Date of Award

Winter 1983

Project Type

Dissertation

Program or Major

Botany

Degree Name

Doctor of Philosophy

Abstract

The kinetics of L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in germinating lettuce (Lactuca sativa cv. Grand Rapids) seeds was investigated. The Km of the enzyme was determined to be 4.2 x 10('-5) M. PAL in lettuce seeds did not show tyrosine ammonia-lyase activity. The nature of inhibition of PAL by various substrate analogues and phenylpropanoid compounds was studied. Substrate analogues like D-phenylalanine, p-fluorophenylalanine, (beta)-phenyllactic acid and tryptophan were found to inhibit PAL competitively whereas tyrosine did not show any inhibition of enzyme activity. Of the phenylpropanoids used, cinnamic acid was found to be a competitive inhibitor whereas chlorogenic acid showed mixed inhibition. Other phenylpropanoid compounds like p-coumaric acid, caffeic acid, coumarin, quercetin, ferulic acid etc. did not show any inhibition of PAL activity in vitro.

The regulation of PAL activity by various substrate analogues, intermediates and endproducts of the phenylpropanoid pathway in relation to the growth of the embryonic axes was studied. The substrate, L-phenylalanine, its D-isomer and (beta)-phenyllactic acid did not show any significant effect on PAL activity at low concentrations (50 and 100 uM), whereas at higher concentrations (500 uM) L-phenylalanine inhibited both PAL activity and radicle elongation. On the other hand, D-phenylalanine promoted both. Other substrate analogues like 2-aminoxy-3-phenylpropionic acid (AOPP), p-fluorophenylalanine and tryptophan showed a strong inhibition of PAL activity, the inhibition being concentration dependent. Another substrate analogue, tyrosine, stimulated both PAL activity and radicle elongation.

The phenylpropanoid compounds were inhibitors of PAL activity at higher concentrations. At lower concentrations, there was a concentration dependent inhibition of PAL activity and radicle elongation. In all these treatments, a strong correlation was observed between PAL activity and radicle length.

PAL was purified 37 fold from 40 h old lettuce seedlings. Excised radicles were used as a source of the enzyme for purification since more than 95% of the PAL activity was localized in the radicles. A combination of four techniques viz. ammonium sulfate precipitation, gel filtration, ion exchange and hydroxylapatite chromatography were used for the purification of PAL from lettuce seedlings.

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