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Modulation of cellular transglutaminase: Isolation and characterization of transglutaminases from guinea pig epidermis

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posted on 2023-09-06, 02:53 authored by Hee Chul Kim

Transglutaminases are calcium-dependent enzymes that catalyze the formation of covalent $\varepsilon(\gamma$-glutamyl)lysine cross-links in proteins. A cationic proenzyme form of epidermal transglutaminase (protransglutaminase E) has been isolated from epidermis of new born mouse and from adult guinea pig. Upon activation, this zymogen is the source of most of the extractable enzyme activity in epidermis and skin. The molecular mass of protransglutaminase E determined by sedimentation equilibrium was 77,800 and was similar to the value estimated by SDS-PAGE. Dispase or thrombin treatment of the proenzyme resulted in an 80-fold activation of transglutaminase activity. In the nondenatured state, the Dispase-activated enzyme was shown by gel permeation and sedimentation equilibrium methods to be the same molecular size as the proenzyme. In the denatured state, the enzyme consisted of two peptide fragments of M$\sb{\rm r}$ 50,000 and M$\sb{\rm r}$ 27,000, as analyzed by SDS-PAGE under non-reducing conditions. The activated epidermal transglutaminase showed a thiol dependency for full enzyme activity as well as a low Ca$\sp{2+}$ ion requirement (Ka $<$ 10$\sp{-10}$ M). High levels of Ca$\sp{2+}$ ion induced a concentration dependent activation of proenzyme in a reversible manner. The purified zymogen from guinea pig skin displayed higher glycine, serine, and alanine contents then guinea pig liver transglutaminase. Westernbolt analysis of three different forms of transglutaminase present in epidermis, with monospecific polyclonal antibodies to protransglutaminase E, showed non-identity with either the membrane-associated transglutaminase or the cytosolic tissue transglutaminase. These results indicate that a substantial portion of the total transglutaminase in skin occurs as proenzyme form of the epidermal transglutaminase and that activation could be induced by neutral protease and Ca$\sp{2+}$ ions that are made available during the terminal differentiation of epidermal calls into the cornified layers of skin.

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American University

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English

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Ph.D. American University 1989.

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http://hdl.handle.net/1961/thesesdissertations:1849

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application/pdf

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Part of thesis digitization project, awaiting processing.

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