title: Activity and distribution of tissue transglutaminase in association with nerve-muscle synapses.
abstract: We have measured, characterized, and localized calcium-dependent protein cross-linking activity in rat skeletal muscle, and in myotubes cultured independently or in coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate-free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross-linking of endogenous proteins, as measured by the content of epsilon-(gamma-glutamyl)lysine isopeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and particulate (B) forms of tTG were separated by ion-exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B1) particulate form. Km values for calcium activation of the three forms of tTG were in the range of 5-15 microM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B1 transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.
authors.affiliation: Department of Zoology, University of Bristol, England.
authors.forename: D
authors.initials: D
authors.lastname: Hand
authors.forename: F J
authors.initials: FJ
authors.lastname: Campoy
authors.forename: S
authors.initials: S
authors.lastname: Clark
authors.forename: A
authors.initials: A
authors.lastname: Fisher
authors.forename: L W
authors.initials: LW
authors.lastname: Haynes
mesh_terms: D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D046508:Culture Techniques; D005260:Female; D008297:Male; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D009469:Neuromuscular Junction / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013569:Synapses / Q000502:physiology*; D014018:Tissue Distribution; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D011503:Transglutaminases