Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.     

In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.     

Sample preparation for two-dimensional blue native/SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes

Ammonium sulfate precipitation was tested as a sample preparation step for BN-PAGE analyses of S. coelicolor cytoplasmic protein complexes. A procedure of sample preparation compatible with two-dimensional BN/SDS-PAGE was established and used to visualize protein complexes. To validate the sample preparation procedure, representative protein complexes were identified. Several previously characterized protein complexes were rediscovered and their reported oligomeric states reconfirmed. In addition, we identified new but plausible interactions that have never been reported before. Our work provides useful reference for the wide application of BN-PAGE in protein interaction study.     

Application of "blue native" electrophoresis in the studies of mitochondrial respiratory chain complexes in physiology and pathology

"Blue Native" polyacrylamide gel electrophoresis (BN-PAGE), originally described by Sch?gger and von Jagow in 1991, is an elegant method to study protein complexes from mitochondrial membranes. BN-PAGE, commonly used in molecular biology to study composition of protein complexes and protein-protein interactions, enables separation of respiratory chain complexes keeping their properties and enzymatic activities unchanged. BN-PAGE, supplemented by other methods, e.g. in gel activity assay, SDS-PAGE (as a first or second dimension) can be successfully adapted for diagnosis of mitochondrial diseases connected with abnormalities of the respiratory chain. Therefore, to make a correct diagnosis of the deficiency of respiratory chain complexes, other methods, as histochemical colorimetric reactions allowing evaluation of the OXPHOS catalytic activity in individual cells and spectrophotometric technique should be used simultaneously with BN-PAGE.     

Coupling of native liquid phase isoelectrofocusing and blue native polyacrylamide gel electrophoresis: a potent tool for native membrane multiprotein complex separation

In this study, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. It is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Finally, each single component can be resolved using denaturing electrophoresis (3D N-LP-IEF-BN-SDS-PAGE). The thylakoid membrane of spinach which contains four big protein complexes was chosen as a model for setting up analytical methods suitable for any membrane proteins. The pI-based MicroRotofor has a number of advantages over BN-PAGE: it does not require the addition of any chemicals, and separation of complexes is based on the protein's real physicochemical properties which inevitably change when dye is added. Results were more easily reproduced than with BN, and the pI of each native complex was also determined. Although some fractions still contained comigrating complexes after MicroRotofor, these were subsequently separated by BN for further analysis. Thus, highly hydrophobic complexes, such as ATP-synthetas and Cyt b6/f, were separated in native form as were various complexes of LHCII trimers, which have different pI but similar molecular masses. SDS-PAGE revealed almost all the subunits from the four photosynthetic complexes, indicating that by using 3D N-LP-IEF-BN-SDS-PAGE it is possible to achieve a greater degree of component identification than with 2D BN-SDS-PAGE.     

Changes in mitochondrial electron transport chain activity during insect metamorphosis

The midgut of the tobacco hornworm (Manduca sexta) is a highly aerobic tissue that is destroyed by programmed cell death during larval-pupal metamorphosis. The death of the epithelium begins after commitment to pupation, and the oxygen consumption of isolated midgut mitochondria decreases soon after commitment. To assess the role of the electron transport chain in this decline in mitochondrial function, the maximal activities of complexes I-IV of the respiratory chain were measured in isolated midgut mitochondria. Whereas there were no developmental changes in the activity of complex I or III, activities of complexes II and IV [cytochrome c oxidase (COX)] were higher in mitochondria from precommitment than postcommitment larvae. This finding is consistent with a higher rate of succinate oxidation in mitochondria isolated from precommitment larvae and reveals that the metamorphic decline in mitochondrial respiration is due to the targeted destruction or inactivation of specific sites within the mitochondria, rather than the indiscriminate destruction of the organelles. The COX turnover number (e- x s(-1) x cytochrome aa3(-1)) was greater for the enzyme from precommitment than postcommitment larvae, indicating a change in the enzyme structure and/or its lipid environment during the early stages of metamorphosis. The turnover number of COX in the intact mitochondria (in organello COX) was also lower in postcommitment larvae. In addition to changes in the protein or membrane phospholipids, the metamorphic decline in this rate constant may be a result of the observed loss of endogenous cytochrome c.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.     

Analysis of chloroplast ribosomes by polyacrylamide gel electrophoresis and electron microscopy

Ribosomal particles were isolated from chloroplasts and cytoplasm of eukaryotes, Euglena gracilis and Spinacia oleracea, and from prokaryotes, E. coli and Anacystis nidulans. The ribosomes were analyzed by polyacrylamide gel electrophoresis and by negative staining in the electron microscope. The prokaryote ribosomes show a slight difference in their electrophoretic mobilities between the two species: E. coli ribosomes migrate ahead of the Anacystis ribosomes. In comparison to eukaryote cytoplasmic ribosomes, chloroplast ribosomes of both species demonstrate a higher electrophoretic mobility and significantly smaller dimensions (about 230 × 187 Å compared to about 197 × 162 Å). Some differences in form were noted for Euglena cytoplasmic ribosomes which may contribute to their high S value. In comparison to prokaryote ribosomes, the mobility of chloroplast ribosomes is similar to the mobility of the prokaryote group of ribosomes, and it specifically coincides with the migrating band of ribosomes from the blue-green alga, Anacystis. Subunits of chloroplast and prokaryote ribosomes have similar mobilities and cannot be distinguished in gels. The similarities in size and in electrophoretic mobilities of chloroplast and blue-green algal ribosomes support the hypothesis of a common phylogenetic origin for the two.     

Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism

Mitochondria play important roles in generation of free radicals, ATP formation, and in apoptosis. We studied the levels of mitochondrial electron transport chain (ETC) complexes, that is, complexes I, II, III, IV, and V, in brain tissue samples from the cerebellum and the frontal, parietal, occipital, and temporal cortices of subjects with autism and age-matched control subjects. The subjects were divided into two groups according to their ages: Group A (children, ages 4-10 years) and Group B (adults, ages 14-39 years). In Group A, we observed significantly lower levels of complexes III and V in the cerebellum (p<0.05), of complex I in the frontal cortex (p<0.05), and of complexes II (p<0.01), III (p<0.01), and V (p<0.05) in the temporal cortex of children with autism as compared to age-matched control subjects, while none of the five ETC complexes was affected in the parietal and occipital cortices in subjects with autism. In the cerebellum and temporal cortex, no overlap was observed in the levels of these ETC complexes between subjects with autism and control subjects. In the frontal cortex of Group A, a lower level of ETC complexes was observed in a subset of autism cases, that is, 60% (3/5) for complexes I, II, and V, and 40% (2/5) for complexes III and IV. A striking observation was that the levels of ETC complexes were similar in adult subjects with autism and control subjects (Group B). A significant increase in the levels of lipid hydroperoxides, an oxidative stress marker, was also observed in the cerebellum and temporal cortex in the children with autism. These results suggest that the expression of ETC complexes is decreased in the cerebellum and the frontal and temporal regions of the brain in children with autism, which may lead to abnormal energy metabolism and oxidative stress. The deficits observed in the levels of ETC complexes in children with autism may readjust to normal levels by adulthood.     

Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse

The purpose of the present study was to examine the role of mitochondria in the aging process by determining whether the activities of various electron transport chain oxidoreductases are deleteriously affected during aging and whether the hypothesized age-related alterations in different tissues follow a common pattern. Activities of respiratory complexes I, II, III, and IV were measured in mitochondria isolated from brain, heart, skeletal muscle, liver, and kidney of young (3.5 months), adult (12-14 months), and old (28-30 months) C57BL/6 mice. Activities of some individual complexes were decreased in old animals, but no common pattern can be discerned among various tissues. In general, activities of the complexes were more adversely affected in tissues such as brain, heart, and skeletal muscle, whose parenchyma is composed of postmitotic cells, than those in the liver and kidney, which are composed of slowly dividing cells. The main feature of age-related potentially dysfunctional alterations in tissues was the development of a shift in activity ratios among different complexes, such that it would tend to hinder the ability of mitochondria to effectively transfer electrons down the respiratory chain and thus adversely affect oxidative phosphorylation and/or autooxidizability of the respiratory components.     

线粒体电子传递系统的组装及其生物学意义

电子传递链亦称呼吸链,由位于线粒体内膜的I、II、III、IV 4种复合物组成,负责电子传递和产生质子梯度。电子主要从复合物I进入电子传递链,经复合物III传递至复合物IV。电子传递系统的组装是一个十分复杂的过程,目前已知主要有约69个结构亚基以及至少16个组装因子参与了人类复合物I、III、IV的组装,这些蛋白质由核基因组与线粒体基因组共同编码。对线粒体电子传递系统的蛋白质组成及其结构已研究得较为清楚,但对它们的组装了解得还比较初步。许多人类线粒体疾病是由于电子传递系统的功能障碍引起的,其中又有许多是由于该系统中一个或多个部件的错误组装引起的。研究这些缺陷不仅能够加深对线粒体疾病发病机理的了解,也有助于揭示线粒体功能的调控机制。将着重对电子传递系统复合物的组装及其与人类疾病关系的研究进展进行综述。     

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Mitochondrially generated ROS increase with age and are a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS, which may impair normal complex activity. This study of heart mitochondria from young, middle-aged, and old mice reveals that there is an age-related decline in complex I and V activity that correlates with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of the aged mouse heart.     

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.     

Analysis of polyacrylamide gel electrophoresis through the PIQUANT image recognition system

Polyacrylamide gels, with separated components which can be made visible by fluorescent or colored dyes and thereafter photographed, can be analyzed at high speed and resolution by a computerized image-recognition device (PIQUANT). The system provides relative mobilities and relative distributions of separated components of multiple samples at the rate of about 1 sec/sample and with greater resolution than can be attained by conventional methods.