Cobley J G, Singer T P, Beinert H, Grossman S
J Biol Chem. 1975 Jan 10;250(1):211-7.
It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th
据报道,在铁限制条件下连续培养的产朊假丝酵母细胞,在存在环己酰亚胺的情况下通气时,以及在生长过程中增加铁供应时,会发生位点1磷酸化,而不会出现对杀粉蝶菌素敏感的现象,并且NADH脱氢酶的铁硫中心也不会发生变化。本研究对这些发现进行了重新调查。在这些转变过程中跟踪的参数和特性包括NADH氧化对杀粉蝶菌素的敏感性、偶联位点1的有无、用NADH或连二亚硫酸钠还原时出现的EPR信号、NADH氧化酶、NADH-铁氰化物还原酶和NADH-5-羟基-1,4-萘醌(胡桃醌)还原酶的比活性,以及铁氰化物测定中NADH脱氢酶的动力学行为。通过监测亚线粒体颗粒中NADH与人工氧化剂的氧化速率、观察铁氰化物测定的动力学以及测量EPR引发的铁硫中心浓度,可以确定不同实验情况下存在的NADH脱氢酶类型及其相对浓度。研究发现,在连续培养过程中逐渐增加铁浓度(从铁限制生长转变为铁和底物限制生长)时,以及对铁限制细胞进行通气时,偶联位点1、对杀粉蝶菌素的敏感性、NADH-铁氰化物活性以及铁硫中心1和2会同时增加,同时NADH-胡桃醌还原酶活性会下降。环己酰亚胺阻止了所有这些变化。在这些转变过程中,铁硫中心3和4的增加相对较少。得出的结论是,在这两种实验条件下,指数期细胞中存在的NADH脱氢酶类型被静止期细胞特有的类型所取代,并且与后一种酶相关的位点1磷酸化、对杀粉蝶菌素的敏感性以及铁硫中心1和2的出现是这种取代的结果。没有发现没有出现对杀粉蝶菌素敏感现象而偶联位点1发生发展的证据。