Glutamate dehydrogenase (NADP-dependent) mRNA in relation to enzyme synthesis in Euglena gracilis. Evidence for post-transcriptional control.
Cells of Euglena gracilis Klebs strain z Pringsheim had high NADP-dependent glutamate dehydrogenase activity when grown on glutamate as nitrogen source but activity was completely repressed in cells grown on ammonium (NH4+). A 120-fold purification of NADPH-glutamate dehydrogenase (subunit Mr = 45 000) was achieved from glutamate-grown cells by affinity chromatography on blue Sepharose CL-6B. Antisera raised against the homogeneously pure protein were used to demonstrate that increase in NADPH-glutamate dehydrogenase activity on transfer from NH4+ to glutamate medium resulted from an increase in the amount of protein. Glutamate NH4+-grown cells were labelled with L-[35S]methionine and anti-(NADPH-glutamate dehydrogenase) used to immunoprecipitate the dehydrogenase from cell extracts. NADPH-glutamate dehydrogenase protein was detected in glutamate-grown but not NH4+-grown cells. Anti-(NADPH-glutamate dehydrogenase) was used to detect NADPH-glutamate dehydrogenase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in glutamate NH4+-grown cells, there being no apparent difference in mRNA abundance between cells showing a tenfold difference in NADPH-glutamate dehydrogenase specific activity. These results indicate that the synthesis of this dehydrogenase is regulated primarily at the post-transcriptional level.
Parker
J E
JE
Javed
Q
Q
Merrett
M J
MJ
D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005056:Euglena gracilis / Q000201:enzymology* / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018086:Glutamate Dehydrogenase (NADP+); D007120:Immunochemistry; D008715:Methionine / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010779:Photofluorography; D011061:Poly A / Q000032:analysis; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*
D003470:Culture Media; D012333:RNA, Messenger; D011061:Poly A; D008715:Methionine; D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+); D009584:Nitrogen