| Popis: |
Nitric oxide is a known regulator of cellular function that can act indirectly via activation of guanylyl cyclase, or directly via the S-nitrosylation of cysteine residues. However, little is known about the breadth of proteins that are regulated. Actin is present in nearly all eukaryotic cells and in a variety of isoforms that differ slightly in amino acid sequence, and more specifically in the number and placement of cysteine residues. It has been shown by others that different cysteines in the various isoforms can be modified by a number of oxidation reactions including S-nitrosylation. We therefore sought to determine the effect of S-nitrosylation on the interactions of skeletal, smooth, and non-muscle actins with myosin. We measured skeletal, smooth, and non-muscle (β and γ) actin nitrosylation in response to in vitro treatment with nitroso-L-cysteine - an endogenous nitrosothiol and NO donor - as well as actin filament velocity over heavy meromyosin (HMM), and the actin activated ATPase rates of HMM. A coumarin switch assay showed that all three isoforms could be nitrosylated by nitroso-L-cysteine. Nitrosylation of each actin isoform significantly decreased the in vitro velocity over HMM by approximately 38% with 50 μM donor compared to untreated controls. In contrast, there was no difference observed in the α-skeletal actin activated ATPase rates of the control and treated filaments. Further studies are being conducted to determine which cysteine residues in skeletal, smooth, and non-muscle actin are modified by nitroso-L-cysteine. Together these data suggest that nitrosylation of actin affects the attached time of actomyosin crossbridges. Given the ubiquitous nature of actin it is possible that nitrosylation is a common regulatory scheme for muscle contraction and various forms of cell motility. |
