Source: https://chemweb.com/articles/SV10541/0008300001
Timestamp: 2019-04-22 02:25:00+00:00

Document:
50+ Years of Protein Folding by A. V. Finkelstein (S3-S18).
The ability of proteins to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured rates of spontaneous folding of single-domain globular proteins range from microseconds to hours: the difference–10-11 orders of magnitude–is the same as between the lifespan of a mosquito and the age of the Universe. This review (based on the literature and some personal recollections) describes a winding road to understanding spontaneous folding of protein structure. The main attention is given to the free-energy landscape of conformations of a protein chain–especially to the barrier separating its unfolded (U) and the natively folded (N) states–and to physical the-ories of rates of crossing this barrier in both directions: from U to N, and from N to U. It is shown that theories of both these processes come to essentially the same result and outline the observed range of folding and unfolding rates for single-domain globular proteins. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control, and explain the observed maximal size of “foldable” protein domains.
Thirty Years of Studies of Qβ Replicase: What Have We Learned and What Is Yet to Be Learned? by A. B. Chetverin (S19-S32).
Qβ replicase (RNA-directed RNA polymerase of bacteriophage Qβ) has an unsurpassed capacity to amplify polynucleotides in vitro. In 1986, the Group of Viral RNA Biochemistry was organized at the Institute of Protein Research in order to exploit this property for the synthesis of messenger RNAs to be used in cell-free translation systems. Although the task has not been implemented in full, this work has led to a number of unexpected important results including uncovering the nature of the “template-free” RNA synthesis by Qβ replicase, discovering the ability of RNA molecules for spontaneous recombination, revealing the unusual mechanism Qβ replicase uses to discriminate between its proper and improper templates, and discovering a new function of the largest ribosomal protein S1, that is also one of the replicase subunits. Finally, our work resulted in the invention of the molecular colonies technique that has become the basis for the next generation sequencing methods and provided a new insight into the origin of life. However, Qβ replicase has not yet revealed all its secrets, and its studies promise further interesting findings.
The Molten Globule Concept: 45 Years Later by V. E. Bychkova; G. V. Semisotnov; V. A. Balobanov; A. V. Finkelstein (S33-S47).
In this review, we describe traditional systems where the molten globule (MG) state has been detected and give a brief description of the solution of Levinthal’s paradox. We discuss new results obtained for MG-mediated folding of “nontraditional” proteins and a possible functional role of the MG. We also report new data on the MG, especially the dry molten globule.
The Institute of Protein Research of the Russian Academy of Sciences Is 50 Years Old by E. S. Nadezhdina (S1-S2).
Here I introduce collection of review articles written by members of the Institute of Protein Research of the Russian Academy of Sciences. This collection commemorates the 50th anniversary of the Institute. The review articles cover a broad range of problems concerning the spatial structure of protein molecules, including the state of the molten globule, protein–RNA interactions, polysome and ribosome structure, the molecular colony method, and the original methods for studying the structure of proteins. Several of the reviews consider the practical use of knowledge about the structure of proteins and protein polymers. They reflect both the long experience of the authors and contemporary scientific data.
Interactions between the Translation Machinery and Microtubules by E. M. Chudinova; E. S. Nadezhdina (-).
Microtubules are components of eukaryotic cytoskeleton that are involved in the transport of various components from the nucleus to the cell periphery and back. They also act as a platform for assembly of complex molecular ensembles. Ribonucleoprotein (RNP) complexes, such as ribosomes and mRNPs, are transported over significant distances (e.g. to neuronal processes) along microtubules. The association of RNPs with microtubules and their transport along these structures are essential for compartmentalization of protein biosynthesis in cells. Microtubules greatly facilitate assembly of stress RNP granules formed by accumulation of translation machinery components during cell stress response. Microtubules are necessary for the cytoplasm-to-nucleus transport of proteins, including ribosomal proteins. At the same time, ribosomal proteins and RNA-binding proteins can influence cell mobility and cytoplasm organization by regulating microtubule dynamics. The molecular mechanisms underlying the association between the translation machinery components and microtubules have not been studied systematically; the results of such studies are mostly fragmentary. In this review, we attempt to fill this gap by summarizing and discussing the data on protein and RNA components of the translation machinery that directly interact with microtubules or microtubule motor proteins.
