Studies on bovine γ-glutamylamine cyclotransferase.

Date

2006-08

Authors

Roca, Maryuri.

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Worldwide access.
Access changed 5/25/11.

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Abstract

The purification and study of proteins are cooperative processes because at least partially purified protein is needed in order to study its properties, and certain information about the protein's properties is required in order to design its purification. Particularly difficult to purify is γ-glutamylamine cyclotransferase (γGACT ) which catalyzes the cyclization of the γ-glutamyl moiety in L-γ-glutamylamines, notably Nε−(γ-glutamyl)lysine. From this last activity the function of the enzyme is speculated to be related to the catabolism of transglutaminase products; although, there is no direct evidence of this. Electrophoretically pure bovine γGACT was obtained using preparative ultracentrifugation, anion exchange chromatography on DEAE-Sepharose, ammonium sulfate fractionation and precipitation, size exclusion chromatography on Sephacryl S100, anion exchange chromatography on Mono-Q under reducing conditions, isoelectric focusing of the alkylated sample, electroelution, electrophoresis, ultrafiltration, and lyophilization. The enzyme was purified more than 2,000 fold to a specific activity of more than 1,300U/mg of enzyme. A monomeric enzyme of molecular mass of 22,000 Daltons was observed. Anion exchange chromatography on a Mono Q GL column revealed two forms of the enzyme with pIs of 6.86 and 6.62 under non-reducing conditions, and a single form of pI 6.62 under reducing conditions. γGACT was then subjected to analytical isoelectric focusing and the active fraction appeared as a single band on SDS-PAGE. Amino acid sequencing of the tryptic digest of the band from SDS-PAGE corresponding to the enzyme was carried out by microcapillary reverse-phase HPLC nano-eletrospray tandem mass spectrometry; 42 proteins and protein fragments of similar mass and pI as that of γGACT were obtained. Analysis of their properties indicates that the unknown protein for MGC:134378 is the most likely protein to be the bovine γGACT enzyme. However, expression of the active enzyme from the cloned gene has to be done in order to assure that this is indeed the γGACT enzyme. An affinity column based on the inhibitor glutarylhexylamine showed binding of the enzyme when loaded at low ionic strength and elution when salt was increased.

Description

Includes bibliographical references (p. 222-233).

Keywords

Transglutaminases., Enzymes -- Purification.

Citation