Partial purification and some properties of phenylalanine ammonia-lyase of tobacco (Nicotiana tabacum)

O'Neal, D. L.; Keller, Carol J.

doi:10.1016/s0031-9422(00)85250-4

Cited by 34 publications

(8 citation statements)

References 16 publications

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“…In a number of studies (3,16,23,24,28,29), it had been found that trans-p-coumaric acid inhibited the PAL activity. The results presented in Table I show that this compound has a similar effect on the enzyme of the gherkin hypocotyl.…”

Section: Resultsmentioning

confidence: 99%

“…Several investigations (3,16,23,24,28,29) lead to the conclusion that the (hydroxy)cinnamic acid(s) in addition to affecting the level of active PAL, have also a more direct effect on the enzymatic activity, probably by end product inhibition. The changes that occur after dark-grown gherkin seedlings are exposed to the light can now be explained by the following mechanism.…”

Section: Resultsmentioning

confidence: 99%

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On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Engelsma¹

1974

Plant Physiol.

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Irradiation with ultraviolet light causes in the hypocotyl of dark-grown gherkin seedlings the partial conversion of transhydroxycinnamic acids to the cis-isomers. The trans-hydroxycinnamic acids inhibit the development of phenylalanine ammonia-lyase activity, and the transformation of these compounds to the much less inhibitory cis-isomers forms a ready explanation for the increase in phenylalanine ammoniaIyase activity in the hvpocotvl of gherkin seedlings irradiated with ultraviolet light. Arguments are advanced that the increase in phenylalanine ammonia-lyase activitv caused by irradiation with blue light is also (at least in part) initiated by trans-cis isomerisation of the hvdroxycinnamic acids.Since Zucker's paper (31) on the induction of PAL2 (EC 4.3. 1 .5) by light in potato tuber tissue the photoinduction of this enzyme has been studied in a great number of other plants, but the mechanism of this process has so far remained obscure (4,22,27,32). Previously (6), I postulated that photoactivation of one or more photoreceptors starts a series of dark reactions that lead to the formation of a factor which promotes PAL synthesis. This concept will probably have to be revised. In more recent investigations (9-14), evidence has been obtained that the PAL level in gherkin hypocotyls is controlled by end products of the shikimic acid pathway: cinnamic acid, p-coumaric, and ferulic acid. and/or glucose esters of the latter two compounds. It appeared that increases in PAL activity similar to those brought about by irradiation were obtained with treatments which caused the release of the cinnamic acid derivatives from the tissue (9) or their conversion into other compounds (13,14). This has led us to the idea that the effect of light on PAL might be due to a temporary diminution in the concentration of (hydroxy)-cinnamic acid(s) in certain cell compartments. The only direct effect of light on these compounds is the trans-cis conversion by the UV part of the spectrum (21). Blue light causes a similar 1 Dedicated to the memory of Milton Zucker who in 1965 was the first to report the photoinduction of phenylalanine ammonialyase. The present author was privileged to have many discussions with him in the years thereafter about the regulatory mechanisms -oncerning this enzyme.2 Abbreviation: PAL: phenylalanine ammonia-lyase.reaction, provided that a suitable photoreceptor is present (25). In this paper, the question will be discussed of whether these reactions play a role in the induction of PAL activity in gherkin hypocotyls. MATERIALS AND METHODSThe experiments were performed with 3-day-old dark-grown gherkin seedlings, Cucumnus sativuis L. cv. 'Venlose niet plekkers," strain Tercken VI (15). The irradiations were carried out at 25 C with UV light of 360 vw/ccrn (Philips HPW, 125 w, Ak,,,x 365 nm) and with blue light of 150 1Aw/cm2 (6). In vivo spectrophotometry of gherkin hypocotyls was carried out in a Cary spectrophotometer with a scattered transmission attachment. In all experiments. one seedling only was ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Engelsma¹

1974

Plant Physiol.

