Publications

• Phosphorylation of histidine in proteins by a nuclear extract of Physarum polycephalum plasmodia.

  Huebner V.D., Matthews H.R.

  A high salt nuclear extract from the true slime mold Physarum polycephalum was used as a source of kinase activity for the incubation of calf thymus histones with [gamma-32P]ATP. A major proportion of the 32P incorporated into histones was acid-labile and alkali-stable. The nature of the alkali-st ... >> More

  A high salt nuclear extract from the true slime mold Physarum polycephalum was used as a source of kinase activity for the incubation of calf thymus histones with [gamma-32P]ATP. A major proportion of the 32P incorporated into histones was acid-labile and alkali-stable. The nature of the alkali-stable phosphorylated component was analyzed by subjecting the phosphorylated protein to total alkaline hydrolysis and separating the resultant phosphoamino acids by anion exchange chromatography. The 32P-labeled material co-chromatographed with phosphohistidine standards and did not co-chromatograph with phosphoserine, phosphothreonine, or phosphotyrosine standards. In similar experiments using reversed phase high-performance liquid chromatography to separate the phosphoamino acids, the 32P-labeled phosphoamino acid behaved like the 1-isomer of phosphohistidine, in not being retained by the column, and unlike 3-phosphohistidine, phosphoserine, phosphothreonine, phosphotyrosine, and phosphoarginine, which were all retained on the column. Histone H4 was a good substrate for the histidine kinase activity and the location of the phosphorylated histidine residue was probed by peptide mapping using chymotrypsin or V8 protease. Both maps were consistent with labeling of histidine 75 and inconsistent with labeling of histidine 18. The data show that Physarum nuclei contain a major kinase activity which produces phosphohistidine. The methods we have developed for studying this kinase activity provide the basis for a complete characterization of the structure and function of the Physarum enzyme and can be applied to the study of similar kinase activities in other systems. << Less

  J Biol Chem 260:16106-16113(1985) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of chemical and enzymatic acid-labile phosphorylation of histone H4 using phosphorus-31 nuclear magnetic resonance.

  Fujitaki J.M., Fung G., Oh E.Y., Smith R.A.

  Phosphorus-31 nuclear magnetic resonance (31P NMR) is used to investigate acid-labile phosphorylation of histone H4. 31P NMR detects phosphorylated histidine residues in in vitro enzymatically phosphorylated H4. The source of kinase is nuclei from either regenerating rat liver or Walker-256 carcin ... >> More

  Phosphorus-31 nuclear magnetic resonance (31P NMR) is used to investigate acid-labile phosphorylation of histone H4. 31P NMR detects phosphorylated histidine residues in in vitro enzymatically phosphorylated H4. The source of kinase is nuclei from either regenerating rat liver or Walker-256 carcinosarcoma. When regenerating rat liver is the source, 31P NMR spectroscopy on the denatured phosphorylated protein exhibits a resonance at 5.3 ppm relative to an 85% orthophosphoric acid external reference. This peak corresponds well with the chemical shift of standard pi-phosphohistidine scanned under similar conditions. Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis confirms acid lability. When the source of kinase is Walker-256 carcinosarcoma, the 31P NMR spectrum contains a resonance at 4.9 ppm which corresponds well with standard tau-phosphohistidine run under the same conditions. Chemical phosphorylation of H4 has been accomplished by using dipotassium phosphoramidate which specifically phosphorylated the imidazole moiety of histidine at neutral pH. NaDodSO4--polyacrylamide gel electrophoresis confirms acid lability, and high-pressure liquid chromatography of protein hydrolysates yields phosphohistidine. 31P NMR of chemically phosphorylated H4 in a structured state reveals two peaks at 4.8 and 7.3 ppm with line widths of 9 and 55 Hz, respectively. These resonances indicate that both histidine residues of H4 (His-18 and His-75) are phosphorylated, the latter relatively immobile and the former relatively free in solution. 31P NMR studies on chemically phosphorylated peptide fragments of H4, namely, H4(1-23) and H4(38-102), confirm this model of H4 structure. << Less

  Biochemistry 20:3658-3664(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.