A method to study the rapid phosphorylation‐related modulation of neutral trehalase activity by temperature shifts in yeast

FEBS Letters

De Virgilio, Claudio¹  Bürckert, Niels¹  Wiemken, Andres¹  Boller, Thomas¹ 

[1] Department of Botany, University of Basel, Basel, Switzerland

关键词: Trehalase;    Heat shock;    Protein phosphorylation;    Trehalose;    Saccharomyces cerevisiae;   

DOI  :  10.1016/0014-5793(91)81319-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0–4°C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30°C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (<1 min) activation during handling at 4°C before homogenization. Activity of the enzyme in these homogenates decreased 75–90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27°C, and very low activities again for cells shifted from 27 to 40°C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.

【 授权许可】

Unknown