Mitochondrial transcription factor A (mtTFA) and diabetes.

Mitochondrial DNA (mtDNA) content decreased in an age-dependent manner and may be one of the causal factors in age-related type 2 diabetes. Mitochondrial transcription factor A (mtTFA), which provides the replication primer, plays a key role for the regulation of mtDNA replication and its level is proportional to mtDNA. Here, we studied on the regulatory mechanism of mtTFA expression and the factors affecting the transcriptional activity of the mtTFA promoter. The promoter of human mtTFA contains 67 CpG dinucleotides. When the plasmids bearing the mtTFA promoter (2378 bp) linked to luciferase were transiently transfected into HepG2 cells, in vitro methylation of NRF-1 site by HhaI methylase abolished the mtTFA promoter activity up to 90%, implying that the CpG methylation of NRF-1 site inactivate mtTFA promoter-driven transcriptional activity. Besides the promoter methylation, the exogenous hydrogen peroxide or glucose also modulates the promoter activity of mtTFA. The bacterially overexpressed mtTFA protein exhibits a strong binding affinity to circular DNA (perhaps to mtDNA in mitochondria in vivo) and the protection of the DNA from cleavage by a hydrogen peroxide attack. Taken all these results together, age-related alterations of oxidative stress may affect mtDNA replication via regulating mtTFA activity. Furthermore, a vicious cycle may be present between mtTFA protein level and oxidative stress in the sense of DNA damage. Further studies were necessary to prove the presence of methyl cytosine in the mtTFA promoter of either an aged or a diabetic person and the effect of oxidative stress on the mtTFA function and expression resulting in a change of the mtDNA copy number.

Correlation of plasma homocysteine and mitochondrial DNA content in peripheral blood in healthy women.

Hyperhomocysteinemia is an independent risk factor of cardiovascular disease and associated with insulin resistance, although their causal relationship remains unclear. A previous report has shown that high concentration of homocysteine damages mitochondrial gene expression, function and structure. As we found recently, the mitochondrial DNA (mtDNA) contents are inversely correlated with insulin resistance parameters. Thus there is possibility that plasma total homocysteine (tHcy) level is somewhat correlated with mtDNA content. Sixty healthy women (mean age 40.3+/-20.9 yr, range 18-78 yr) were recruited to investigate the correlation of plasma tHcy level and mtDNA content in peripheral blood. A significant negative correlation was found between plasma tHcy levels and mtDNA content (r=-0.507, P<0.01). Plasma tHcy and mtDNA content have an independent effect on each other and on insulin resistance (HOMA-insulin resistance (IR) score) respectively in multiple regression model. Plasma tHcy showed positive correlations with age (r=0.407), W/H ratio (r=0.370), total cholesterol (r=0.338), LDL-cholesterol (r=0.317) and insulin resistance (HOMA-IR score) (r=0.261); and a negative correlation with folate (r=-0.273). MtDNA content showed negative correlations with age (r=-0.407), BMI (r=-0.440), W/H ratio (r=-0.659), SBP (r=-0.350), total cholesterol (r=-0.340), triglyceride (r=-0.376), LDL-cholesterol (r=-0.349), fasting plasma insulin (r=-0.483), and insulin resistance (HOMA-IR score) (r=-0.423); and a positive correlation with folate (r=0.299). In this study, there was a significant inverse correlation between plasma tHcy level and mtDNA content. Further study will be warranted to elucidate the mechanism by which two factors are associated.

The orphan nuclear receptor small heterodimer partner as a novel coregulator of nuclear factor-kappa b in oxidized low density lipoprotein-treated macrophage cell line RAW 264.7.

Small heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but was suppressed by oxidized low density lipoprotein (oxLDL) and its constituent 13-hydroxyoctadecadienoic acid, a ligand for peroxisome proliferator-activated receptor gamma. Furthermore, SHP acted as a transcription coactivator of nuclear factor-kappa B (NF kappa B) and was essential for the previously described NF kappa B transactivation by palmitoyl lysophosphatidylcholine, one of the oxLDL constituents. Accordingly NF kappa B, which was transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF kappa B in oxLDL-treated, resting macrophage cells.

