Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex

Abstract

The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue-specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by approximately 50% reduced, mitochondrial genome-encoded COX I and II mRNAs and nuclear genome-encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50-80% reduction in mitochondrial transcription and translation rates in heme-depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme-depleted tissues contained a 30-50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme-depleted tissues. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.

Accumulation of Amyloid Precursor Protein in the Mitochondrial Import Channels of Human Alzheimer’s Disease Brain Is Associated with Mitochondrial Dysfunction

“…Cytochrome c oxidase activity in mitochondria from the frontal cortex of AD1, AD11, AD16, NC2, NC15, and NC17 was measured as described previously (Vijayasarathy et al, 1999). The rate of oxidation of ferrocytochrome c was measured by a decrease in absorbance at 550 nm.…”

3D model of RNA polymerase and bidirectional transcription

“…Mitochondrial D-loop region; In-vitro transcription; mt RNA polymerase; gel-mobility shift assay; 3D model Both human and mouse mitochondrial (mt) in-vitro reconstituted transcription system and mt lysate were shown to transcribe bidirectionally from D-loop region containing H-strandpromoter/L-strand-promoter (HSP/LSP) of mitochondrial genome .These transcripts ranged between >100bp and >3000bp in both human and mouse [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The D-loop distal genes were also transcribed in this system [16].…”

“…The D-loop distal genes were also transcribed in this system [16]. The nuclear coded RNA polymerase, RNase P and RNase MRP as well as several transcription factors have now been proposed to function in a co-ordinate fashion for the synthesis of the polycistronic RNA and its processing into individual RNA molecules [1][2][3][4][5][6][7][8][9][10][11][12]. The ideal human transcription complex [13] was described to constitute RNA polymerase, transcription factor TFB1M (or TFB2M having weaker activity than TFB2M), LSP/HSP template (1-741bp) and mt general transcription factor TFAM.…”

“…The purification of mitochondrial cytochrome c oxidase (EC 1.9.3.1) was described before [22,27,28]. Subunits (I-IV) of cytochrome c oxidase were gel purified for raising antiserum/ antibody in rabbit as described before [1]. …”

“…Both RNA polymerase and RNase MRP are involved for mt DNA replication from a RNA-D-loop DNA hybrid [1][2][3][4][5][6][7][8][9][10], whereas these two enzymes in addition to RNase P are involved in transcription as well as RNA (mRNA, rRNA and tRNA) processing of human and mouse mt.RNA [1][2][3][4][5][6][7][8][9][10][11][12]23]. RNase MRP cleaves mRNA substrate at an adjacent conserved decamer sequence 5′-CGACCCCUCC-3′ (relative to CSB2 in D-loop), whereas RNase P is involved in 5′ and 3′ end-processing of mt tRNA [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. This conserved decamer sequence 5′-CGACCCCUCC-3′ is also present in MRP RNA [3,4,7,[11][12][13].…”