High-Pressure Scanning Microcalorimetry – A New Method for Studying Conformational and Phase Transitions by S. A. Potekhin (-).
The development of high-pressure scanning microcalorimetry and the first results studying transitions in proteins, lipids, and model polymers are reviewed. Special attention is given to changes (increments) in volume parameters upon transitions as well as the nature of these changes. It is demonstrated that the use of the model of compound transfer reaction in its purest form for assessment of denaturation volume effects failed due to serious difficulties.
Analysis of Insulin Analogs and the Strategy of Their Further Development by O. M. Selivanova; S. Yu. Grishin; A. V. Glyakina; A. S. Sadgyan; N. I. Ushakova; O. V. Galzitskaya (-).
We analyzed the structural properties of the peptide hormone insulin and described the mechanism of its physiological action, as well as effects of insulin in type 1 and 2 diabetes. Recently published data on the development of novel insulin preparations based on combining molecular design and genetic engineering approaches are presented. New strategies for creation of long-acting insulin analogs, the mechanisms of functioning of these analogs and their structure are discussed. Side effects of insulin preparations are described, including amyloidogenesis and possible mitogenic effect. The pathways for development of novel insulin analogs are outlined with regard to the current requirements for therapeutic preparations due to the wider occurrence of diabetes of both types.
Investigations of Photosensitive Proteins by Serial Crystallography by G. K. Selikhanov; M. S. Fando; M. V. Dontsova; A. G. Gabdulkhakov (-).
This review contains recent data on serial femtosecond X-ray crystallography (SFX), based on a femtosecond X-ray free electron laser, as well as on the possibilities of its application for studying photosensitive proteins. Development of this method began rather recently, and it has already shown its effectiveness and some unique advantages over conventional X-ray structural analysis. This technology is especially promising for structural studies of membrane proteins and for kinetic studies. The main principle of the method, the possibility of its application in structural biology, its advantages and disadvantages, as well as its prospects for further development are analyzed in this review. Special attention is given to publications in which the SFX method has been used to study photosensitive proteins.
Three-Dimensional Organization of Polyribosomes–A Modern Approach by Z. A. Afonina; V. A. Shirokov (S48-S55).
Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed tworow forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.
Archaeal Flagella as Biotemplates for Nanomaterials with New Properties by S. N. Beznosov; M. G. Pyatibratov; O. V. Fedorov (S56-S61).
At the end of 1980s, regions of the polypeptide chain of bacterial flagella subunits (flagellins) responsible for different properties of these protein polymers were identified by structural studies. It was found that the N-and C-terminal regions are responsible for the polymerization properties of subunits, and the central region is responsible for antigenic properties of the flagellum. Soon after that, it was proposed to use variability of the central flagellin domain for directed modification to impart new properties to the flagellum surface. Such studies of flagella and other polymeric structures of bacterial origin thrived. However bacterial polymers have some shortcomings, mainly their instability to dissociating effects. This shortcoming is absent in archaeal flagella. A limiting factor was the lack of the three-dimensional structure of archaeal flagellins. A method was developed that allowed modifying flagella of the halophilic archaeon Halobacterium salinarum in a peptide that connects positively charged ions. Later, corresponding procedures were used that allowed preparing the anode material for a lithiumion battery whose characteristics 4-5-fold exceeded those of batteries commonly used in industrial production. We describe other advantages of archaeal flagella over bacterial analogs when used in nanotechnology.
Analysis of Insulin Analogs and the Strategy of Their Further Development by O. M. Selivanova; S. Yu. Grishin; A. V. Glyakina; A. S. Sadgyan; N. I. Ushakova; O. V. Galzitskaya (S146-S162).
Investigations of Photosensitive Proteins by Serial Crystallography by G. K. Selikhanov; M. S. Fando; M. V. Dontsova; A. G. Gabdulkhakov (S163-S175).