Peripheral blood mitochondrial DNA content is related to insulin sensitivity in offspring of type 2 diabetic patients.

OBJECTIVE: To investigate whether the peripheral blood mtDNA (pb-mtDNA) content is decreased and linked to insulin resistance in the offspring of type 2 diabetic patients. RESEARCH DESIGN AND METHODS: A total of 82 offspring of type 2 diabetic patients and 52 age-, sex-, and BMI-matched normal subjects from the Mokdong, Korea, population were selected for this study by stratified, randomized sampling. Of the offspring of diabetic patients, 52 had normal glucose tolerance (NGT), 21 had impaired glucose tolerance (IGT), and 9 had newly diagnosed type 2 diabetes. The pb-mtDNA content was measured by real-time polymerase chain reaction with a mitochondria-specific fluorescent probe, normalized by a nuclear DNA, 285 rRNA gene. The associations between pb-mtDNA content and several parameters of insulin resistance were studied. RESULTS: The pb-mtDNA contents tended to be lower in the 82 offspring of type 2 diabetic patients (1,084.7 +/- 62.6 vs. 1,304.0 +/- 99.2 in the offspring and control subjects, respectively, P = 0.051) and was significantly lower in the combined NGT and IGT offspring group (NGT+IGT, 1,068.0 +/- 67.8, P < 0.05) than in the control subjects. In NGT+IGT offspring, the pb-mtDNA content was significantly correlated with logarithmically transformed insulin sensitivity (r = 0.253, P < 0.05) and was the main predictor of insulin sensitivity. CONCLUSIONS: Quantitative mtDNA status might be a hereditary factor associated with type 2 diabetes and could serve as an indicator for insulin sensitivity.

Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells.

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.

The angiopoietin-tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis.

OBJECTIVES: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery. METHODS: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. RESULTS: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. CONCLUSION: These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.

Role of endocytosis in the transactivation of nuclear factor-kappaB by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivation by the peroxisome proliferator-activated receptor (PPAR)-gamma and by nuclear factor-kappaB (NF-kappaB). In the present study, the oxLDL signalling pathways involved in NF-kappaB transactivation were investigated by utilizing a reporter construct driven by three upstream NF-kappaB binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase in intracellular calcium and stimulated NF-kappaB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. NF-kappaB activation by oxLDL or lysoPC was inhibited by inhibitors of protein kinase C or by a chelator of intracellular calcium. Tyrosine kinase or phosphatidylinositol 3-kinase inhibitors did not block NF-kappaB transactivation. Furthermore, oxLDL-induced NF-kappaB activity was abolished by PPAR-gamma ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, NF-kappaB transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-kappaB in resting macrophages via protein kinase C- and/or calcium-dependent pathways, and that this does not involve the endocytic processing of oxLDL. The endocytosis-dependent activation of PPAR-gamma by oxLDL may function as a route of inactivation of the oxLDL-induced NF-kappaB signal.

Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B.

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (NF-kappaB) (p40-kappaB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the kappaB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-kappaB binding to the kappaB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-kappaB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-kappaB-mediated activation in IL-12 p40 promoter. The NF-kappaB components p50 and p65 directly bound peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-gamma inhibited the NF-kappaB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-kappaB-DNA interactions and physical interactions between NF-kappaB and PPAR-gamma.

Identification of retinoic acid receptor element in human cholesteryl ester transfer protein gene.

