Metabolism of myosin heavy chain in steady-state chick skeletal muscle cultures

Abstract

Synthesis, accumulation and breakdown of the 200000-mol.wt. heavy subunit of myosin were analysed over an 11 day period in muscle cell cultures isolated from the leg muscle of 12-day chick embryos. Muscle cells accumulated myosin heavy chain rapidly from days 2 to 5 and maintained a maximum, constant myosin-heavy-chain concentration between days 7 and 11. Myosin-heavy-chain content and breakdown rate were compared in steady-state muscle cultures grown either in the presence of an optimum batch of horse serum (control) or in the presence of horse serum that had been pre-selected for its ability to inhibit several-fold the rate of synthesis of myosin heavy chain (inhibitory). The quantity of myosin heavy chain in the inhibited cultures was decreased in direct proportion to the decrease in the rate of synthesis of myosin heavy chain; however, the half-lives of myosin heavy chain (control, 17.7h; inhibitory, 17.0h) were virtually identical. In contrast, the absolute rate of breakdown of myosin heavy chain, expressed as molecules/min per nucleus, was approx. 5-fold lower in the inhibited cultures (4.3 X 10(3) molecules/min per nucleus) than in the control cultures (21.7 X 10(3) molecules/min per nucleus). Thus, inhibition of myosin-heavy-chain synthesis in this case was accompanied by diminished myosin-heavy-chain concentration and absolute breakdown rate at the altered steady state, but relative myosin-heavy-chain breakdown rates were unchanged.

Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes

“…Previously published estimates of MHC half-lives in cultured myotubes vary from 20-200 hours (Rubenstein et al, 1976;Walker and Strohman, 1978;Zani et al, 1978;Carvatti and Perriard, 1981;Young and Dombroske, 1981). These studies all involved isolation of leucine-labelled fractions containing myosin or actomyosin followed by SDS-PAGE and counting radioactivity within gel slices.…”

Measuring protein breakdown rate in individual proteins in vivo

“…the difference between synthesis and breakdown rates). $$\frac{d{P}^{\ast }}{dt}=\stackrel{•}{P^{\ast }}=-k_d{P}^{\ast }$$ $$\frac{dP}{dt}=\stackrel{•}{P}=r_{italic\text{net}}$$ $$\begin{array}{cc}left\frac{d\left(\frac{P^{\ast }}{P}\right)}{dt}=\frac{\stackrel{•}{P^{\ast }}P-P^{\ast }\stackrel{•}{P}}{P^2}=\frac{(-k_d{P}^{\ast })P-P^{\ast }{r}_{\text{net}}}{P^2}=-k_d\left(\frac{P^{\ast }}{P}\right)-\frac{r_{\text{net}}}{P}\left(\frac{P^{\ast }}{P}\right)& center\text{where}E=\frac{P^{\ast }}{P}\\ left\Downarrow & center\\ left\frac{dE}{dt}=-(k_d+\frac{r_{\text{net}}}{P})E& center\end{array}$$ As it is the case for determination of the fractional synthesis rate [39], a steady protein mass is required during these experiments as well [29,31,40]. When the protein mass rightly can be assumed to be unchanged over the course of the FBR-assessment ( r net = 0), the change in protein enrichment is described by the mono-exponential decay with the FBR, k d , as the disappearance rate: $$\frac{dE}{dt}=-k_{d}E\iff E\left(t\right)=e^{-kd}$$…”

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

“…Although elegant approaches were developed (e.g., see Ref. 49), it became apparent that the reutilization of the recently released tracer by reuptake via new protein synthesis results in an underestimated breakdown rate, which seems hard to circumvent and avoid (1,34). To avoid reincorporation of tracer amino acids, the tracer amino acids must be quickly removed via metabolism after release from protein breakdown or not reutilized at all.…”

“…If protein can be prelabeled with one or more labeled amino acids, then the rate of disappearance of labeled amino acids from protein can be measured to determine protein breakdown (14,32,34,46,49). Historically, this approach has been used in vitro on bacteria (14,27,36,46), in cell cultures (41), and in chick skeletal muscle cells (49) as well as in human muscle cells (34).…”

“…If protein can be prelabeled with one or more labeled amino acids, then the rate of disappearance of labeled amino acids from protein can be measured to determine protein breakdown (14,32,34,46,49). Historically, this approach has been used in vitro on bacteria (14,27,36,46), in cell cultures (41), and in chick skeletal muscle cells (49) as well as in human muscle cells (34). In all approaches, an isotope-labeled amino acid was introduced into proteins, and the average rate of protein breakdown was obtained by measuring the rate of release of labeled amino acids from the prelabeled protein structure.…”