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As expected, conventional phosphorylation of rabbit liver with [gamma-32P]ATP in the presence of Mg2+ resulted in hundreds of phosphorylated proteins. For the specific detection of proteins phosphorylated on histidine, a method was utilised which exclusively detects histidine phosphoproteins. Phosphorylation of rabbit liver with [gamma-32P]ATP in the presence of EDTA labeled only three proteins with molecular masses of about 20, 40 and 110 kDa under denaturing electrophoretic conditions. Addition of protein histidine phosphatase (PHP) resulted in the dephosphorylation of the 110 kDa protein. In contrast, histidine phosphoproteins of 20 and 40 kDa were not affected by PHP.
Following its phosphorylation in the presence of EDTA the 110 kDa protein was purified and detected by autoradiography. The three chromatographic steps of the purification were anion exchange, affinity chromatography and gelfiltration. Gelfiltration revealed the surprising result that we were dealing with a protein of ~450 kDa. The discrepancy in its molecular mass depending on native conditions (gelfiltration: 450 kDa) versus denaturing conditions (SDS-PAGE: 110 kDa) was indicative of a multimeric enzyme.
Sequence information revealed peptides of ATP-citrate lyase (ACL). The identity of ACL was further verified by taking advantage of the fact that phosphate bound to ACL can be removed by ADP but not by GDP, AMP and ATP.
In summary, the ATP-citrate lyase was identified as the first physiological substrate of protein histidine phosphatase.
Molecular/biological techniques were established to get information concerning the structural aspects of PHP. Recombinant baculoviruses as vectors were used to express the PHP in cultured insect cells. Afterwards the protein was purified with metal affinity resins. There were no differences between the recombinant PHP and the native enzyme due to their activities. In analogy mutants of the PHP were expressed and purificated. In the first case Cys(73) was mutated to Ala(73). Another mutation to Ala was the complete change of the nucleotide-binding region (G(75)xG(77)xxS(80)). By deleting ten amino acids at the N- as well as at the C-terminus further mutations were made.
Assays with ACL as the substrate showed that not only the expressed wildtype was active but also the mutation Cys(73). Therefore, Cys(73) is not essential for the catalytic activity. The other mutations showed a loss of the enzymatic activity. The autophosphorylated ACL was not dephosphorylated. This means there could be important amino acids for the catalytic reaction at both ends of the PHP or in the region of the nucleotide-binding region. To identify these amino acids is an interesting project.
With the results of these important regions of the amino acid sequence you can get further insights about the specificity and the regulation of the PHP.