The transcriptional regulation of the human cholesteryl ester transfer protein (CETP) gene by retinoic acid was investigated by a transient transfection assay. A series of deleted vectors generated from the 5'-upstream region (3434 bp) of the CETP gene linked to the luciferase reporter gene was individually transfected to HepG2 cells. Promoter analyses revealed essential regulatory machinery in the -110/-40 region of the upstream sequence of the human CETP gene. When the cells transfected with the reporter vectors were stimulated with all-trans retinoic acid (tRA), the hormone response was drastically reduced when the -165/-110 region was deleted, thereby suggesting that there may be a retinoic acid receptor element (RARE) in the region. A footprinting analysis showed that the DNA segment at the -165/-134 is protected by the HepG2 nuclear extract. A competition analysis on the gel mobility shift assay using consensus RARE and a purified retinoic acid receptor confirmed the -165/-134 region as being RARE.

Oxidation-dependent effects of oxidized LDL: proliferation or cell death.

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.

End-organ resistance to growth hormone and IGF-I in epiphyseal chondrocytes of rats with chronic renal failure.

We tested the hypothesis that there is direct end-organ resistance to growth hormone (GH) and IGF-I in chronic renal failure (CRF) independent of circulating inhibitors. Male Sprague-Dawley rats underwent 5/6 nephrectomy and were pair-fed with weight matched (100 g) sham operated controls for two weeks. Rats with CRF had significantly higher serum creatinine and blood urea nitrogen (P < 0.01 in both cases) and gained significantly less weight and length (P < 0.01 in both cases) compared with controls. Epiphyseal chondrocytes were grown in 10% fetal calf serum (FCS). Both CRF cells and control cells maintained chondrogenic phenotypes, and showed immunohistochemical staining with antibodies to collagen II and proteoglycan (aggrecan). Distribution of the cell subpopulations according to cell size (by flow cytometry) and alkaline phosphatase activity of CRF and control chondrocyte cultures were not different. Growth responses of CRF chondrocytes were reduced (P < 0.01) compared with control chondrocytes when grown in 10% FCS and 10% normal rat serum. Under serum free conditions, growth responses of CRF chondrocytes were reduced to GH and IGF-I at concentrations of 10, 30 and 100 ng/ml, and to insulin at 100, 300 and 1,000 ng/ml compared with controls cells (P < 0.01). To show that this resistance is specific for the GH/IGF system, growth responses to fibroblast growth factor and transforming growth factor beta 1 were studied and showed no difference between CRF and control cells. Thus, the present study provides direct evidence of specific end-organ resistance to GH, IGF-I in CRF chondrocytes in the absence of circulating factors.

Activation of LDL receptor gene expression in HepG2 cells by hepatocyte growth factor.

The effect of recombinant human hepatocyte growth factor (HGF) on low density lipoprotein (LDL) receptor gene expression was studied in the human hepatoma cell line HepG2. HepG2 cells were incubated with serum-free media in the presence and absence of HGF for various times and 125I-labeled LDL specific binding at 4 degrees C, uptake at 37 degrees C, and the levels of LDL receptor mRNA were measured. Incubation with HGF produced time- and concentration-dependent increases in 125I-labeled LDL binding (2-fold), uptake (2.5-fold), and LDL receptor mRNA (6-fold). HGF increased the rate of LDL receptor gene transcription 4- to 5-fold relative to that of several "house-keeping" genes as measured by nuclear run-on transcription. The half-life of LDL receptor mRNA, measured with actinomycin D, was not increased in HGF-treated cells. The stimulation of LDL receptor expression occurred independently of changes in cellular cholesterol or DNA biosynthesis or total cell protein. HepG2 cells were transiently transfected with plasmids bearing either three copies of repeats 2 and 3 (pLDLR(23)3LUC) or one copy of the LDL receptor promoter from -556 to +53 (pLDLR600LUC) linked to firefly luciferase. Incubation of pLDLR(23)3LUC, or pLDLR600LUC-transfected cells with HGF for 4 or 24 h at 37 degrees C produced a concentration-dependent increase in luciferase activity. A maximal stimulation of 3 to 6-fold was achieved for each construct at an HGF concentration of 100 ng/ml. In contrast, HGF had little or no effect on reporter activity in HepG2 cells transfected with a luciferase reporter plasmid bearing the HMG-CoA reductase promoter extending from -325 to +22. Thus, when compared to the native LDL receptor promoter, multiple copies of repeats 2 and 3 of the LDL receptor promoter can fully support activation of the luciferase reporter gene by HGF, demonstrating that the effect of HGF is mediated through the SRE-1. The lack of HGF effects mediated through the HMG-CoA reductase sterol regulatory element suggests, however, that sterol depletion may not be responsible for the induction of the LDL receptor promoter by growth factors. The signalling pathways or effectors responsible for activation of the LDL receptor and HMG-CoA reductase genes thus differ in their response to HGF. These data suggest that the level of SREBP's reaching the nucleus may be determined by as yet unidentified second messengers as well as by sterols.

