Effect of leupeptin of protein turnover in normal and dystrophic chicken skeletal muscle cells in culture

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

Kay¹,

Siemankowski²,

Siemankowski³

et al. 1982

“…Breakdown of the myofibrillar protein fraction in skeletal muscle is sensitive to nutritional stress and rehabilitation, muscle hypertrophy, denervation, neuromuscular diseases, hormone administration, altered intracellular Ca2+ concentrations and proteinase inhibitors (Fulks et al, 1975;Goldberg, 1969a,b;Kameyama & Etlinger, 1979;Laurent et al, 1978a,b,c;Maruyama et al, 1978;Millward et al, 1974Millward et al, , 1975aRiebow & Young, 1980;Rourke, 1975a,b;Segal et al, 1974). However, the intracellular signalling mechanisms that co-ordinate synthesis and breakdown of specific polypeptides are unknown.…”

mentioning

confidence: 99%

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

Young¹,

Dombroske²

1981

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.

“…The radioactivity incorporated into total cellular protein was determined by precipitation of samples of the initial cell homogenate with 5% (w/v) trichloroacetic acid (Riebow & Young, 1980). Radioactivity was then measured by liquid-scintillation spectrometry.…”

Section: Incorporation Of Radioactive Amino Acids Into Proteinmentioning

confidence: 99%

Effect of creatine on contents of myosin heavy chain and myosin-heavy-chain mRNA in steady-state chicken muscle-cell cultures

Young¹,

Denome²

1984

Embryonic-chick muscle cells reach a steady state with respect to protein metabolism after approx. 1 week in cell culture. To determine if this steady state could be altered by the administration of agents that have been reported to stimulate myosin heavy-chain synthesis, 7-day muscle-cell cultures were treated with 0-1 mM-creatine. Incorporation of [3H]leucine into myosin heavy chain was stimulated by 30-40% at the optimum creatine concentration (0.2 mM), but this stimulation was blocked when actinomycin D (10 micrograms/ml) was also present. However, the quantity of myosin-heavy-chain mRNA as measured by hybridization in vitro was only 15% higher in creatine-treated cultures, and was therefore not entirely responsible for the observed effect. It is important to note that creatine only exerted its action on myosin-heavy-chain synthesis rate in steady-state cultures; creatine was ineffective in altering this rate in rapidly differentiating 3-day muscle cultures. Finally, muscle-cell cultures that had been grown for the entire 7-day culture period in the presence of 0.2 mM-creatine were assayed for quantity of myosin heavy chain. Control and creatine-treated cultures contained 12.7 +/- 1.5 and 20.5 +/- 1.8 micrograms/dish respectively. In conclusion, creatine apparently enhances the quantity of myosin heavy chain in steady-state embryonic muscle-cell cultures, but it probably does not mediate regulation of myosin content in adult skeletal muscle.