The effect of thiamin on the activation of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in Euglena gracilis.
The effect of thiamin on thiamin pyrophosphate-dependent 2-oxoglutarate (2-OG) decarboxylase activity in Euglena gracilis was investigated. The total activity of 2-OG decarboxylase in thiamin-sufficient cells in 3 times that in thiamin-deficient cells. The addition of thiamin to thiamin-deficient cells causes the total enzyme and holoenzyme activities to increase and reach similar levels to that in thiamin-sufficient cells. Cycloheximide and chloramphenicol, inhibitors of protein synthesis, have no effect on the total enzyme activity. Immunochemical titration and determination of 2-OG decarboxylase mRNA by using an antibody directed against Euglena 2-OG decarboxylase indicate that the increase in the holoenzyme activity of 2-OG decarboxylase is due to activation of pre-existing protein and does not require synthesis of new proteins in thiamin-deficient cells. During the period of the increase in the total activity, the apoenzyme increases and reaches a temporary peak in 2 h. Immunoblot analysis demonstrates that the precursor form (a 65 kDa subunit) of 2-OG decarboxylase in thiamin-deficient cells is more abundant than that in thiamin-sufficient cells and the increase in the apoenzyme by addition of thiamin results from the conversion of the precursor form into the mature form (a 62 kDa subunit).
Shigeoka
S
S
Department of Food and Nutrition, Kinki University, Nara, Japan.
Nakano
Y
Y
D000818:Animals; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005056:Euglena gracilis / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D013831:Thiamine / Q000494:pharmacology*
D011756:Diphosphates; D004792:Enzyme Precursors; D012333:RNA, Messenger; D002262:Carboxy-Lyases; C021474:2-oxoglutarate decarboxylase; D013831:Thiamine