Table I. Kinetic and immunological properties of wild-type and mutant myosin light chain kinases.
Kinetic parameters were determined from double-reciprocal plots. Each assay was performed in duplicate; the means ± S.E. obtained from multiple assays are presented. Immunoblots were performed as described previously (Herring et al. 1990). Examples of the blots are shown in Fig. 3. The reactivity of each monoclonal antibody to the mutant kinases is summarized; a plus sign indicates detectable reactivity, and a minus sign indicates that no reactivity was detected.
| Kinase | Km | Vmax | Antibody reactivity |
||||
|---|---|---|---|---|---|---|---|
| 16a | 14a | 12a | 9a | 2a | |||
| μM | pmol32P/min/ng | ||||||
| WTa | 3.5 ± 0.6 (n = 5) | 22.8 ± 4.6 | + | + | + | + | + |
| LCB1 | 9.4 ± 0.9 (n = 12) | 18.3 ± 1.4 | − | + | + | − | + |
| LCB2 | 43.7 ± 4.6 (n = 5) | 32.1 ± 3.9 | + | + | − | + | − |
| LCB3 | 30.7 ± 2.3 (n = 6) | 20.9 ± 1.4 | − | + | − | − | − |
| Nt | 0 | + | + | + | + | + | |
a
WT, wild-type myosin light chain kinase expressed in COS cells; Nt, truncated kinase (residues 1–294).