Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation.
Xanthine oxidoreductase (XOR) has been purified from lactating mouse mammary tissue and its properties and developmental expression have been characterized. XOR was purified 80-fold in two steps using benzamidine-Sepharose affinity chromatography. The purified enzyme had a specific activity of 5.7 U/mg and an activity to flavin ratio of 192. SDS-polyacrylamide gel electrophoresis showed that it was composed of a single (150 kDa) band and N-terminal sequence analysis verified that it was intact mouse XOR. Isoelectric focusing showed that purified XOR was composed of three catalytically active, electrophoretic variants with pI values of 7.55, 7.65, and 7.70. The majority of the XOR activity in both pregnant and lactating mammary glands was shown to exist as NAD+-dependent dehydrogenase (XD form), while the enzyme in freshly obtained mouse milk exits as O2-dependent oxidase (XO form). The activity and protein levels of XOR selectively increased in mammary tissue during pregnancy and lactation. The time course of these increases was biphasic and correlated with the functional maturation of the mammary gland. These results indicate that XOR may have novel, mammary gland-specific functions, in addition to its role in purine metabolism.
McManaman
J L
JL
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. jim.mcmanaman@UCHSC.edu
Neville
M C
MC
Wright
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001550:Benzamidines; D002846:Chromatography, Affinity; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008667:Metalloproteins / Q000096:biosynthesis / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D008982:Molybdenum; D009243:NAD; D011247:Pregnancy; D012098:Reproduction / Q000502:physiology*; D014968:Xanthine Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification; D014969:Xanthine Oxidase / Q000096:biosynthesis* / Q000302:isolation & purification
D001550:Benzamidines; D007527:Isoenzymes; D008667:Metalloproteins; D009243:NAD; D008982:Molybdenum; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; C032157:benzamidine