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).
Department of Food and Nutrition, Kinki University, Nara, Japan.
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S
Shigeoka
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Nakano
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