Department of Biology, Brandeis University, Waltham, Massachusetts 02154.
Plant Physiol. 1968 Apr;43(4):547-54. doi: 10.1104/pp.43.4.547.
A cell-free system from Chlorella pyrenoidosa Chick (Emerson strain 3) which produces acid-volatile radioactivity from (35)SO(4) (2-) is described. A high speed supernatant from cells broken in the French Press at pH 7.0 shows maximal activity when fortified with ATP, an ATP-generating system (creatine phosphate and creatine phosphokinase), TPN, a TPN-reducing system (glucose-6-phosphate and glucose-6-phosphate dehydrogenase) and MgCl(2). This system is quite labile and is not stable to dialysis. Addition of low concentrations of 2,3,-dimercaptopropan-1-ol (BAL) to the buffers used for enzyme preparation stabilize the extracts and permit them to be dialyzed for 4 hours without loss of activity. If additional BAL is also added to the incubation mixtures it can replace TPNH as a reductant. DPNH also shows appreciable acticity.The system prepared with BAL-containing buffers shows maximal activity at pH 9.0. At this pH, the system requires only ATP, Mg(2+) and additional BAL and has high activity and stability compared with the other conditions tried. The optimum concentrations of these reactants has been determined and the kinetics of production of acid-volatile radioactivity are described. Nucleoside triphosphates other than ATP are not appreciably active in this system. In all cases, anaerobic conditions are required for maximal activity, the enzyme extracts are labile to heat, and no unequivocal requirement for thioctic acid can be demonstrated.
从 Chlorella pyrenoidosa Chick（Emerson 株 3）中提取的无细胞体系可以将（35）SO（4）（2-）转化为酸不稳定放射性物质。在 pH 值为 7.0 的法国压榨细胞破碎后，高速上清液在添加 ATP、ATP 生成系统（磷酸肌酸和肌酸磷酸激酶）、TPN、TPN 还原系统（葡萄糖-6-磷酸和葡萄糖-6-磷酸脱氢酶）和 MgCl（2）时表现出最大活性。该系统非常不稳定，不能耐受透析。向酶制剂缓冲液中添加低浓度的 2,3,-二巯基丙醇（BAL）可稳定提取物，并允许它们在不损失活性的情况下透析 4 小时。如果在孵育混合物中也添加额外的 BAL，它可以代替 TPNH 作为还原剂。DPNH 也表现出明显的活性。用含 BAL 的缓冲液制备的系统在 pH 9.0 时表现出最大活性。在该 pH 值下，该系统仅需要 ATP、Mg（2+）和额外的 BAL，与尝试的其他条件相比，具有较高的活性和稳定性。已确定这些反应物的最佳浓度，并描述了酸不稳定放射性物质产生的动力学。除了 ATP 以外的核苷三磷酸在该系统中没有明显的活性。在所有情况下，都需要无氧条件才能获得最大活性，酶提取物对热不稳定，并且不能明确证明硫辛酸的需求。
