A specific and reversible macromolecular inhibitor of phenylalanine ammonia-lyase and cinnamic acid-4-hydroxylase in gherkins.
A non-dialysable inhibitor of phenylalanine ammonia-lyase (L-phenylalanine ammonia-lyase, EC 4.3.1.5) has been partially purified from dark-grown gherkin hypocotyls. On extraction of tissue it is found both in the soluble (106 000 X g supernatant) and microsomal (106 000 X g pellet) fractions and can be extracted from the microsomal membranes with 10 mM sodium cholate and 1 M KCl. The soluble and microsomal fractions have similar properties, suggesting the presence of the same active component. The inhibitor is small (Mr less than 20 000), thermolabile, sensitive to proteolytic digestion, and apparently hydrophobic. Purification of the inhibitor was achieved by chromatography on DEAE-cellulose by gel filtration on Sephadex G-50. The inhibitor preparations inhibit phenylalanine ammonia-lyase isolated from a number of plant tissues and also cinnamic acid-4-hydroxylase (trans-cinnamate, NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11) from gherkins and peas, but not a wide range of other enzymes. The evidence suggests that inhibition of the two enzymes is due to the same substance, but this has not yet been confirmed. Kinetic experiments show that the inhibitor is competitive with phenylalanine for the lyase and that its association with the lyase is reversible. Further, a mixture of inhibitor and lyase can be separated on non-denaturing polyacrylamide gels without loss of lyase activity. The activities of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase are often concurrently regulated and both have regulatory roles in phenol metabolism; it is suggested that the inhibitor may be specifically involved in controlling their activities in vivo.
D000642:Ammonia-Lyases / Q000037:antagonists & inhibitors*; D002934:Cinnamates; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D008970:Molecular Weight; D010650:Phenylalanine Ammonia-Lyase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D010940:Plant Proteins / Q000302:isolation & purification / Q000502:physiology*; D010944:Plants / Q000201:enzymology*
D002934:Cinnamates; D010940:Plant Proteins; D006899:Mixed Function Oxygenases; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase