Activation of transglutaminase and production of protein-bound gamma-glutamylhistamine in stimulated mouse mast cells.
The identification of transglutaminase in the growth-factor-dependent mouse mast cell line PT18 was accomplished through its characteristic catalytic properties (specificity, calcium dependency, and inhibition by iodoacetamide); and by both immunoprecipitation and Western blot analysis using affinity purified antibody. The enzymatic activity in these cells increased in association with the release of histamine from the cells induced by an IgE-dependent mechanism or by exposure to the ionophores A23187 or Br-x537A. The increase in transglutaminase activity was paralleled by a marked increase in the level of protein-bound gamma-glutamylhistamine, determined in radiolabeled form in mast cells that were either metabolically labeled with [3H]histidine or incubated with [3H]histamine before degranulation. The highest level of bound gamma-glutamylhistamine was found in the immunologically stimulated cells. Enzymatic activity and the gamma-glutamyl derivative were associated primarily with the cells, both before and after stimulation. Separation of gamma-glutamylhistamine in a proteolytic digest of these cells was carried out using a combination of ion exchange chromatography and high performance liquid chromatography. The gamma-glutamyl compound was identified and quantitated through the enzymatic production of histamine with the use of gamma-glutamylamine cyclotransferase, an enzyme specific for the disassembly of gamma-glutamylamines.
Fesus
L
L
Szucs
E F
EF
Barrett
K E
KE
Metcalfe
D D
DD
Folk
J E
JE
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D006632:Histamine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D006636:Histamine Release; D006639:Histidine / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism*; D011503:Transglutaminases / Q000032:analysis* / Q000502:physiology
D011506:Proteins; C013541:gamma-glutamylhistamine; D006639:Histidine; D006632:Histamine; D011503:Transglutaminases