Stability of the complex formed between French bean (Phaseolus vulgaris) phenylalanine ammonia-lyase and its transition-state analog

Dh, Jones; Dh, Northcote

doi:10.1016/0003-9861(84)90265-0

Cited by 8 publications

(4 citation statements)

References 17 publications

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“…3 A). This activity was not regained after removal of CA by dialysis or gel filtration through Sephadex G 25, nor was it regained during extended incubations of the enzyme in the presence of substrate or by dialysis against saturated L-phenylalanine at 37 ~ C, treatments known to overcome the more potent in-vitro inhibition of PAL by AOPP (Jones and Northcote 1984). This irreversible effect of CA in vivo is clearly different from the reversible effect of the acid observed in vitro, and could be the result of one or a combination of the following factors : (a) inhibition of transcription-translation, which could be sufficient to account for the decrease in extractable activity if PAL turnover were sufficiently rapid, (b) increased degradation of PAL subunits and (c) inactivation of the enzyme.…”

Section: Resultsmentioning

confidence: 95%

“…The mechanism of CA-mediated PAL inactivation has been investigated by titration of the activesite concentration of PAL using site-specific tritiation (Jones and Northcote 1984) of partially purified enzyme from cells treated with CA in vivo or of purified PAL treated with inactivation factor in vitro. These results strongly indicate that an irreversible modification of the enzyme active site occurs.…”

Section: Discussionmentioning

confidence: 99%

“…Aliquots (5 gl) of 100 mM NaBH4 in ice-cold 100 mM NaOH were then slowly added to the protected enzyme and to an equivalent batch of enzyme not pre-treated with AOPP, until the activity of the unprotected enzyme was fully inhibited. Both PAL samples were then extensively dialysed against 50 mM Tris-HC1, pH 8.0, before displacement of AOPP from the protected PAL with L-phenylalanine as described by Jones and Northcote (1984). The deprotected enzyme was then treated with 5-gl aliquots of NaB3H4 until the enzyme was fully inhibited.…”

Section: Growthmentioning

confidence: 99%

“…The active site of PAL contains the unusual amino acid dehydroalanine, and this may be reduced to alanine, with subsequent enzyme inactivation, following treatment with NaBH4 (Jones and Northcote 1984). We therefore investigated whether the ability to tritiate PAL with NaBaH4 could be lost, either following exposure of the enzyme to extracts from CA-treated cells in vitro, or during CA-mediated enzyme removal in vivo.…”

Section: Effects Of Ca Treatments On the P A L Active Sitementioning

confidence: 99%

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L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

Bolwell

Cramer

Lamb

et al. 1986

Planta

110

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The extractable activity ofL-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [(35)S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of(35)S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.

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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Growthmentioning

confidence: 99%

Section: Effects Of Ca Treatments On the P A L Active Sitementioning

confidence: 99%

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