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Purification and activation properties of pyruvate carboxylase from the fungus Aspergillus nidulans
| Content Provider | Semantic Scholar |
|---|---|
| Author | Marston, Fiona A. O. Osmani, Stephen A. Scrutton, Michael C. |
| Copyright Year | 1982 |
| Abstract | Previous studies on a partially purified pyruvate carboxylase from the related fungus, Aspergillus niger. have shown that this enzyme is fully active in the absence of acetyl-CoA but is inhibited by L-aspartate (Bloom & Johnson, 1961; Feir & Suzuki, 1969). We have recently reported that activation of a partially purified preparation of A . nidulans pyruvate carboxylase by acetyl-CoA can be observed in the presence of L-aspartate or of a-oxoglutarate (Osmani et al., 198 1). Homogeneous preparations of A . nidulans pyruvate carboxylase have been obtained by subjecting the partially purified preparation described previously (Osmani et al., I98 1) to chromatography on hydroxylapatite, followed by gel filtration on Sepharose 6B and then chromatography on DEAESephadex A-50 (Sod2form). The purified enzyme migrates as a single band when subjected to polyacrylamide-gel electrophoresis in the absence and presence of sodium dodecyl sulphate. Under the former conditions a single band of enzymic activity, determined as described by Scrutton & Fatabene (1979, is coincident with the protein band. The subunit molecular weight of A. nidulans pyruvate carboxylase obtained as described by Weber & Osborn (1969) is 125000, in agreement with that found for other pyruvate carboxylase that are subject to activation by acyl derivatives of coenzyme A and inhibition by dicarboxylic acids (Utter et al., 1975). An antibody raised to the purified enzyme in rabbits gives a single precipitin line in Ouchtorlony diffusion analysis and causes complete inhibition of enzymic activity. By using this homogeneous enzyme preparation we have confirmed that acyl-CoA activation is not observed in the absence of the regulatory inhibitors and in the presence of saturating substrate concentrations. However 2-3-fold activation by acetyl-CoA is observed at pH 7.4 in the presence of non-saturating concentrations of MgATP2and pyruvate. This activation results primarily from a decrease in the apparent K , for pyruvate and a decrease in the Hill coefficient (h) describing the relationship between initial rate and pyruvate concentration. With MgATP2as varied substrate, addition of acetyl-CoA causes an increase in apparent V,,,,,, without affecting the apparent K , or h for this substrate. The properties of interaction of A. nidulans pyruvate carboxylase with HC0,are unaffected by addition of acetyl-CoA. Variation of MgATP2or HC0,concentration has no significant effect on the apparent K , or h for acetyl-CoA. These studies demonstrate that pyruvate carboxylase from A . nidulans has both molecular and activation properties resembling those of other regulated pyruvate carboxylase. However, the activating effect of acetyl-CoA is weaker than that described for any other pyruvate carboxylase that shows this response. |
| Starting Page | 132 |
| Ending Page | 132 |
| Page Count | 1 |
| File Format | PDF HTM / HTML |
| DOI | 10.1042/bst0100132 |
| Volume Number | 10 |
| Alternate Webpage(s) | http://www.biochemsoctrans.org/content/ppbiost/10/2/132.1.full.pdf |
| Alternate Webpage(s) | https://doi.org/10.1042/bst0100132 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
