NADH-ferricyanide reductase of rat liver plasma membrane and HeLa plasma membrane: A novel target for antitumor agents
Abstract
The objectives of the study were to purify, and characterize the anticancer drug-responsive NADH-ferricyanide reductase of rat liver plasma membrane and HeLa plasma membrane. The effect of the antitumor drugs, doxorubicin and LY181984, an antitumor sulfonylurea, on the enzyme activity was studied. Plasma membranes were highly purified (90% or greater) by aqueous two phase partition from rat liver homogenate. Initially, an NADH-ferricyanide reductase was purified from rat liver plasma membrane 2230 fold with an apparent molecular weight of 31.5 kDa. Partial amino acid sequence of the enzyme showed that it had identical sequence homology with rat NADH-cytochrome b$\sb5$ reductase. This enzyme was insensitive to antitumor drug doxorubicin. This enzyme was removed from the plasma membrane by cathepsin D treatment of the membranes. The remaining NADH-ferricyanide reductase activity was sensitive to doxorubicin and was purified 200 fold over the cathepsin D treated rat liver plasma membranes with an apparent molecular weight of 57 kDa. It had also an NADH-coenzyme Q$\sb0$ (CoQ$\sb0)$ reductase (NADH: External Acceptor (Quinone) Reductase (EC 1.6.99..)) activity. Partial amino acid sequence of the enzyme showed that it is unique and had no known sequence homology to any known redox enzyme. Antibody against the enzyme was produced and the affinity purified antibody immunoprecipitated both the doxorubicin-sensitive NADH-ferricyanide reductase activity and NADH-CoQ$\sb0$ reductase activity. Using the antibody-coupled affinity column, NADH-CoQ$\sb0$ reductase of rat liver plasma membrane and HeLa plasma membrane was purified. The NADH-CoQ$\sb0$ reductase was found to be a transmembrane glycoprotein. The doxorubicin-inhibited enzyme activity was inhibited as well by an antitumor drug sulfonylurea LY181984 half maximally at 1$\mu$M. Thus the enzyme may be a target for the action of certain antitumor drugs.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Biochemistry|Oncology
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