胱硫醚酶一起孵育也会失活。然而，当γ-胱硫醚酶用特异性抑制剂D，L-炔丙基甘氨酸预处理时，未观察到这种情况。在肝脏胞质中观察到的PFK的胱氨酸依赖性失活主要是由结合硫与酶之间的反应引起的，而不是由直接的硫醇/二硫键交换引起的。因此，胱氨酸与γ-胱硫醚酶生成的结合硫对胞质酶的体外修饰具有高效力和相对特异性。
