RNA synthetic activity of glutamate dehydrogenase: determination of enzyme purity, RNA characteristics, and deamination/amination ratio.
The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.
Osuji
Godson O
GO
Cooperative Agricultural Research Center, Prairie View A&M University, Texas A&M University System, PO Box 4079, Prairie View, TX 77446, USA. Godson_Osuji@PVAMU.edu
Konan
Jonas
J
M'Mbijjewe
Gitonga
G
D000586:Amination; D010367:Arachis / Q000201:enzymology*; D001483:Base Sequence; D048271:Chitosan; D018076:DNA, Complementary / Q000235:genetics; D003641:Deamination; D020869:Gene Expression Profiling; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D036226:Seedlings / Q000201:enzymology; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D012313:RNA; D048271:Chitosan; D005969:Glutamate Dehydrogenase