Absence of pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in Morris hepatoma 7777

Abstract

Morris hepatoma 7777 previously has been shown to have no detectable pyridoxine- (pyridoxamine-) 5'-phosphate oxidase activity [Thanassi, J. W., Nutter, L. M., Meisler, N. T., Commers, P., & Chiu, J.-F. (1981) J. Biol. Chem. 256, 3370-3375]. In order to determine if this enzyme was missing in the hepatoma, we purified rat liver oxidase and raised antibodies to it in rabbits. Final purification of rat liver oxidase for use as an antigen was accomplished by affinity chromatography and gel electrophoresis. The rat liver enzyme is similar to rabbit liver oxidase [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436-3442] having two noncovalently linked subunits with molecular weights in the range of 25 000-28 000. Evidence indicating that inactive enzyme was simultaneously purified with native enzyme was obtained. The IgG fraction was purified from the serum of a rabbit that had been immunized with rat liver oxidase. This was used in the development of ELISA and immunoblot analyses for the presence of antigenically active pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in cytosolic preparations from normal rat liver and Morris hepatoma 7777. The results indicated that there was no immunologically detectable oxidase protein in the tumor. An alternate pathway of pyridoxal 5'-phosphate synthesis, involving oxidation of pyridoxine to pyridoxal followed by phosphorylation, was ruled out. The implications of these findings with respect to acquisition of nutrients by tumors are discussed.

Diseases Associated with Defects in Vitamin B6 Metabolism or Utilization

“…Thanassi and coworkers (120,163,164) have established that pyridoxine (pyridoxamine) 5' -phosphate oxidase is low or absent from hepatomas, which implies that these cells must obtain PLP from circulation. Hepatocarcinogens decrease the levels of enzymes involved in PLP synthesis without altering the activity of the phosphatase (59).…”

Effect of tryptophan metabolites on the activities of rat liver pyridoxal kinase and pyridoxamine 5-phosphate oxidase in vitro

“…Pyridoxamine 5-phosphate oxidase has previously been purified 1375-fold by Nutter et al (1983) from rat liver, but its properties have not been elucidated. The optimum pH of rabbit liver enzyme has been reported to be 9-10 with 0.2M- Tris/HCl (Kazarinoff & McCormick, 1975), and that in pig brain enzyme was pH 8.4 in 0.1 M-pyrophosphate buffer (Kwok & Churchich, 1980).…”

“…However, the properties of rat liver pyridoxamine 5-phosphate oxidase have not been elucidated. Only Nutter et al (1983) purified this enzyme, in order to prepare the antibody, and they reported that the purified enzyme preparation contained both active and inactive enzymes, and the subunit Mr of the oxidase was 25000-28000.…”

Absence of Pyridoxine-5‘-Phosphate Oxidase (PNPO) Activity in Neoplastic Cells: Isolation, Characterization, and Expression of PNPO cDNA

“…Remarkably, few studies of the status of PLP and the enzymes involved in its metabolism have been conducted in reference to neoplasia and are limited to those using a rodent hepatocellular carcinoma model (19)(20)(21)(22). Interestingly, PNPO enzyme activity and protein were not detectable in several rat hepatomas (19)(20)(21)(22).…”

“…Remarkably, few studies of the status of PLP and the enzymes involved in its metabolism have been conducted in reference to neoplasia and are limited to those using a rodent hepatocellular carcinoma model (19)(20)(21)(22). Interestingly, PNPO enzyme activity and protein were not detectable in several rat hepatomas (19)(20)(21)(22). It was found that the lack of PNPO activity in these hepatic tumors followed an oncofetal developmental pattern; that is, certain liver tumors exhibited undetectable and/or low PNPO activity, comparable to or even lower than those found in fetal liver.…”