title: Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard.
abstract: The kinetic parameters and some enzymatic characteristics of human platelet and chicken gizzard transglutaminases were determined. Activity of the transglutaminases was regulated by calmodulin. These enzymes co-isolated with alpha-actinin and were dissociated from alpha-actinin by gel filtration and absorption onto a calmodulin affinity column. Silver-stained polyacrylamide gels showed that the protein peak eluted by EGTA from this column contained polypeptides of Mr approximately 58,000 and 63,000. The transglutaminases required Ca2+ for incorporation of monodansylcadaverine into casein and actin substrates. Activity was enhanced 3-fold by calmodulin with a biphasic effect, showing stimulation at 10-200 nM and inhibition at concentrations higher than 300 nM. In the presence of 200 nM calmodulin, half-maximal transglutaminase stimulation was obtained with 2.5 microM free [Ca2+]. Chlorpromazine inhibited calmodulin enhancement of the transglutaminases. Activity of the transglutaminases was independent of proteolytic activation, since inhibitors for Ca2+-dependent proteases failed to inhibit filamin cross-linking. For comparison, factor XIIa, a plasma and platelet transglutaminase, required both Ca2+ and thrombin for activation and was insensitive to calmodulin. The cross-linking pattern of fibrin, fibrin monomers, and fibrinogen by the calmodulin-regulated transglutaminases showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disappearance of fibrinogen alpha-chains with no decrease of beta- and gamma-chains or formation of gamma-gamma dimers. By autoradiography, cross-linked products of 125I-fibrinogen revealed heavily labeled high molecular weight polymers and polypeptides of Mr 98,000, 116,000, and 148,000; the latter appeared to be a transient species. However, when fibrin, fibrin monomers, and fibrinogen were used as factor XIIIa substrates, gamma-gamma dimers and alpha-polymers were formed. Formation of gamma-gamma dimers was slower with fibrinogen than with fibrin. Iodoacetamide blocked activity of factor XIIIa but not of the calmodulin-regulated transglutaminases.
authors.forename: E G
authors.initials: EG
authors.lastname: Puszkin
authors.forename: V
authors.initials: V
authors.lastname: Raghuraman
mesh_terms: D000185:Actinin / Q000032:analysis; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003285:Contractile Proteins / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005176:Factor XIII / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D064448:Filamins; D005895:Gizzard, Avian / Q000201:enzymology*; D005779:Immunodiffusion; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism; D008970:Molecular Weight; D011108:Polymers / Q000032:analysis; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D011108:Polymers; D000185:Actinin; D004533:Egtazic Acid; D005337:Fibrin; D005340:Fibrinogen; D005176:Factor XIII; D011503:Transglutaminases; D002118:Calcium; D007460:Iodoacetamide