Energy-Linked Synthesis and Decay of Membrane Proteins in Isolated Rat Liver Mitochondria<sup>*</sup>

Wheeldon, L. W.; Lehninger, Albert L.

doi:10.1021/bi00875a021

Cited by 131 publications

(31 citation statements)

References 45 publications

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“…Results presented in this paper support the idea that energy for mitochondrial protein synthesis is derived from ATP itself, and not from a "high energy" intermediate or state of the mitochondria [6,21,371. Thus : a) Oligomycin (5 pg) did not inhibit incorporation when an ATP-generating system was used as the energy source while it did inhibit substrate-supported incorporation.…”

Section: Metabolic Inhibitorssupporting

confidence: 73%

Incorporation in vitro of [¹⁴C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Williams¹,

Birt²

1971

European Journal of Biochemistry

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The energy requirements for incorporation in vitro of [14C]leucine into sterile mitochondria isolated from thoracic flight muscles of newly-emerged Lucilia cuprina have been studied.There is an absolute requirement for addition of either ADP or ATP. Oxidisable substrates (pyruvate, DL-a-glycerophosphate, proline), or an ATP-generating system increased incorporation above that observed with added ADP or ATP alone. Both the rate and duration of incorporation are strongly dependent on the concentration of adenine nucleotides used. Experiments with oligomycin and 2,4-dinitrophenol suggest that energy for incorporation is provided as ATP.With non-sterile preparations, incorporation was, independent of added adenine nucleotides, and almost constant for up to four hours with no added substrate or adenine nucleotide. It was inhibited by added substrate and also by added amino acids, especially isoleucine and valine.A previous report [l] has demonstrated the ability of sterile preparations of mitochondria from the thoracic flight muscle of newly-emerged Lucilia cuprina t o incorporate [14C]leucine into trichloroacetic acid insoluble material in vitro. A number of variables affecting the behaviour of the system in vitro was studied in that report. Because of the paucity of information on mitochondrial protein synthesis in insects [2,3], it was decided to continue the study by characterising, in some depth, the energy requirements for incorporation in vitro.Hence in this paper the requirements for adenine nucleotides and oxidisable substrates, the effectiveness of an ATP-generating system compared with oxidisable substrates, and the effects of inhibitors of energy supply on [14C]leucine incorporation have been studied.Results are compared with non-insect systems (e.g. Neurospora crassa [4] MATERIALS AND METHODS Tissue Preparations Incubation Conditions for [ '*C]Leucine IncorporationIncubations were conducted in 5-ml test tubes covered with aluminium caps a t 30 "C in a shaking water bath; the final incubation volume was 1.0 ml containing approximately 1 .O mg (three thoraces) of mitochondrial protein. After 5 min preincubation, incubations were commenced by adding 0.5 pCi [14C]leucine (344 mCi/mmole).All experiments (except that described in Fig. 9) were done with the following "basic mixture" (final concentrations in 1 ml): 1 mM EDTA, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES) buffer pH 7.4,iO mM potassium phosphate pH 7.4,65 mM KC1,lS mM MgSO,, amino acid mixture lacking leucine (19 amino acids, each 0.05 mM, i.e. 143 pg total [l]). Preparation of Samples for Scintillation CountingSamples were prepared for counting as described previously [i]. Results are not corrected for observed counting efficiency which was 84O/,.

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Section: Metabolic Inhibitorssupporting

confidence: 73%

Incorporation in vitro of [¹⁴C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Williams¹,

Birt²

1971

European Journal of Biochemistry

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“…(Kroon, 1965 ;Mahler et al ., 1968, Wheeldon andLehninger, 1966 . ) All three antibiotics chloramphenicol, lincomycin, and erythromycin strongly inhibit in vivo particulate mitochondrial cytochrome synthesis, albeit not completely like euflavine and ethidium .…”

