Isolation and incubation conditions to study heart mitochondrial protein synthesis

Abstract

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

Nonclinical and Pharmacokinetic Assessments To Evaluate the Potential of Tedizolid and Linezolid To Affect Mitochondrial Function

“…An in vitro study using isolated rat heart mitochondria was conducted to compare the tedizolid and linezolid concentrations that inhibit 50% (IC 50 ) of MPS. The assay, sample preparation techniques, and data analysis were previously described in detail (9,36). In brief, intact, highly coupled mitochondria isolated from normal rat heart were incubated in a medium containing […”

“…The rate of MPS was expressed as picomoles of methionine incorporated per milligram of mitochondrial protein. Dose-response curves were analyzed with Sigma Plot, version 12.5 (Systat Software Inc., San Jose, CA), using the best-fit slope for each concentration of compound tested, expressed as a percentage of the vehicle control best-fit slope, plotted against the test compound concentration (9,36). Data shown represent means and standard errors of the means (SEM) of the best-fit slopes at each concentration (n ϭ 6); the best-fit hyperbolic decay regression line of these data (using the least-squares method) was used to determine the MPS IC 50 .…”

Oxazolidinones Inhibit Cellular Proliferation via Inhibition of Mitochondrial Protein Synthesis

“…The procedure for mitochondria preparation and the protein synthesis assay was adapted from that described by McKee et al (29). All experiments involving animals were carried out in compliance with national legislation and subject to local ethical review.…”

Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism

“…Mitochondria from heart and liver were isolated, and shuttle assays were performed as described in detail previously (28) based on methods described by McKee et al (17). Briefly, hearts were retrograde-perfused with ice-cold isolation buffer containing (in mM) 220 mannitol, 70 sucrose, and 2 EGTA at pH 7.0 (MSE) followed by perfusion with MSE plus 0.4 mg/ml Nagarse (Sigma protease XXVII).…”