Mechanism of Action of Guinea Pig Liver Transglutaminase I. PURIFICATION AND PROPERTIES OF THE ENZYME: IDENTIFICATION OF A FUNCTIONAL CYS- TEINE ESSENTIAL FOR ACTIVITY

Guinea pig liver transglutaminase has been purified 230fold 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 f 5,000 (sedimentation and diffusion), 90,000 f 4,000 (sedimentation equilibrium), and 90,000 f 10,000 (14Ciodoacetamide incorporation), show some variation. The s&+ and Dzs,w values for transglutaminase were determined as 5.40 x 10-u and 6.44 x lo* cm* set-l, 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 i4C-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.