Developmental expression of vitamin K-dependent gamma-carboxylase activity in zebrafish embryos: effect of warfarin.
Vitamin K-dependent gamma-carboxylation is an essential posttranslational modification required for the functional activity of coagulation proteins such as factors VII, IX, X, and prothrombin. Warfarin, an inhibitor of vitamin K-dependent gamma-carboxylation, was used in earlier work on adult zebrafish to provide evidence for the presence of vitamin K-dependent carboxylase in zebrafish. Here we demonstrate the presence of vitamin K-dependent carboxylase activity in zebrafish by directly assaying the microsomal fraction prepared from adult, unfertilized eggs, and embryos from different developmental stages. Gamma-carboxylase activity was detected both before and after fertilization of embryos and the activity levels remained relatively constant from 6 h postfertilization (hpf) through other advanced stages of development. The expression of activity in the early embryos (0-6 hpf) may be due to the presence of maternal protein since the activity was detected even in the unfertilized eggs. Gamma-carboxylase activity in the eggs as well as early embryos suggested that vitamin K-dependent carboxylase is important throughout development. The detection of vitamin K-dependent carboxylase mRNA by RT-PCR and inhibitor studies using warfarin confirmed these activity results. Further, these studies provide a basis for selecting warfarin-resistant zebrafish mutants in order to find genes regulating gamma-carboxylase activity including the yet unidentified vitamin K-epoxide reductase.
Hanumanthaiah
R
R
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Thankavel
B
B
Day
K
K
Gregory
M
M
Jagadeeswaran
P
P
D000595:Amino Acid Sequence; D000818:Animals; D019735:Carbon-Carbon Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000196:embryology / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010063:Ovum / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D014859:Warfarin / Q000494:pharmacology; D015027:Zebrafish
D012333:RNA, Messenger; D014859:Warfarin; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase