• Journal of Life Science
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• v.19 no.9
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• pp.1226-1231
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• 2009

Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.298-303
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• 2005

This experiment was carried to investigate the optimum time and times of abscisic acid (ABA) treatment for the coloration in 'Kyoho' grapes. The L-phenylalanine ammonia-lyase (PAL) enzyme activities were showed highly in both ABA treatments at veraison and 10 days after veraison. However, PAL enzyme was slightly higher in ABA treatment at 10 days after veraison than in that at veraison. Anthocyanin content showed a tendency that were increased during fruit development after veraison in all treatments, and was the highest in ABA treatment at 10 days after veraison. Fructose and glucose as soluble sugars were detected by HPLC and showed little differences in all treatments. In times of ABA treatment, PAL activity showed a tendency that decreased after increased in all treatments. PAL activity in 2 times treatment of ABA was higher than other treatments. Also, anthocyanin content was highest in 2 times treatment of ABA, as 5 folds of control and folds of 1 time treatment of ABA. Both fructose and glucose contents in all ABA treatments was slightly higher than control.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.34-38
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• 1999

We observed the structure of phenylalanine ammonia-lyase gene (PAL) which is one of the best studied plant defense-related genes responding to pathogen infection by producing suberin, lignin, and phytoalexins. In tomato, at least 5 different genetic loci have been identified by genomic southern blot hybridization and nucleotide sequence analyses of partially cloned gene fragments (Lee et al. 1992). However, our results suggest that two other isoforms designated as PAL X1 and PAL X2 are located on the chromosome in tomato plant. Furthermore, the preliminary results obtained from southern blot hybridization analyses of subcloned fragment digested with several restriction endonuclease indicated that PAL X1 and PAL X2 clones contain at least two copies of PAL gene and partial nucleotide sequence analyses of each subcloned fragment with the same primer taken from known nucleotide sequence of PAL5 gene indicated that they are located side by side on the same chromosome.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.205-212
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• 2011

Phenylalanine ammonia-lyase (PAL) from the maize pathogen Ustilago maydis was analysed immunologically to obtain insights into the structural relationships between plant PAL and fungal PAL and between PAL and histidine ammonia-lyase (HAL). Cross-reactivity was found among all the PAL proteins from different species tested, using antibodies raised against both plant and fungal PALs. Both anti-Alfalfa and anti-popular PAL antibodies strongly recognized plant PALs but only weakly recognized fungal PALs. Antibodies raised against U. maydis PAL only weakly recognized the Rhodotorula glutinis yeast PAL. The anti-U. maydis PAL antibodies showed low affinity for the plant PALs but they bound strongly to Pseudomonas bacterial HAL. Significant cross-reactivity between the two plant PAL antibodies and the bacterial HAL was also observed. Both the anti-Ustilago PAL and the anti-poplar PAL antibodies displayed similar enzyme inhibition patterns, including moderate inhibition of bacterial HAL activity. However, the bacterial HAL antibody inhibited only Ustilago PAL. The PAL and HAL antibodies tested showed no inhibition against yeast PAL. This is first report on the immunological relationships between PAL and HAL.

• Current Research on Agriculture and Life Sciences
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• v.20
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• pp.65-70
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• 2002

This study was conducted to investigate allelopathic potential of various crosses of rice cultivars using their inhibitory effect on barnyardgrass growth under field conditions, and to determine PAL activity and phenolic compounds involved in phenylpropanoid pathway from selected crosses of rice cultivars. Under field conditions, Kouketsumochi, Woo co chin yu possessed higher allelopathic potential inhibiting over 90% of barnyardgrass growth. Crosses of Kouketsumochi/Woo co chin yu, Dongjinbeyo/Kouketsumochi, Dongjinbeyo/Woo co chin yu showed over 80% inhibitory effects on barnyardgrass growth. The highest PAL activity, $63.46{\mu}kats/kg$ proteins was detected in Kouketsumochi which is the most important enzyme in phenylpropanoid pathway and also higher PAL activity in crosses with Kouketsumochi. Content of cinnamate was $2.64{\mu}g/g$ f.w. in Kouketsumochi which was 2 to 5 times higher than other rice cultivars tested, indicating that higher PAL activity resulted in more cinnamate. The similar trends in cinnamate content and PAL activity were observed in crosses of rice cultivars with Kouketsumochi.

