Brain Mitochondrial Protein Research Articles

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5‐Triiodothyronine (T3) inhibited L‐[14C]leucine uptake into synaptosomes. Inhibition was competitive with a Ki of 3.1 × 10−5m. Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l‐Thyroxine and analogues inhibited the incorporation of l‐[14C] leucine into cerebral synaptosome protein. At 50 μm, the triiodo‐compounds were more inhibitory than tetraiodo‐>3,5‐triiodo‐l‐thyronine >3,3′,5‐triiodothyropro‐pionic> l‐thyroxine >3,5‐diiodo‐l‐tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5‐Triiodothyronine had no effect on respiratory control or 2,4‐DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [14C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm‐ouabain. 3,3′,5‐Triiodothyronine had no effect on synaptosome fraction ATPase or Na‐K ATPase. Addition of T3 induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm) or cycloheximide (50μg/ml). [14C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5‐triiodothyronine. There was no inhibition of [14C]proline uptake or incorporation.The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.

Inhibition of memory formation in the imprinting period: Irreversible action of cycloheximide in young chickens

1. (1) A single intracranial, 40 μg dose of cycloheximide, administered during the first week of life, renders a chicken permanently incapable of learning at its normal rate. 2. (2) The onset of the period of greatest sensitivity to cycloheximide's effect on subsequent visual learning is delayed until the end of the first day of life just as is the critical period for visual imprinting. 3. (3) The time course of susceptibility for subsequent retarded auditory learning precedes that for visual learning in the same way that the ability to imprint in the auditory system precedes that for the visual system. treatment at 6 h is maximally effective while injection on day 3 does not produce auditory retardation though it still produces visual retardation. 4. (4) The permanent learning deficit is not produced by brain mitochondrial protein synthesis inhibition using chloramphenicol and does not occur after subcutaneous injection of cycloheximide. 5. (5) Complete protection from the retardation effect has been conferred by manipulating early sensory experience while the drug is acting. Reduction of either visual or auditory input can prevent the chicken from becoming a retarded learner in the deprived modality. 6. (6) The permanent learning deficit is a consequence of the effects, in the metabolically deranged brain, of the sensory input that is normally necessary for early learning.

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria.

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 +/- 0.1, 0.6 mM NaHCO(3), had an electrophoretic mobility of - 2.88 +/- 0.01 micro/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 +/- 0.02, of rat liver mitoplasts was - 1.22 +/- 0.07, and of rat cerebral cortex mitoplasts - 0.91 +/- 0.04 micro/sec per v per cm. Treatment of these particles with 50 microg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in micro/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 microg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK(a) approximately 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.

Protein synthesis in isolated rat brain mitochondria and nerve endings

The relationship of brain mitochondrial protein synthesis to nerve ending protien synthesis was investigated by isolating both fractions simultaneously from young rats and comparing the incorporation of [ 14C]leucine under identical incubation conditions. Both fractions had similar requirements for sodium and potassium activation with concentrations of100mMand10mMrespectively producing maximum levels of incorporation. Calcium, magnesium, insulin, and EDTA inhibited both systems in a parallel fashion. Chloramphenicol had almost no effect on either fraction. Cycloheximide depressed protein synthesis to approximately 25% of control values at concentrations of 10 μg/ml. The data suggest that the mechanism of protein synthesis in nerve endings and brain mitochondria is similar if not identical, and that this mechanism is sensitive to cycloheximide and resistant to chloramphenicol.

Protein synthesis in isolated rat brain mitochondria

Protein synthesis in a rat brain mitochondrial preparation purified by centrifugation through a ficoll gradient is inhibited by chloramphenicol and oxytetracycline but not by cycloheximide. This establishes that rat brain mitochondrial protein synthesis is similar to liver mitochondria in its sensitivity to these antibiotics. The possible nature of the cyclo-heximide-sensitive protein synthesis found with other brain mitochondrial preparations is discussed.

Brain and liver mitochondrial protein synthesis: Potassium dependent chloramphenicol inhibition

Protein synthesis in vitro in mitochondrial preparations from mouse brain and mouse liver is sensitive to inhibition by chloramphenicol if potassium is the predominant cation in the incubations, but not when the media are enriched with respect to sodium ion and are potassium ion poor. All preparations of brain mitochondria examined to date also exhibit sensitivity to inhibition by cycloheximide, whereas this is not found with liver mitochondria.

Isolation and partial characterization of beef heart proteolipid

A rapid procedure for the preparation of beef heart proteolipid by solvent fractionation of the total lipid extract of the tissue is described. The product is composed of two-thirds protein and one-third lipid, nearly all of which is phospholipid. Cardiolipin accounts for over half of the lipid moiety and both this lipid and phosphatidylinositol are enriched several-fold in the proteolipid, as compared to the phophsholipids of whole beef heart. The protein moiety may be freed of more than 90% of associated phospholipid by treatment of the proteolipid with Dowex 1 (formate) followed by precipitation of the apoprotein with diethyl ether. The apoprotein is soluble in acidified chloroform-methanol and contains 14–15% N and 0.2% P. N-terminal amino acid determinations and polyacrylamide-gel electrophoresis indicate that the apoprotein is heterogeneous. The principal N-terminal amino acid is aspartic acid, but nine other N-terminal amino acids can also be detected. The amino acid composition of the proteolipid apoprotein is indistinguishable from that of the intact proteolipid, but differs significantly from both brain white matter proteolipid and mitochondrial structural protein.