Epidermal transglutaminase. Identification and purification of a soluble substrate with studies of in vitro cross-linking.

A Mr = 36,000 substrate for epidermal transglutaminase was identified immunochemically in buffer extracts of bovine snout epidermis, using antiserum to isolated high molecular weight substrate proteins recovered after cross-linking by transglutaminase. The high molecular weight proteins were not present in soluble epidermal extracts in EDTA prior to cross-linking. The substrate was purified by DEAE-Sepharose CL-6B and ACA-34 gel filtration, where it demonstrated an apparent molecular weight of 36,000. In the presence of Ca2+ and transglutaminase, the purified protein was converted to high molecular weight polymers which, under conditions of high protein concentration, included a protein aggregate insoluble in urea, sodium dodecyl sulfate, or beta-mercaptoethanol. Cross-linking did not occur with Ca2+ alone or in the presence of EDTA or putrescine, a competitive inhibitor. The epsilon-(gamma-glutamyl) lysine isodipeptide was identified in high molecular weight products of cross-linking but not in the Mr = 36,000 precursor. We postulate the intracellular assembly of Mr = 36,000 substrate, with subsequent cross-linking by transglutaminase and insolubilization into the keratinocyte membrane.