Abstract
Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.
Highlights
Class III myosins were originally discovered in Drosophila eyes and designated as the NINAC protein
Because calmodulin is thought to be involved in the phototransduction process, concentrating calmodulin may be a major role for myosin III in fly photoreceptors
Protein Expression and Purification—The expression yields of human myosin IIIA 2IQ co-expressed with calmodulin (MIII) in the baculovirus insect cell (Sf9) system were ϳ0.5–1 mg/liter of Sf9 cells at a density of 2.0 ϫ 106 cells/ml
Summary
Reagents—All reagents were the highest purity commercially available. ATP and ADP were prepared fresh from powder. Samples from sequential time points for each specific ATP concentration were run on SDS-PAGE, transferred to nitrocellulose membrane, blocked with 3% bovine serum albumin and probed with anti-phosphothreonine primary antibodies (Zymed Laboratories Inc.). Mant-labeled nucleotides were excited by energy transfer from tryptophans by exciting at 295 nm or by direct excitation at 355 nm, and the emission was measured through a 400 nm long pass filter Both methods of exciting mant-ATP and mant-ADP gave similar results. Phosphate release was measured with a sequential mix experiment in which 2 M myosin III was mixed with 1.75 M ATP and aged for 5 s to allow myosin III to bind and hydrolyze the ATP, and the MIII1⁄7ADP1⁄7Pi complex was mixed with actin (0 – 40 M) [29, 36] (final concentrations after mixing 1.0 M MIII, 0.875 M ATP, 2.7 M MDCC-PBP). All concentrations mentioned in the stopped-flow experiments are final concentrations unless stated otherwise
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