Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers

Abstract

Thermogenesis by resting muscle varies with conditions and plays an active role in homeostasis of body weight. The low metabolic rate of living resting muscles requires that ATP turnover by myosin be inhibited relative to the purified protein in vitro. This inhibition has not been previously seen in in vitro systems. We used quantitative epifluorescence microscopy of fluorescent nucleotides to measure single nucleotide turnovers in relaxed, permeable skeletal muscle fibers. We observed two lifetimes for nucleotide release by myosin: a fast component with a lifetime of ≈20 s, similar to that of purified myosin, and a slower component with a lifetime of 230 ± 24 s. We define the latter component to be the "super relaxed state." The fraction of myosins in the super relaxed state was decreased at lower temperatures, by substituting GTP for ATP or by increased levels of myosin phosphorylation. All of these conditions have also been shown to cause increased disorder in the structure of the thick filament. We propose a model in which the structure of the thick filament modulates the nucleotide turnover rates of myosin in relaxed fibers. Modulation of the relative populations of the super relaxed and conventional relaxed states could have a profound effect on muscle thermogenesis, with the capacity to also significantly alter whole-body metabolic rate. metabolic rate | thick filament | phosphorylation | fluorescent nucleotides T he metabolic activity of resting skeletal muscle is of interest because it plays a significant role in the whole-body restingenergy expenditure (1). Muscle metabolism is involved in coldinduced thermogenesis, in consumption of calories from excess food intake, and is a major regulator of blood-sugar levels (1-5). The mechanism of muscle thermogenesis and its regulation remain an active area of investigation. Here we identify another mechanism that plays a role in thermogenesis of resting skeletal muscle, the modulation of myosin ATPase activity by the structure of the thick filament.Our study also addresses a long-standing discrepancy concerning myosin kinetics. Ferenczi et al. (6) noted an inconsistency between the ATP turnover rate of purified frog myosin and the rate of oxygen consumption of living, resting frog muscle. The low metabolic activity of living, resting muscle (6, 7) sets an upper limit on the myosin ATPase activity, and this limit was less than one-fifth of the rate observed for myosin in vitro. A similar difference is seen in mammalian fibers (8, 9). The ATP turnover rate of rabbit myosin [0.16 s −1 at in vivo temperatures (6, 9) would also have to be inhibited by more than a factor of 5 to be compatible with the resting-energy consumption of rabbit muscle [0.7 J × L −1 s × −1 (8, 9)]. These observations show unambiguously that myosin in living, resting vertebrate muscle is inhibited by a large factor relative to purified myosin measured in vitro.Although more than 30 years have passed since the original observation, the mechanism of myosin inhibition, unambiguously required to...

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Phosphorylation of myosin regulatory light chain has minimal effect on kinetics and distribution of orientations of cross bridges of rabbit skeletal muscle

“…For isolectric focusing, a sample was diluted to 2.5 mg/ml in a buffer [9 M urea, 130 mM DTT, 20% glycerol, 250 l of 40% carrier ampholines pH 4 -6 (Bio-Rad, Hercules, CA), 10% Triton X-100]. Denaturing isoelectric focusing gel (10 M urea, 18% acrylamide/bis-acrylamide, 20% glycerol, 10% Triton-X 100, 1 ml of 40% ampholines, 60 l of 10% APS, and 30 l of TEMED) was run overnight at constant 350 V and fixed with 7% TCA, 50% methanol for 2 h following the procedure of Cooke and colleagues (56,67). The gel was washed with double-distilled water for 10 min and stained with Diamond Pro-Q phosphoprotein gel stain (Invitrogen) for 3 h in the dark.…”

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