Mechanism of Action of Guinea Pig Liver Transglutaminase

Abstract

Abstract Guinea pig liver transglutaminase has been purified 230-fold in high yield by means of diethylaminoethyl cellulose chromatography of liver homogenate supernatant fluid, precipitation of the enzyme with protamine, selective extraction with ammonium sulfate solution, and rechromatography on the cellulose absorbent. Estimates of molecular weight made by several procedures, 76,900 ± 5,000 (sedimentation and diffusion), 90,000 ± 4,000 (sedimentation equilibrium), and 90,000 ± 10,000 (14C-iodoacetamide incorporation), show some variation. The s25,w0 and D25,w values for transglutaminase were determined as 5.40 x 10-13 and 6.44 x 10-7 cm2 sec-1, respectively. The —SH content, 16 to 17 residues/90,000 g, was determined on native and denatured enzyme by two procedures. The amino acid composition of transglutaminase is reported. The effects of several —SH reagents on enzymatic activity have been investigated. Irreversible inhibition by 14C-iodoacetamide at pH 6.8 occurs only in the presence of the essential cation, Ca++. One mole of 14C-carbamidomethyl is incorporated per mole of enzyme with the complete loss of enzymatic activity. Substrate affords efficient protection against inactivation and 14C-carbamidomethyl incorporation. Analyses of the 14C-iodoacetamide-inactivated enzyme show that inactivation results from alkylation of 1 cysteine residue.