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 from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.
Felix
C F
CF
Departamento de Bioquímica Médica, ICB/CCS/UFRJ, Rio de Janeiro, RJ,Brazil.
Moreira
C C
CC
Oliveira
M S
MS
Sola-Penna
M
M
Meyer-Fernandes
J R
JR
Scofano
H M
HM
Ferreira-Pereira
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002241:Carbohydrates / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology; D013696:Temperature; D013997:Time Factors; D014199:Trehalose / Q000737:chemistry* / Q000494:pharmacology; D015003:Yeasts / Q000201:enzymology
D002241:Carbohydrates; D010713:Phosphatidylcholines; D010743:Phospholipids; C024990:asolectin; D014199:Trehalose; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases