Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: VII. SOLUBILIZATION PROPERTIES OF MENADIONE REDUCTASE

Abstract

Abstract When NaCl concentration was lowered, membrane vesicle-bound menadione reductase of Halobacterium cutirubrum showed 6- to 7-fold increased activity. The enzyme with this increased activity was initially found in a transient, membrane-bound form, followed by release from the membranes. Several detergents caused solubilization and a similar increase in enzyme activity, but sodium dodecyl sulfate, which increased activity, gave only limited solubilization. Unlike mitochondrial DPNH dehydrogenase, the halophilic system showed only minor changes in substrate affinity upon these treatments. Conditions that resulted in highly active, membrane-bound enzyme also released trapped horseradish peroxidase, which had been added as a marker of the interior of these vesicles. These results suggest that menadione reductase is attached to the interior surface of the vesicles and becomes accessible to the substrates upon the breakage of the vesicles during the course of solubilization. Half of the menadione reductase content of the vesicles was released into the supernatant in 1 m NaCl but was still attached to large membrane fragments. As NaCl concentration was further lowered the enzyme in this fraction was detached and could be eluted from an Agarose column in a position corresponding to 36,000 molecular weight. In addition, at NaCl concentrations lower than 1 m, large membrane fragments containing the second portion of the enzyme were released. Tween 80 solubilized half of the total enzyme in the vesicles; the solubilized enzyme was eluted at 48,000 molecular weight, which probably corresponds to a protein-detergent complex. The sequential action of lowered NaCl concentration and Tween 80 resulted in the recovery of all the enzyme in this low molecular weight fraction.