The incorporation of glycerol into mitochondrial lipids during neonatal renal compensatory growth

[¹⁴C]glycerol incorporation into isolated inner and outer mitochondrial membrane is enhanced in the remaining kidney after unilateral nephrectomy. Serum from neonatal rabbits taken 24 hours after unilateral nephrectomy and added to tissue slice incubations appears to stimulate incorporation of [¹⁴C]glycerol into mitochondrial lipids of normal kidney cortex. Post-nephrectomy serum, however, depresses incorporation of [¹⁴C] glycerol and [³H]leucine into mitochondria when added to kidney cortex from animals in which uninephrectomy was performed 24 or 48 hours previously.     

Mitochondrial heterogeneity in foetal and suckling rat liver: differential leucine incorporation into the proteins of two mitochondrial populations.

Iodination of intact mitochondria with ¹²⁵I results in the labeling of essentially one polypeptide with an approximate MW of 30 000. This polypeptide seems to be a component of the inner boundary membrane as it can not be removed from the mitochondria by procedures which destroy the outer membrane (e.g. incubation with digitonin). The amount of the radio-active label which can be bound to this polypeptide is determined by ADP, atractylate, and bongkrekate, components which act on the functional state and the position of the $\text{ADP}\text{ATP}$ carrier in the membrane. [1,2]     

Effects of a leucine analog on growth hormone processing and secretion by cultured cells

Bovine and rat growth hormones (bGH and rGH, respectively) possess signal peptides that direct the hormone to the secretory pathway and are proteolytically cleaved prior to secretion. Previous in vitro translation studies indicated that incorporation of the polar leucine analog beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal peptide function. To test the effects of this analog on GH secretion by cultured animal cells, transfections of mouse L-cells with a bGH expression plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells was performed in the absence or presence of beta-hydroxyleucine. Transient expression of bGH in mouse L-cells or endogenous expression of rGH in anterior pituitary cells resulted in an accumulation of GH in the culture medium. Treatment with beta-hydroxyleucine resulted in a block in secretion as evidenced by an accumulation of GHs within these cells. Amino-terminal sequencing of the intracellular form of the analog-substituted GHs demonstrated accurate signal peptide cleavage. In contrast, in vitro translations of bGH RNA performed in the presence of beta-hydroxyleucine and microsomal membranes resulted in the inhibition of signal peptide cleavage. The results suggest that beta-hydroxyleucine can uncouple signal peptide processing and protein secretion in cultured cells.     

Double labelling study of the spreading depression effect on the incorporation of labelled leucine into the proteins of the brain mitochondrial subfractions

T opical application of potassium chloride solutions (above 0·25 M) as well as some other agents onto the surface of the brain cortex provokes typical changes in the basic performance of the brain. Serious impairment of learning as well as depression of spontaneous activity, depolarization of the cortical cells, disappearance of the evoked responses, changes in unit activity and alteration of energy metabolism are among the detectable features of this phenomenon called cortical spreading depression (CSD) by its discoverer LEÃ (1944). For reviews on CSD see M arshall (1959); B urést (1962); O chs (1962). The inhibitory effect of CSD on the incorporation of the labelled amino acids into the proteins of the rat cerebral cortex has been reported (R uščák 1964; B ennett and E delman , 1969; K řivánek , 1969, 1970) and the distribution of the effect among the crude subcellular fractions was studied by K řivánek (1970). Possible involvement of macromolecules, particularly proteins in memory formation and storage has been claimed. It was tempting to see whether the proteins of some mitochondrial subfractions namely nerve endings particles and pure mitochondria, could reveal a differential susceptibility to the potassium action which, in vivo , induces such profound changes in the brain cortex function.     

Amino acid incorporation into the protein of mitochondrial preparations from cerebral cortex and spinal cord

1. Washed guinea-pig cerebral-cortex mitochondria incorporate [(14)C]leucine into their protein at a rate comparable with the rates reported for liver or heart mitochondria only if the mitochondria are separated from myelin and nerve endings by density-gradient centrifugation. 2. The non-mitochondrial components (myelin and nerve endings) of brain mitochondrial preparations incorporated [(14)C]leucine at a negligible rate. 3. The mitochondria do not require an exogenous supply of energy or a full supply of amino acids to support the process. 4. The incorporation rate was linear up to 2hr. aerobic incubation at 30 degrees and was inhibited by chloramphenicol, only slightly by actinomycin D and not by penicillin or pretreatment with ribonuclease. The observed incorporation is considered to be unlikely to be due to contaminating cytoplasmic ribosomes or bacteria. 5. The process was also studied in mitochondrial preparations from rabbit cerebral cortex and spinal cord.     

Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucine into Plasmodium falciparum in vitro

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [3H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [3H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.