Properties, Development and cellular-localization of cinnamic acid 4-hydroxylase in cut-injured sweet potato

In sweet potato root tissue, cinnamic acid 4-hydroxylase activity increased markedly in response to cut injury, and reached a maximum after 1 day of incubation. The patterns of development and successive decline were similar to those for phenylalanine ammonia-lyase activity. The development of both enzyme activities was inhibited by cycloheximide. The activity was strictly dependent on pH of the homogenizing and reaction media. The optimum pH of the reaction was 8.0. The respective Km values for trans-cinnamic acid and NADPH were 2.6×10-5 and 1.8×10-6M. The activity was not affected by β-mercaptoethanol and the intermediates and product of the polyphenol biosynthetic pathway. Carbon monoxide inhibited strongly the activity and its inhibition was partially prevented by light. Thus, the enzyme may be involved in the cytochrome P-450 mediated electron transport system. Studies using differential centrifugation and sucrose density gradient centrifugation, showed that the intracellular distribution of 4-hydroxylase activity differed distinctly from that of the mitochondrial marker enzyme and was not in accord with that of NADPH-cytochrome c reductase activity.