• Korean Journal of Plant Tissue Culture
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• v.26 no.1
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• pp.41-47
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• 1999

Phenylalanine ammonia-lyase (PAL; EC 4, 3, 1, 5) genomic clones were isolated from tomato(Lycopersicon esculentum L.) genomic DNA libraries using tomato PAL5 cDNA sequences as probes. The nucleotide sequences of tPAL1, tPAL4 and tPAL5 were compared. tPAL5 contains an open reading frame encoding a polypeptide of 722 amino acids, interrupted by a 710 bp intron in the codon for the amino acid 139. tPAL1 encodes a polypeptide of 249 amino acids which is much shorter than tPAL5 gene due to a premature stop codon and does not contain an intron. tPAL4 encodes a polypeptide of 357 amino acids, interrupted by a 305 bp intron in the codon for the amino acid 138. Premature stop codons observed in tPAL1 and tPAL4 gene produce a short polypeptide rather than a normal polypeptide (722 aa). tPALl shows 87.2% homology with tPAL4 and 85.3% homology with tPAL5 gene whereas tPAL4 showes 91.4% homology with tPAL5 at nucleotide level. In general, phylogenetic analysis showed that genes isolated from tomato, potato, and sweet potato were belong to the same group and another dicot plants such as parsley, bean, soybean, pea and alfalfa formed another group. PAL genes isolated from rice and yeast showed very low homology with other PAL genes and formed the other group.

• Journal of Life Science
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• v.12 no.3
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• pp.274-280
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• 2002

The metabolites related to phenylpropanoid pathway play an important role in the self-defense of plants and induced by environmental stress like wounding, pathogen attack, UV-irradiation and so on. The mRNA level of rite phenylalanine ammonia lyase (PAL) was increased at 12 h to 48 h, however it was gradually decreased 48 h to 60 h after UV irradiation. The PAL enzyme activities in rice were peaked at the time of 24 h after UV irradiation, on the other hand, it was not affected by wounding. The PAL enzyme activities in pepper were raised high at 24 h and 10 h by UV irradiation and wounding respectively. The cinnamic acid 4-hydroxylase (C4H) activities were increased by wounding treatment and were detected from 12 h to end time point of experiment, while UV-irradiation didn't affect the C4H activity in rice and pepper. These results were assumed that the action of isoflavonid has an alternative effect on the defenses which include wounding and UV irradiation and on the diverse roles in rice and hot pepper.

• Journal of Life Science
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• v.15 no.2 s.69
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• pp.293-297
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• 2005

This experiment was carried to investigate the optimum concentration of abscisic acid (ABA) treatment for enhancing fruit coloration of 'Kyoho' grapes. Cluster and berry weights showed a tendency that increased in proportion to concentration of ABA treatment, but were not significant in all treatments. Also, soluble solids and titratable acidity were not significant during fruit development in all treatments. L-phenylalanine ammonia-lyase (PAL) activity showed a tendency that decreased after rapidly increased in all treatments, and was the highest in 1000 mg/l ABA treatment at final harvest. Anthocyanin and total phenolics contents were high in proportion to ABA treatment concentration, and anthocyanin content in 1000 mg/l ABA treatment was 2.5 folds of that in control. Fructose and glucose as soluble sugars were detected, but sucrose was not detected. Both fructose and glucose contents increased during fruit development, but showed little difference in all treatments.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.20 no.2
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• pp.37-43
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• 2020

Phenylketonuria is the most prevalent disorder caused by an inborn error in aminoacid metabolism. It results from mutations in the phenylalanine hydroxylase (PAH) gene. If untreated or late treated, results in profound and irreversible mental disability. Newborn screening test identify patients with phenylketouria. The early initiation of a phenylalanine restricted diet very soon prevents most of the neuropsychiatric complications. However, the diet therapy is difficult to maintain and compliance is poor, especially in adolescents and adulthood. Since 2015, American Medical College of Medical Genetics and Genomics (ACMG) recommended more strong restrictive diet therapy for target blood level of phenylalanine (<360 umol/L). For over four decades the only treatment was a very restrictive low phenylalanine diet. This changed in 2007 with the approval of cofactor therapy (Tetrahydrobiopterin, BH4) which is effective in up to 30% of patients. Data from controlled clinical trials with sapropterin dihydrochloride indicate a similar occurrence of all-cause adverse events with this treatment and placebo. Large neutral aminoacids (LNAA) competes with phenylalanine for transport across the blood-brain-barrier and have a beneficial effect on executive functioning. A new therapy has just been approved that can be effective in most patients with PAH deficiency regardless of their degree of enzyme deficiency or the severity of their phenotype. Phenylalanine ammonia lyase (PAL-PEG) was approved in the USA by FDA in May of 2018 for adult patients with uncontrolled blood phenylalanine concentrations on current treatment. Nucleic acid therapy (therapeutic mRNA or gene therapy) is likely to provide longer term solutions with few side effects.