Section: Mitochondrial Amino Acid Incorporationmentioning

confidence: 99%

Biogenesis of Mitochondria

Kellerman

Biggs

Linnane

1969

The Journal of Cell Biology

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Growth under conditions of oxygen restriction results in a generalized decrease in the definition of the mitochondrial membranes, a decrease in the mitochondrial cytochromes, and a decrease in citric acid cycle enzymes of the obligate aerobic yeast Candida parapsilosis. Addition of unsaturated fatty acids and ergosterol to cultures exposed to limited oxygen results in improved definition of the mitochondrial membranes and an increase in the total mitochondrial cytochrome content of the cells. Euflavine completely inhibits mitochondrial protein synthesis in vitro. Its in vivo effect is to cause the formation of giant mitochondrial profiles with apparently intact outer membranes and modified internal membranes; the cristae (in-folds) appear only as apparently disorganized remnants while the remainder of the inner membrane seems intact. Cytochromes a, a3, b, and c1 are not synthesized by the cells in the presence of euflavine. Ethidium appears to have effects identical to those of euflavine, whereas chloramphenicol, lincomycin, and erythromycin have similar effects in principle but they are less marked. The effects of all the inhibitors are freely reversible after removal of the drugs. The results are discussed in terms of a functionally three-membrane model of the mitochondrion. In addition, the phylogenetic implications of the observed differences between this organism and the facultative anaerobic yeasts are considered.

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“…Mitochondria were isolated by the method of Greenawalt et al [14] employing enzymic digestion of cell walls. The mitochondria were introduced into the amino acid incorporation system of Wheeldon and Lehninger [15], containing ~~-['~C]leucine, which employs an external source of ATP (phosphoenolpyruvic acid, ATP, and pyruvate kinase). The mitochondria were precipitated with 10 % trichloroacetic acid containing 20 mM unlabelled oL-leucine and then washed and counted by the method of Hawley and Greenawalt [8].…”

Section: Resultsmentioning

confidence: 99%

“…Incorporation of [14C]leucine was performed using the medium of Wheeldon and Lehninger [15]. Incubations were carried out for 15 min at 30 "C and incorporation of radioactivity determined as described before [S].…”

Section: Incorporation Of [14c]leucine By Mitochondria In Vitromentioning

confidence: 99%

Biogenesis of Mitochondrial Membranes in Neurospora crassa

Hawley

Greenawalt

1975

European Journal of Biochemistry

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The conidia of Neurospora crassa entered logarithmic growth after a 1‐h lag period at 30°C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3‐h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide‐sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0–8‐h‐old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 μg cycloheximide/ml, increased from low levels in the conidia to peak levels at 3–4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0–4‐h‐old cells, but declined after 15 min of incorporation in 6 and 8‐h‐old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12200 to 80000). Within the high‐molecular‐weight class, proteins of between 12000 and 21500 molecular weight were preferentially labelled early in germination, whereas after 8 h of growth proteins of 27500 to 80000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000‐molecular‐weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.

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Energy-Linked Synthesis and Decay of Membrane Proteins in Isolated Rat Liver Mitochondria^*

Cited by 131 publications

References 45 publications

Incorporation in vitro of [¹⁴C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Incorporation in vitro of [¹⁴C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Biogenesis of Mitochondria

Biogenesis of Mitochondrial Membranes in Neurospora crassa

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Energy-Linked Synthesis and Decay of Membrane Proteins in Isolated Rat Liver Mitochondria*

Cited by 131 publications

References 45 publications

Incorporation in vitro of [14C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Incorporation in vitro of [14C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Biogenesis of Mitochondria

Biogenesis of Mitochondrial Membranes in Neurospora crassa

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Product

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Energy-Linked Synthesis and Decay of Membrane Proteins in Isolated Rat Liver Mitochondria^*

Incorporation in vitro of [¹⁴C]Leucine into the Mitochondrial Protein of Lucilia cuprina

Incorporation in vitro of [¹⁴C]Leucine into the Mitochondrial Protein of Lucilia cuprina