Inactivation of rice bran thiamine-binding protein by N,N'-dicyclohexylcarbodiimide.
The addition of a carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) to thiamine-binding protein isolated from rice bran resulted in a remarkable loss of its binding activity with [14C]thiamine. Thiamine and chloroethylthiamine substantially protected the protein against inactivation by DCCD, whereas thiamine phosphates did not. Another carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inactivated rice bran thiamine-binding protein. Inactivation of the thiamine-binding protein was accompanied by covalent binding of DCCD to the protein as shown by the use of [14C]DCCD. The binding of [14C]DCCD to the thiamine-binding protein was specific, and significantly inhibited by the addition of thiamine. The loss of thiamine-binding activity was proportional to the specific binding of [14C]DCCD. For complete inactivation of the thiamine-binding activity, the binding of 2.46 mol of [14C]DCCD per mol of thiamine-binding protein was required. Furthermore, limited proteolysis of the binding protein by trypsin yielded two polypeptides with molecular weights of 35,000 (large polypeptide) and 12,500 (small polypeptide) which were separated by SDS-polyacrylamide gel electrophoresis. The binding sites of [14C]DCCD were found to be located on the large polypeptide. These results suggest that a specific carboxyl residue in the large polypeptide releasable from rice bran thiamine-binding protein by trypsin digestion when modified by DCCD is involved in the binding of thiamine.
D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D012275:Oryza; D010944:Plants / Q000378:metabolism*; D011485:Protein Binding; D011804:Quinolines / Q000494:pharmacology; D013831:Thiamine / Q000378:metabolism*
D002234:Carbodiimides; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D011804:Quinolines; C026989:thiamine-binding protein; D004024:Dicyclohexylcarbodiimide; C000470:EEDQ; D013831:Thiamine