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“…These difficulties are overcome by using the direct assay method of recording the absorbance of cinnamic acid or p-coumaric acid under acidic conditions. A similar method was used by O'Neal & Keller (1970) who estimated cinnamic acid after stopping the reaction with trichloroacetic acid.…”

Section: Discussionmentioning

confidence: 99%

Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

Kalghatgi

Rao

1975

Biochemical Journal

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1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (a) and 90000 (,B) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from MichaelisMenten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18mm to as high as 5.0mm with the increase in the concentration of the substrate and during this process the Vmax. increases by 2-2.5-fold. 10. The value ofHill coefficient is 0.5. 11. Steadystate rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

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“…Furthermore, downregulation of C4H by antisense gene expression in transgenic tobacco leads to a corresponding decrease in PAL activity, suggesting that transcinnamic acid is sensed as a metabolic regulator of phenylpropanoid pathway flux in vivo (J. Blount and R.A. Dixon, unpublished results). Tobacco PAL is particularly sensitive to direct inhibition by trans-cinnamic acid in vitro (O'Neal and Keller, 1970). Tight coupling between PAL and C4H therefore could maintain, in the microsomal "compartment," a low trans-cinnamic acid pool that would avoid feedback inactivation of PAL, as suggested by Noe et al (1980).…”

Section: Implications Of Metabolic Channeling For Phenylpropanoid Patmentioning

confidence: 93%

Transgene-Mediated and Elicitor-Induced Perturbation of Metabolic Channeling at the Entry Point into the Phenylpropanoid Pathway

1999

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3 H-L -Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of 3 H-trans -cinnamic acid formed from 3 H-L -phenylalanine did not equilibrate with exogenous trans -cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans -cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase ( PAL ) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL -overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans -cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes. INTRODUCTIONThe phenylpropanoid pathway is involved in the biosynthesis of a wide variety of natural products from plants. Many of these products have important functions in plant development and in interactions of the plant with its environment (Hahlbrock and Grisebach, 1979;Hahlbrock and Scheel, 1989;Dixon and Paiva, 1995). Many studies have addressed the transcriptional regulation of genes encoding enzymes of the phenylpropanoid pathway and subsequent changes in extractable enzyme activities in response to developmental and environmental cues Lawton and Lamb, 1987;Hahlbrock and Scheel, 1989). Far less attention has been paid to how the cell regulates flux into different end products of the pathway once all of the enzymatic machinery is assembled.The first committed step in the biosynthesis of phenylpropanoid compounds is the conversion of L -phenylalanine (Phe) to trans -cinnamic acid by L -Phe ammonia-lyase (PAL; EC 4.3.1.5; Figure 1). PAL is a tetrameric enzyme whose subunits are encoded by a multigene family in most species that have been studied (Cramer et al., 1989;Nagai et al., 1994; Wanner et al., 1995;Fukasawa-Akada et al., 1996).PAL genes are transcriptionally activated after microbial infection or treatment of plant cells with microbial elicitors (Edwards et al., 1985;Lawton and Lamb, 1987). The second step in the phenylpropanoid pathway, the hydroxylation of trans -cinnamic acid to 4-coumaric acid, is catalyzed by a cytochrome P450 monooxygenase, cinnamic acid 4-hydroxylase (C4H; EC 1.14. 13.11;Russell and Conn, 1967;Fahrendorf and Dixon, 1993;Teutsch et al., 1993). C4H is induced by light, elicitors, and wounding (Fahrendorf and Dixon, 1993;Buell and Somerville, 1995;Batard et al., 1997;Bell-Lelong et al., 1997), and its induction often is closely coordinated with PAL induction (Mizutani et al., 1997).Enzymes of complex metabolic pathways may...

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Partial purification and some properties of phenylalanine ammonia-lyase of tobacco (Nicotiana tabacum)

Cited by 34 publications

References 16 publications

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

Transgene-Mediated and Elicitor-Induced Perturbation of Metabolic Channeling at the Entry Point into the Phenylpropanoid Pathway

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