There was no peak of 14C corresponding to free polyamines (Figure 2B), suggesting that polyamines in CST fractions were covalently incorporated into axonal tubulins. In vivo stabilization of neuronal MTs by polyamines could occur by covalent posttranslational modification of tubulins. To confirm modification of tubulins with polyamines and evaluate a potential role for transglutaminase, an enzyme known to catalyze polyamination, purified mouse brain tubulin/MTs

were incubated with recombinant transglutaminase in vitro. In vitro transglutaminase activity was controlled by addition of Ca2+. Transglutaminase polyaminated both tubulin and polymerized MTs via a Ca2+-dependent INK1197 reaction. Modification was visualized in two ways: First, MDC, a fluorescent diamine and transglutaminase substrate (Lorand et al., 1971), was used as a polyamine analog, giving a fluorescent band at the apparent MW of tubulin in SDS-PAGE. This band was observed whether the substrate was unassembled tubulin or assembled MTs (Figure 3A; see also Figure S2). Second, modified tubulins were visualized by immunoblot when physiological polyamines (SPM and SPD) were used. Bands were identified as tubulin using the DM1A antibody against α-tubulin, and polyamine modification was identified with an anti-SPM/SPD antibody that recognizes both SPM and SPD

(Figure 3B). Both free tubulin and tubulin in MTs were modified by transglutaminase and SPM/SPD.

Polyaminated tubulins/MTs showed reduced solubility as the bulk of modified tubulins Obeticholic Acid solubility dmso were pelleted. When transglutaminase was incubated with tubulin without polyamines, we observed tubulin crosslinking, but crosslinked tubulin aggregates remained in supernatants rather than pelleting, and much crosslinked tubulin failed to enter the gel (Figures 3C and 3D). The presence of polyamines in reactions largely eliminated crosslinking. To determine Megestrol Acetate whether modification of tubulin by transglutaminase favors disassembled or polymerized tubulin, similar amounts of free tubulins and taxol-stabilized MTs were incubated in vitro with transglutaminase and polyamines. There was no significant difference in incorporation of either MDC (Figures 3C and 3D) or SPM/SPD (data not shown). Patterns of polyamine modification on unassembled tubulins (Figures 3A–3D), polymerized MTs (Figure 3A), and taxol-stabilized polymerized MTs (Figures 3C and 3D) were similar. Electron microscopy showed polyaminated tubulins polymerized in vitro at 37°C (Figure 3E). In contrast, tubulin treated with transglutaminase without polyamines formed large aggregates with no evidence of MT formation (Figure 3F). Since intracellular polyamine levels are high, crosslinking is unlikely to occur. These data suggest that transglutaminase-catalyzed polyamination of tubulin occurs on both tubulin and MTs, and that polyaminated tubulins polymerize normally.