Partial Purification and Characterization of Arginine Decarboxylase from Avocado Fruit, A Thermostable Enzyme

Abstract

ABSTRACrA partially purified preparation of arginine decarboxylase (EC 4.1.1.19), a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained trom the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60°C, in the presence of 1.2 millimolar MnCl2, 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The Km of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fuHly developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of Pl,P,,., of these enzymes was 1613 and 68 nanomoles of C02 produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at I millimolar under similar conditions. The effect ofdifferent inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed.It is well established that the intracellular concentration of polyamines changes markedly in response to conditions which affect growth and development of plant tissue (1 1, 12). Hence, processes like cell division (7) or cell differentiation (4, 9) are deemed to be regulated by polyamines. The properties of the enzymes involved in the biosynthesis of polyamines and their degradation, and the mode by which the activities of these enzymes are regulated, may therefore contribute to our understanding of the overall processes of development and senescence of fruits.Whereas in mammalian cells putrescine is synthesized from Lornithine by ornithine decarboxylase (22), there have been a number of reports indicating that putrescine in plants is formed from L-arginine (19). Arginine is converted by ADC2 to agmatine which is then converted to putrescine (1,11,12). In recent studies by Heimer et al. (10) and Cohen et al. (6) on tomato ovaries and fruits, it was observed that the activity of ODC changed during fruit development, whereas the relatively low '

Interrelationship of Polyamine and Ethylene Biosynthesis during Avocado Fruit Development and Ripening

“…These results suggest that the main pathway for PA synthesis during the early stages of fruit growth is through ornithine, while at the later stages PA synthesis is negligible. In contrast, Winer et al (30) reported higher levels of ADC activity in 'Fuerte' avocadoes during fruit development and ripening. They attributed the high ADC activity to the elimination of an unidentified inhibitor by dialysis and heat inactivation.…”

Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.)

“…Activity of both ODC and ADC have been found to correlate with growth rates during fruit development in mandarin orange [29], tobacco [39], tomato [8,16,42] and avocado [48]. In pea, an increase in putrescine content and a transient increase in ADC activity was detected after gibberellic acid (GA3) induction of fruit development in unpollinated ovaries [6,32].…”

Characterization of arginine decarboxylase from Dianthus caryophyllus

“…1). ADC has been partially purified from several species, includ- ing pea [22], oat [26], rice [6], avocado fruit [28], broad bean [17], soybean [19], and carnation [5]. Among these, the post-translational processing of ADC has been reported to occur in the oat, A. thaliana and carnation.…”

“…ADC has been partially purified from several species, including carnation, Brassica campestris, Vicia faba, soybean, Lathyrus sativus, Oryza sativa, oats, and avocado fruit [5,7,17,19,22,23,26,28], but its molecular mass remains controversial. It was recently reported that Ca 2+ and CO 2+ inhibit the activity of partially purified ADC from rat liver and brain Abbreviations: ADC, arginine decarboxylase; FPLC, fast performance liquid chromatography; GST, glutathione S-transferase; HRP, horseradish peroxidase; IPTG, isopropyl b-D -thiogalactopyranoside; NTA, nitrilotriacetic acid; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis.…”