Table 2.

Transglutaminase-catalyzed formation of labeled insoluble aggregates from labeled R₅O₁₈R₅ and polypeptide amine donors

	14C incorporation into aggregates, dpm × 10−5
	Exp. 1	Exp. 2	Exp. 3
Polylysine + purified transglutaminase
+ Prelabeled R5Q18R5	0.62	1.06	0.78
+ Unlabeled R5Q18R5 + 14C - GEE	0.003	0.001
+ Prelabeled R5Q18R5 + EDTA	0.08	0.12	0.06
− R5Q18R5 + 14C - GEE	0.006	0.002
− Polylysine	0.06	0.15
Brain extract + purified transglutaminase
+ Prelabeled R5Q18R5	2.5	3.4
+ Unlabeled R5Q18R5 + 14C - GEE	0.18
+ Prelabeled R5Q18R5 + EDTA	0.12	0.07
− R5Q18R5 + 14C - GEE	0.007
− Brain extract	0.12
Brain extract + any residual purified transglutaminase from preincubation
+ Prelabeled R5Q18R5	0.21	0.21
+ Unlabeled R5Q18R5 + 14C - GEE	0.001	0.0009
+ Prelabeled R5Q18R5 + EDTA	0.014	0.033
− R5Q18R5 + 14C - GEE	0.0024	0.0014

R₅Q₁₈R₅ was labeled by preincubation with 2 μCi of ¹⁴C glycine ethyl-ester (GEE) for 3 h in the presence of purified transglutaminase. Subsequent incubation with amine donors (brain proteins or polylysine) resulted in the formation of aggregates insoluble in the presence of 20 mM 2-mercaptoethanol and either 4 M urea or 1% SDS at 100°C for 5 min. The aggregates were centrifuged, washed, and counted. Included among the negative controls was one in which the R₅Q₁₈R₅ was not prelabeled with ¹⁴C glycine ethyl-ester, but the latter was incubated together with the unlabeled R₅Q₁₈R₅ during the enzymatic coupling to polylysine or brain protein.