Evidence for Transglutaminase Activity in Plant Tissue

Abstract

An extract prepared from the apical meristematic region of etiolated pea seedligs was able to catalyze the incorporation of putrescine into trchloroacetic acid precipitable material. The enzyme was found to be soluble and followed a typical Michaelis-Menten kinetics when N-Ndimethyl casein was used as a substrate. Its activity was promoted by Ca2 We present here, for the first time, evidence indicating that an enzyme with characteristics similar to those found in most transglutaminases described so far is found in plant meristematic tissues. MATERIALS AND METHODSSeeds of Pisum sativum (var 'Kelvedon Wonder') were soaked for 6 h in tap water, planted in pots containing moist vermiculite, and grown in the darkness at 23°C and 80% RH. The apical meristematic hook regions from 7-d-old etiolated pea seedlings were excised and homogenized in a chilled mortar and pestle with 4 to 5 volumes of Tris 0.1 M (pH 8.5). The homogenate was centrifuged at 2000 rpm at 4°C for 10 min and the supernatant was assayed for transglutaminase activity. Transglutaminase activity was assayed by following the rate of incorporation of 3[HI putrescine into TCA precipitable protein. The assay was conducted following the modified filter paper method described by Lorand et al. (1 1). The assay contained, in a total volume of 150 ,ul, 45 mM putrescine containing 2 uCi of [3H]putrescine (18.6 iACi/mmole), up to 600 ,ug of plant protein in 75 ,l homogenization buffer, up to 0.95 mg N-N-dimethylated casein, 1 ,mole CaCl2, and 62.5 ,mol Caps' (pH 8.5). Extracts were incubated at 35°C for 30 min, then 60-ul aliquots were spotted on Whatman No. 1 paper presoaked in 10% TCA and immediately immersed in ice-cold TCA (10%). The filters were washed twice in 5% TCA and once in an ethanol/acetone 1:1 mixture, and once in acetone. The filters were then dried, placed in scintillation vials containing 3 ml Aqualuma (Lumac B.V., Holland), and counted in a Kontron scintillation counter. The transglutaminase activity was expressed as nmol of3[H]putrescine incorporated per mg ofplant protein per h. The activity was linear up to 45 min at 35°C in the range of protein content between 5 and 600 usg per assay. Blank values (up to 200 dpm) of acid-treated or boiled enzymes preparation were always subtracted. The data presented are from single experiments, which are representative of a group of three experiments, each involving triplicate sets of experimental treatments and controls.

Binding of Spermidine to a Unique Protein in Thin-Layer Tobacco Tissue Culture

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Isolation, purifi cation and characterisation of transglutaminase from rosemary (Rosmarinus officinalis L.) leaves

“…TGase activity in plants was fi rst observed in pea seedlings [Icekson and Apelbaum 1987], and subsequently found in organs of both lower and higher plants. Signorini et al [1991] identifi ed TGase activity in the leaves of silver beet (Beta vulgaris L.).…”

Transglutaminases: recent achievements and new sources

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