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Delta-Aminolaevulinate synthase expression in muscle after contractions and recovery.

ABSTRACT: The synthesis of haem has been postulated to be a key regulatory step in muscle mitochondrial biogenesis. We examined the expression of delta-aminolaevulinate synthase (ALAs), the regulatory enzyme of haem metabolism, in 10 Hz electrically stimulated and non-stimulated control rat tibialis anterior (TA) muscle. ALAs activity and mRNA levels were measured at 0, 18 and 48 h of recovery after 3 h of acute stimulation, or after 7 days of stimulation (3 h/day). ALAs activity in control muscles averaged 7.8 +/- 0.8 nmol/h per g (n = 30). After 3 h of stimulation and during recovery, no change in ALAs activity occurred. ALAs mRNA during the same time was unchanged except at 48 h of recovery, when it increased 1.3-fold above control (P < 0.05). After 7 days of stimulation, ALAs activity was unchanged at 0 h, but increased at 18 and 48 h of recovery to 2.0- and 1.8-fold above control (P < 0.05). ALAs mRNA was also increased, but to a level averaging 1.6-fold above control (P < 0.05) at all times, indicating an increased mRNA stability or synthesis. No change in the haem-containing enzyme cytochrome c oxidase (CYTOX) activity occurred after 3 h of stimulation in the red section of the TA. After 7 days of stimulation, the increase in CYTOX activity averaged 1.7-fold above control (P < 0.05) at all times. Thus the induction of ALAs during recovery after 7 days was regulated by factors which not only change ALAs mRNA content, but which also affect ALAs mRNA at translational or post-translational steps. This induction occurred despite a 1.7-fold increase in CYTOX, implying that a precursor-product relationship does not always exist.

SUBMITTER: Takahashi M

PROVIDER: S-EPMC1132505 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Project description:1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta-aminolaevulinate synthase in vitro. These results suggest that haemin represses the synthesis of delta-aminolaevulinate synthase by decreasing the amount of mRNA for the enzyme available for translation.

Project description:Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber's oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may entail important implications for muscle function and fatigue resistance.

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Delta-Aminolaevulinate synthase expression in muscle after contractions and recovery.

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