Effect of heat shock on amino acid metabolism in cultured cowpea cells: Changes in the metabolism of gamma-aminobutyric acid
Abstract
The effects of heat shock and heat adaptation on amino acid metabolism in cowpea suspension cultures were studied. Heat shock (from 26 to 42$\sp\circ$C) resulted in a several-fold increase in the accumulation of gamma-aminobutyric acid (GABA) within 4 hours. Experiments with gabaculine, a potent inhibitor of GABA aminotransferases (GABA AT), confirmed that this increase is due to an increase in GABA biosynthesis, and indicated that GABA utilization also increases. However, heat shock did not result in increased levels of GABA:pyruvate AT, GABA:2-oxoglutarate AT, or glutamate decarboxylase (GAD), the major enzyme of GABA biosynthesis. Measurements of GABA biosynthesis and utilization in the presence and absence of cycloheximide suggested that protein synthesis is not required for the maintenance of levels of these enzymes during heat shock. In 38$\sp\circ$C-adapted cells, GAD level was decreased, even though GABA was somewhat increased. When ($\sp{15}$N) -GABA was fed to cells incubated at 26 or 42$\sp\circ$C, alanine, glutamate and aspartate were the most heavily labeled amino acids. No amino acids other than GABA were labeled in the presence of gabaculine. GAD from heat-shocked cells had a K$\sb{\rm m}$ for glutamate ($\sim$3.5 mM), activity versus pH curve (pH optimum $\sim$5.1), and activity versus temperature curve (Q$\sb{10}\ \sim2$) identical to enzyme from control cells. Comparison of GAD activity in extracts before and after removal of small solutes revealed no evidence of enzyme activation or inhibition by low molecular weight effectors. Increased GABA biosynthesis during heat shock could possibly result from combined effects of cytoplasmic acidification, a 3-fold increase in reaction velocity due to the 16$\sp\circ$C increase in temperature, and altered compartmentation of glutamate.
Degree
Ph.D.
Advisors
Rhodes, Purdue University.
Subject Area
Botany
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