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 Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-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 alpha-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 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway.
Ling
V
V
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138.
Snedden
W A
WA
Shelp
B J
BJ
Assmann
S M
SM
D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D010946:Plants, Medicinal; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D005968:Glutamate Decarboxylase