Serum response element-like sequences of the human low density lipoprotein receptor promoter: possible regulation sites for sterol-independent transcriptional activation.

Serum factors stimulate low density lipoprotein receptor (LDLR) gene expression in HepG2 cells through sterol-independent pathways. Promoter element other than sterol regulatory element-1 (SRE-1) seems to be necessary. Protein binding activity of the human LDLR promoter fragment (550bp) beyond the SRE-1 was determined by DNase I footprint assay. Five different promoter regions were protected from DNase I digestion; -226 to -258, -291 to -304, -324 to -336, -360 to -373, and -521 to -528. The regions of -324 to -336 and -521 to -528 showed serum response element (SRE)-like consensus sequence of CC(A/T)6GG. Serum incubation affected the protection degree of the SRE-like elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 showed serum response elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 in human LDLR gene may be responsible for the rapid activation of the gene transcription by serum factor in a sterol-independent manner.

Identification of protein-receptor components required for the import of prealdehyde dehydrogenase into rat liver mitochondria.

Mitochondrial aldehyde dehydrogenase is synthesized as a high-molecular-weight precursor in cytosol and transported into mitochondrial matrix space where it is processed to the mature enzyme. To identify components of the transport machinery on liver mitochondria, anti-idiotypic antibodies against the rabbit anti-prealdehyde dehydrogenase signal peptide antibodies were produced in chicken eggs and rabbit. Both anti-idiotypic antibodies inhibited the import of prealdehyde dehydrogenase (pALDH) into isolated rat liver mitochondria. The rabbit anti-idiotypic antibody could recognize by Western blotting five mitochondrial membrane proteins with apparent molecular weights of 66, 60, 42, 34, and 29 kDa. The anti-idiotypic antibodies were cross-linked to mitochondrial membrane proteins using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate which is an iodinatable, heterofunctional, and photoreactive cross-linker. Mitochondrial proteins with apparent molecular weights of 66, 60, and 42 kDa were identified using the chicken antibody. The 66- and 34-kDa proteins were cross-linked to the rabbit antibody as the major components and the 42-kDa protein as a minor one. Antibodies against the 60- and 42-kDa proteins, as well as Fab fragments, inhibited the import of pALDH, suggesting that these proteins are receptor/translocator components for pALDH import.

Location and regulation of low-density lipoprotein receptors in intestinal epithelium.

The expression, distribution, and some aspects of the regulation of low-density lipoprotein (LDL) receptors in rat intestinal epithelial cells were examined. Cells prepared by a perfusion technique provided a pure preparation of epithelial cells and could be manipulated to produce crypt-villus units or villi alone. On a total protein basis, the abundance of LDL receptors in villus cell membranes was half that in hepatic membranes. The level of receptors in both tissues was reduced by feeding an atherogenic diet but was increased only in the liver by ethinyl estradiol-induced hypocholesterolemia. The level of LDL receptor mRNA in intestinal epithelial cells was somewhat lower than in liver. Regulation of LDL receptor mRNA was similar to that of protein. Judged by the ratio of mRNA in villus cells to the villus-crypt unit and nuclear run-on assay for LDL receptor gene transcription, we conclude that LDL receptor mRNA is produced in the villus cells. The effect of fat feeding was regulated at the level of transcription. Expression in villus cells in ileum was severalfold higher than in jejunum and higher than in the liver. Together the results suggest serum cholesterol level is not the prime determinant of LDL receptor level in intestine, but LDL degradation in this organ may be regulated by factors in the lumen.

