ANALYSIS OF A SOLUBLE CALMODULIN-BINDING PROTEIN FROM FAVA-BEAN ROOTS - IDENTIFICATION OF GLUTAMATE-DECARBOXYLASE AS A CALMODULIN-ACTIVATED ENZYME

The identity of a soluble 62-kD Ca2+-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using I-125-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the a-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 mu M Ca2+, a 100% stimulation by the addition of 100 mu M Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca2+-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca2+-mediated signal transduction pathway.