High-Frequency 94 GHz ENDOR Characterization of the Metal Binding Site in Wild-Type Ras·GDP and Its Oncogenic Mutant G12V in Frozen Solution

Abstract

The preparation of affinity adsorbents for menadione reductase (EC 1.6.99.2), using CNBr-activated Sepharose 4B and activated CH-Sepharose 4B, is described. The adsorbents used are the sepharoses modified by 4-anilino-5-methoxybenzoquinone-1,2. Bound menadione reductase is specifically eluted with NAD(P)H, while NAD(P) has no effect on the enzyme. The enzyme yield of 83--95% and a 102-fold purification are achieved. Disc-electrophoresis reveals one protein fraction identified with menadione reductase and a separate inactive fraction. It is assumed that the specific binding of menadione reductase to the adsorbent is due to the formation of a complex between the oxidized enzyme and the quinone. This complex breaks down in the presence of NAD(P)H.