Characterisation of a calcium dependent transglutaminate in Pisum sativum leaf and root tissue

Lilley, GR, 1999. Characterisation of a calcium dependent transglutaminate in Pisum sativum leaf and root tissue. PhD, Nottingham Trent University.

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Abstract

Two colorimetric assays for transglutaminase activity involving protein cross-linking were developed. The assays were used to screen protein extracted from root and leaf tissue of the dicotyledons Pisum sativum and Vicia faba and the monocotyledons Triticum aestivum and Hordeum vulgare for transglutaminase activity. In one assay, biotin labelled casein was cross-linked to chemically modified casein bound to a microtiter plate via ϵ-(β-glutamyI) lysine isodipeptide bonds and the biotin labelled reaction product was detected by conjugation to extravidin peroxidase. In a second assay, microtiter plate bound N',N'-dimethylcasein was enzymically modified using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein was then cross-linked into the immobilized polyamines by the transglutaminase under assay resulting in the formation of N',N'-bis(γ-glutamyl) polyamine linkages. The crude plant protein preparations were also screened for the ability to catalyse the production of N-(γ-glutamyl) polyamine bonds using a biotin labelled cadaverine incorporation assay and a radiolabelled putrescine incorporation assay. Crude plant extracts were shown to catalyse the cross-linking of biotin labelled casein to microtiter plate bound chemically modified casein and the incorporation of biotin labelled cadaverine into microtiter plate bound N',N'-dimethylcasein in a calcium dependent manner. The cross-linking of casein and the incorporation of biotin labelled cadaverine into N',N'-dimethylcasein were time dependent reactions with a pH optimum of 7.9. Transglutaminase activity was shown to increase over a 2 week growth period in both the roots and leaves of Pisum sativum. A partially purified transglutaminase from the root tissue of Pisum sativum had an estimated molecular mass of 36 kDa and a Km of 190?M and 0.2μml-1 for biotin labelled cadaverine and biotin labelled casein respectively. Calcium dependent transglutaminase activity was also detected in detergent treated Pisum sativum membrane preparations, implying that a membrane bound form of transglutaminase is also present in this tissue. The ϵ-(γ-glutamyl) lysine isodipeptide product of the transglutaminase reaction was detected in the root and leaf protein of Pisum sativum at a level of 510 pmol mg-1 and 210 pmol mg-1 respectively.

Item Type:	Thesis
Creators:	Lilley, G.R.
Date:	1999
ISBN:	9781369323580
Identifiers:	Number Type PQ10290110 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	02 Oct 2020 11:39
Last Modified:	29 Sep 2023 13:31
URI:	https://irep.ntu.ac.uk/id/eprint/41108

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