Table I. Kinetic properties of recombinant smooth muscle myosin light chain kinases.

Recombinant and mutant smooth muscle myosin light chain kinases were expressed in COS cells, and activities were measured in lysates for the various kinetic parameters as described under “Materials and Methods.” The values are means ± S.E. for three to six experiments.

Kinase	Ca2+/CaMa	EGTAa	[Ca2+].0.5b	KCaMc	Kmd	Vmaxd
	pmol/min/mg		μm	nm
Wild-type	28.8 ± 3.1	0.0	0.66 ± 0.04	1.0	5.3 ± 1.2	36.9 ± 1.7
K979E	10.9 ± 2.6	0.0	3.50 ± 0.05	15 ± 1.0
RRK974-976EED	3.0 ± 0.3	0.0	8.53 ± 1.3	41 ± 5	4.3 ± 0.7	4.3 ± 0.4
KK969-970EE	24.5 ± 1.5	0.0	0.80 ± 0.05	4 ± 3	3.4 ± 0.4	33.0 ± 1.7
K965E-R967De	0.0	0.0
KK961-962EE	20.3 ± 2.1	0.0	0.29 ± 0.01	0.20 ± 0.12	3.1 ± 0.6	24.8 ± 2.2
KK961-962EE/KK969-970EE	5.1 ± 0.1	0.0	0.51 ± 0.06	0.55 ± 0.15

^aThe specific activities measured in the presence of Ca²⁺ and calmodulin or EGTA are expressed as pmol of ³²P incorporated/min/ng of kinase.

^bThe concentration of Ca²⁺ (μm) required for half-maximal activation of smooth muscle myosin light chain kinase.

^cK_CaM values (nm) were calculated from the data presented in Fig. 3 as described under “Materials and Methods.”

^dKinetic values (K_m and V_max) were determined from double-reciprocal plots (Lineweaver-Burk) of data obtained from experiments with varying concentrations of myosin light chain.

^eMutants K965E-R967D/KK969-970EE, KK961-962EE/K965E-R967D, and KK961-962EE/K965E-R967D/KK969-970EE were also catalytically inactive.