Conformational and molecular responses to pH variation of the purified membrane adenosine triphosphatase of Micrococcus lysodeikticus

Biochim Biophys Acta. 1975 Dec 16;413(3):394-414. doi: 10.1016/0005-2736(75)90123-6.

Abstract

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.

MeSH terms

  • Adenosine Triphosphatases* / metabolism*
  • Cell Membrane / enzymology*
  • Cell Membrane / ultrastructure
  • Circular Dichroism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Micrococcus / enzymology*
  • Micrococcus / ultrastructure
  • Microscopy, Electron
  • Molecular Weight
  • Osmolar Concentration
  • Protein Conformation
  • Spectrophotometry, Ultraviolet
  • Tryptophan / analysis
  • Tyrosine / analysis*

Substances

  • Tyrosine
  • Tryptophan
  • Adenosine Triphosphatases