Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast - Synergetic effect between trehalose and phospholipid environment

Yeast cells have had to develop mechanisms in order to protect themselves f rom chemical and physical agents of the environment to which they are expos ed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to hea t shock. In this work, we have studied the effect of trehalose on the prote ction against thermal inactivation of purified plasma membrane H+-ATPase fr om Schizosaccharomyces pombe, in the solubilized and in the reconstituted s tate. We observed that after 1 min of incubation at 51 degrees C in the pre sence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was complete ly abolished. The t(0.5) for the enzyme inactivation increased from 10 to 5 0 s after reconstitution into asolectin liposomes. Curiously, in the presen ce of 1 M trehalose, the t(0.5) for inactivation of the reconstituted enzym e was further increased to higher than 300 s, regardless of whether trehalo se was added inside or outside the liposome. Additionally, the concentratio n that confers 50% for the protection by trehalose (K-0.5) decreased from 0 .5 M, in the solubilized state, to 0.04 M in the reconstituted state, sugge sting a synergetic effect between sugar and lipids. Gel electrophoresis rev ealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M tr ehalose was present in the buffer. It is suggested that both in a soluble a nd in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E-2-like form.