| Autor: |
Richard B. Loeble, John L. Aull, R. Bruce Dunlap |
| Rok vydání: |
1974 |
| Předmět: |
|
| Zdroj: |
Journal of Biological Chemistry. 249:1167-1172 |
| ISSN: |
0021-9258 |
| DOI: |
10.1016/s0021-9258(19)42956-6 |
| Popis: |
SUMMARY Carboxypeptidase A treated with diisopropylphosphorofluoridate (DFP) present at catalytic levels causes the complete inactivation of thymidylate synthetase. The rate of this inactivation is shown to be proportional to the concentration of carboxypeptidase A-DFP. Carboxypeptidase B-DFP also inactivates thymidylate synthetase, but at a much slower rate than carboxypeptidase A-DFP. That the inactivation of thymidylate synthetase observed was due solely to carboxypeptidase activity was illustrated as follows: (a) carboxypeptidase A-DFP and carboxypeptidase B-DFP samples were found to exhibit no chymotryptic or tryptic activity; (b) when present in equal molar ratios (2: 1) with respect to thymidylate synthetase, carboxypeptidase A-DFP caused total inactivation of thymidylate synthetase after 2’7 min of incubation, while less than 10% inactivation of the enzyme was found after 2 hours of incubation with either chymotrypsin or trypsin; (c) the presence of 5 x lop5 M L-benzylsuccinic acid, a potent competitive inhibitor of carboxypeptidase A, was sufficient to prevent both the carboxypeptidase A and the carboxypeptidase B-dependent inactivation of thymidylate synthetase. Native thymidylate synthetase and carboxypeptidase-inactivated enzyme behave in an identical fashion upon electrophoresis on polyacrylamide gels in the presence and absence of sodium dodecyl sulfate. Results of the Stark method suggested that the COOHterminal residue of thymidylate synthetase is either valine or phenylalanine. High voltage electrophoresis performed on incubation mixtures of thymidylate synthetase and carboxypeptidase A-DFP indicated that valine was rapidly released, followed by alanine. Analysis of the amino acids released by the carboxypeptidase A-DFP treatment of thymidylate synthetase in the presence of sodium dodecyl sulfate gave 1.76 moles of valine and 2.00 moles of alanine per mole of enzyme. Analysis of the amino acids released by the carboxypeptidase A-DFP treatment of native thymidylate synthetase as a function of the extent of inactivation showed that only valine was released under these conditions and that total inactivation corresponded closely to the release of 1 mole of valine per mole of thymidylate synthetase. These results are considered in light of the fact that thymidylate synthetase is thought to be composed of |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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