Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine

Journal Article · Tue Jan 14 00:00:00 EST 1992 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00116a009· OSTI ID:5445328

Kvedar, J C; Pion, I A; Bilodeau, E B; Baden, H P; Greco, M A ^[1]

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1. Harvard Medical School, Boston, MA (United States)

A method providing more sensitive detection of transglutaminase substrates were developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when ({sup 14}C)putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r{sup 2} = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.

• OSTI ID: 5445328
• Journal Information: Biochemistry; (United States), Vol. 31:1; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
UPTAKE
SUBSTRATES
ANTIGEN-ANTIBODY REACTIONS
TRANSFERASES
ENZYME IMMUNOASSAY
CADAVERINE
ELECTROPHORESIS
IN VITRO
IN VIVO
KERATIN
MONOCLONAL ANTIBODIES
PUTRESCINE
TRITIUM COMPOUNDS
AMINES
ANTIBODIES
BIOASSAY
CARBON COMPOUNDS
ENZYMES
HYDROGEN COMPOUNDS
IMMUNOASSAY
LABELLED COMPOUNDS
ORGANIC COMPOUNDS
PROTEINS
SCLEROPROTEINS
550201* - Biochemistry- Tracer Techniques