2D NMR and structural model for a mitochondrial signal peptide bound to a micelle.

The 19 amino acid signal peptide of rat liver aldehyde dehydrogenase, possessing a lysine substitution for an arginine and containing 3 extra amino acid residues at the C terminus, was studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment, the peptide contains two alpha-helical regions, both of which are amphiphilic, separated by a hinge region. The helix located closer to the C terminus is more stable than is the helix located near the N terminus. This suggests that the hydrophobic face of the C-terminal helix is buried within the hydrophobic region of the micelle. On the basis of these results a general model for protein translocation is presented in which the C-terminal amphiphilic helix of the signal region in the preprotein first binds to the mitochondrial membrane and then diffuses to the translocation receptor. The receptor then recognizes the N-terminal helix of the signal region, which is not anchored to the membrane. To explain how this signal peptide was imported into isolated mitochondria in the absence of energy or receptor protein [Pak, Y. K., & Weiner, H. (1990) J. Biol. Chem. 265, 14298-14307], a model for signal peptide translocation across a membrane barrier without the need for auxiliary membrane proteins is proposed. In this model the faces of the two helices fold upon each other, resulting in the mutual shielding of positively charged residues by the complementary hydrophilic face of the other amphiphilic helix.

Effects of alcohol on the import of aldehyde dehydrogenase precursor into rat liver mitochondria.

We previously showed that incubation of rat liver mitochondria with alcohols resulted in the inhibition of the import of aldehyde dehydrogenase precursor but not that of ornithine transcarbamylase precursor (Wang TTY, Farrés J, and Weiner H: Arch Biochem Biophys 272:440-449, 1989). The time required for inhibition of import to occur was now measured with ethanol (200 mM) and butanol (100 mM) at 0 degree and 30 degrees C. It required approximately 30 min to achieve 50% inhibition with butanol and 50 min with ethanol. To further substantiate the membrane perturbing effects of alcohols, we also examined the effect of oleic acid on import. We found that incubation of mitochondria with oleic acid (0-100 microM) resulted in inhibition of aldehyde dehydrogenase precursor import in a dose response fashion. In addition to in vitro effects of alcohols on import, we conducted a preliminary study on import of protein into liver mitochondria isolated from rats fed ethanol. We found that the rate of aldehyde dehydrogenase precursor import into liver mitochondria isolated from ethanol fed rats was identical to that from control. The results are consistent with finding that the activity and amount of aldehyde dehydrogenase was the same in mitochondria isolated from the alcohol-fed or control animals.

Import of chemically synthesized signal peptides into rat liver mitochondria.

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.

Purification and characterization of beef and pig liver aldehyde dehydrogenases.

Beef liver cytosolic, mitochondrial, and pig liver mitochondrial aldehyde dehydrogenases (ALDH) had been purified to homogeneity. The two mitochondrial enzymes as with other mammalian mitochondrial enzymes had properties very similar to that of the corresponding human enzyme. These include immunological as well as basic kinetic properties such as low Km for aldehyde, activation by Mg2+ ions, and lack of inhibition by disulfiram. A major difference between these two enzymes and the human mitochondrial enzyme was that they contained an N-terminal-blocked amino acid. Cytosolic ALDHs from human and horse liver have been shown to possess an N-acetyl serine as the N-terminal residue; beef cytosolic ALDH was also found to be blocked. Tissue preparations and subcellular fractions from beef or pig liver could be used to study acetaldehyde oxidation. This is the subject of the accompanying paper (Cao Q-N, Tu G-C, Weiner H, Alcohol Clin Exp Res 12:xxx-xxx, 1988).