Amppnp

Drosophila full-length CaMKII (R3 isoform;^{58 (link)}) was a gift of Leslie Griffith (Brandeis University, Massachusetts, U.S.A.) while full-length Drosophila EAG (GenBank: M61157) was a gift of Dianne Papazian (UCLA, California, U.S.A.). Nucleotides (ATP (Sigma), AMPPNP (Jena Biosciences), ADP (Sigma) and AMPCP (Sigma)) were processed according to manufactures’ instructions. Stock solutions at 50 mM of ATP, AMPPNP and ADP were freshly prepared in either 1 M Tris pH 7.5 or 1 M HEPES pH 7.5. Stock solution of AMPCP at 50 mM in 1 M HEPES pH 7.5 was stored at −20 °C. λ-phosphatase (NEB) was processed according to manufacturer’s instructions, aliquoted and stored at −80 °C. Stock solutions of reagents used in the ATPase assay were prepared as follows: phosphoenolpyruvate (PEP; Sigma) at 50 mM in 50 mM HEPES pH 7.5; nicotinamide adenine dinucleotide, reduced (NADH; Sigma) at 6 mM in 50 mM HEPES pH 7.5; peptide syntide (GenScript) was prepared in water at 2.5 mM. All these solutions were kept aliquoted at −80 °C. The pyruvate kinase/lactate dehydrogenase mix (Sigma) was at 600–1000 units ml⁻¹ and 900–1400 units ml⁻¹ activity, respectively, and kept at −20 °C.

MCAK constructs were thawed and 5 μM samples were prepared in assembly buffer (10 mM K-PIPES, 100 mM KCl, 1 mM EGTA, 1 mM MgCl₂, pH 6.8) containing 2 mM nucleotide, held on ice until analysis. ATP, ADP (Sigma) and AMPPNP (Jena Bioscience) were used. Deuterium labeling was conducted by incubating 2.5 μM MCAK in 30 % D₂O (v/v)(Sigma) in assembly buffer + nucleotide for a given time at 25 °C. Kinetic analysis was done on EGFP-MFU-MCAK for 10, 30, 100, 300, and 1000 seconds, whereas the other constructs (MFU-MCAK and FL-MCAK) were labeled for 300 seconds. Labeling was quenched and digestion conducted at 10 °C for 2.5 minutes by addition 8 μL of Nepenthesin (Rey et al., 2013 (link)), 100 mM glycine-HCl, pH 2.35. The sample was injected into an LC-MS system for analysis.