Characterization of proteinase activity in stratified Douglas-fir seeds.
We investigated the effect of stratification on the proteinase activity involved in mobilization of the major soluble approximately 45 kDa storage protein during germination of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seeds. Complete hydrolysis of the approximately 45 kDa protein was observed approximately 7 days after exposure of stratified seeds to germination conditions. Coincident with the onset of mobilization, proteinase activity was detected primarily in microsomal extracts from stratified seeds. Microsomal-associated proteinase activity was most active at pH 8.0 and had a molecular mass > 175 kDa as determined by gelatin SDS-PAGE gels. In vitro digestion of soluble protein extracts indicated that, following stratification, there was a significant increase in proteinase activity and hydrolysis of the approximately 45 kDa storage protein. Whether this increase was a result of activation of preexisting proteinase(s) or de novo synthesis remains unknown. In vitro digestion of soluble protein extracts in the presence of various proteinase inhibitors showed that digestion of the approximately 45 kDa storage protein is mediated primarily by a metalloproteinase and to a lesser degree by a serine proteinase. The accumulation of approximately 25 kDa protein products following in vitro digestion suggests that mobilization of the approximately 45 kDa soluble storage protein is mediated by a multi-step process involving the action of different classes of proteinases.
Forward
B S
BS
Department of Biochemistry and Microbiology and Centre for Forest Biology, University of Victoria, Victoria, B.C., V8W 3P6, Canada.
Tranbarger
T J
TJ
Misra
S
S
D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000502:physiology*; D018525:Germination / Q000502:physiology; D010940:Plant Proteins / Q000502:physiology; D028224:Pseudotsuga / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*; D013347:Subcellular Fractions / Q000502:physiology; D014197:Trees / Q000201:enzymology*
D010940:Plant Proteins; D010450:Endopeptidases