Mitochondrial Enzyme Function Research Articles

Established human renal cell lines: Phenotypic characteristics define suitability for use in in vitro models for predictive toxicology

The phenotypes of the human renal epithelial cell lines, SK-NEP-1 and G401 (Wilm's tumour lines) and ACHN, A498, A704, Caki-1 and Caki-2 (renal adenocarcinomas), were investigated in order to develop as toxicological model systems, human renal cell lines showing properties similar to those found in discrete renal tubular segments. All cell lines, except G401, demonstrated significant ( P < 0.05) stimulation of adenyl cyclase activity by calcitonin. Alkaline phosphatase activity was not detectable in any cell line except for G401. None of the cell lines tested was capable of forming epithelial layers characteristic of ‘tight’ epithelia. The G401 cell line displayed several characteristics of the proximal nephron including a receptor for hPTH and detectable levels of the brush-border enzymes alkaline phosphatase (0.18 ± 0.02 mU/mg protein), leucine aminopeptidase (14.0 ± 5.1 mU/mg protein), glutathione transferase (8.61 ± 2.53 mU/mg protein) and γ-glutamyl transpeptidase (24.0 ± 2.1 mU/mg protein). hPTH (0.01–1 μ m) stimulated adenyl cyclase activity in homogenates of G401 cells in a dose-dependent manner, and this stimulation was reversed by 10 μ m of the specific PTH antagonist Nle, Tyr-PTH 3–34 amide. The addition of 10 μ m antagonist to unstimulated G401 cell homogenate reduced the basal activity of adenyl cyclase from 87.3 ± 17.4 to 45.9 ± 13.2 pmol cAMP/mg protein · 15 min. The effect of known nephrotoxic agents was tested on G401 cells by measuring basic mitochondrial enzyme function (MTT assay). The antibiotic gentamicin (5 m m), significantly ( P < 0.001) inhibited MTT activity in a dose-dependent manner with a maximum inhibition to23.2 ± 9.2% of untreated G401 cells. S- (1,1,2,2,tetrafluoroethyl)- l-glutathione (4 m m) and its cysteine conjugate (2.5 m m) reduced MTT activity to 44.0 ± 10.9% and 33.3 ± 2.6% of control untreated G401 cells, respectively. We suggest that the G401 cell line may be a useful model of the human proximal tubule in predictive toxicology.

A reversible component of cerebral injury as identified by the histochemical stain 2,3,5-triphenyltetrazolium chloride (TTC)

The extent of histochemical change following middle cerebral artery occlusion was quantitatively determined in three groups of Sprague-Dawley rats with 2,3,5-triphenyltetrazolium chloride (a marker of mitochondrial oxidative enzyme function). In group I (n = 7) occlusion was maintained for 3 h, with immediate sacrifice. In group II (n = 7) occlusion was maintained for 5 h, with immediate sacrifice. In group III (n = 7) occlusion was maintained for 3 h, followed by a 2-h period of reperfusion prior to sacrifice. The area of injury was significantly larger (P less than 0.05) in the 5-h occlusion group [15 +/- 4% (mean +/- SD)] compared to the 3-h occlusion group (9 +/- 2%); indicating a time-dependent worsening of the histochemical detection of injury. However, the area of injury was significantly less in the reperfusion group (5 +/- 2%) compared to the group that was evaluated after 3 h of occlusion without reperfusion (9 +/- 2%); indicating that some component of the injury revealed by 2,3,5-triphenyltetrazolium chloride is potentially reversible. These data suggest that contrary to previous understanding, the histochemical abnormality revealed by 2,3,5-triphenyltetrazolium chloride is reversible in some circumstances and does not necessarily represent inevitable infarction.

An E. coli aminoacyl-tRNA synthetase can substitute for yeast mitochondrial enzyme function in vivo

We have investigated the function of an E. coli aminoacyl-tRNA synthetase in S. cerevisiae strains that are respiration-deficient because of a mutation or a gene disruption in the nuclear encoded gene for the mitochondrial tyrosyl-tRNA synthetase. Although the yeast mitochondrial and E. coli tyrosine tRNAs differ significantly in sequence, expression of the E. coli tyrosyl-tRNA synthetase from a gene fusion restores respiration. The fusion gene contains a presumptive sequence for mitochondrial import from the mitochondrial tyrosyl-tRNA synthetase gene fused to the E. coli coding region. The fusion protein is incorporated into mitochondria. This incorporation and the rescue of the respiratory defect require the presumptive sequence for mitochondrial import. These experiments suggest a more limited definition of the identify of a tyrosine tRNA.

Properties and function of mitochondrial malate enzyme from bass liver

1. 1. Malate enzyme ( l-malate: NADP + oxidoreductase oxaloacetate decarboxylating, EC 1.1.1.40) from bass liver mitochondria was purified to over 90% of homogeneity by gel filtration, affinity and ion exchange chromatographies. 2. 2. The apparent molecular weight estimated by gel filtration was 316,000. Analysis of the enzyme on sodium dodecylsulphate-polyacrylamide disc gel electrophoresis was shown to be a tetramere protein. 3. 3. The enzyme required bivalent cations for catalysis, (Mn 2+ or Mg 2+) and displayed a narrow pH optimum (8.4–8.6 for Tris-HCl buffer) and was inactivated by p-chloromercuribenzoate. 4. 4. The double reciprocal initial velocity plots of both of the substrates, NADP and malate, were linear and intercepting at a point that suggests a sequential mechanism. 5. 5. Product inhibition studies with NADP and malate as variable substrate are consistent with an ordered Bi-Ter mechanism.