Interactions between the Translation Machinery and Microtubules by E. M. Chudinova; E. S. Nadezhdina (S176-S189).
High-Pressure Scanning Microcalorimetry – A New Method for Studying Conformational and Phase Transitions by S. A. Potekhin (S134-S145).
Structural Aspects of Ribosomal RNA Recognition by Ribosomal Proteins by A. D. Nikulin (S111-S133).
This review is focused on the structural aspects of interaction between ribosomal proteins and ribosomal RNA in bacterial ribosomes and complexes of ribosomal proteins with specific fragments of ribosomal RNA. Special attention is given to the recognition of specific spatial architecture of the double-stranded ribosomal RNA by ribosomal proteins and to the role of unstructured protein regions in stabilization of distant ribosomal RNA segments.
Studies of the Process of Amyloid Formation by Aβ Peptide by O. V. Galzitskaya; E. I. Galushko; O. M. Selivanova (S62-S80).
Studies of the process of amyloid formation by Aβ peptide have been topical due to the critical role of this peptide in the pathogenesis of Alzheimer’s disease. Many articles devoted to this process are available in the literature; however, none of them gives a detailed description of the mechanism of the process of generation of amyloids. Moreover, there are no reliable data on the influence of modified forms of Aβ peptide on its amyloid formation. To appreciate the role of Aβ aggregation in the pathogenesis of Alzheimer’s disease and to develop a strategy for its treatment, it is necessary to have a well-defined description of the molecular mechanism underlying the formation of amyloids as well as the contribution of each intermediate to this process. We are convinced that a combined analysis of theoretical and experimental methods is a way for understanding molecular mechanisms of numerous diseases. Based on our experimental data and molecular modeling, we have constructed a general model of the process of amyloid formation by Aβ peptide. Using the data described in our previous publications, we propose a model of amyloid formation by this peptide that differs from the generally accepted model. Our model can be applied to other proteins and peptides as well. According to this model, the main building unit for the formation of amyloid fibrils is a ring-like oligomer. Upon interaction with each other, ring-like oligomers form long fibrils of different morphology. This mechanism of generation of amyloid fibrils may be common for other proteins and peptides.
Methods for Screening Live Cells by A. A. Gordeev; A. B. Chetverin (S81-S102).
Cell screening or, in other words, identification of cells with certain properties is now increasingly used in scientific and medical research, e.g., in diagnostics, drug testing, and production of cell clones with desired characteristics. In this review, we discuss existing methods of cell screening and their classification according to the cell presentation format. We describe the principles of the one-dimensional and two-dimensional formats and compare the main advantages and drawbacks of these formats. The first part describes the methods based on the 2D-format of cell presentation, when cells are immobilized in the same plane by various techniques. The second part describes the methods of the 1D-screening, when cells are aligned in a line in a stream of fluid and scanned one-by-one while passing through a detector. The final part of the review describes the method of high-performance cell analysis based on the merged gel technique. This technique combines the advantages of both 1D and 2D formats and, according to the authors, might become an effective alternative to many modern methods of cell screening.
Chirality and Handedness of Protein Structures by A. V. Efimov (S103-S110).
In proteins, the polypeptide chain forms a number of right-and left-handed helices and superhelices, right-and left-turned hairpins, and some other structures that are nonsuperimposable, although they are not mirror images of each other as the Lamino acids are not converted to the Damino acids. This property of protein structures will be referred to here as pseudo-chirality–or handedness. It has been shown that there are two kinds of handedness in proteins–helical handedness and handedness of arrangement. Some protein structures exhibit both the kinds of handedness. Handedness is observed at all levels of protein structural organization–from α-helices, β-strands, hairpins, βαβ-units up to complex structural motifs, superhelices, and supramolecular structures in fibrous and polymer proteins. There are several structures that have unique handedness in proteins, for example, α-helices, αα-corners, βαβ-units, abcd-units, and so on. This property of the polypeptide chain is of particular value in protein folding and protein modeling, because it drastically reduces the number of possible folds.
Structural Aspects of Ribosomal RNA Recognition by Ribosomal Proteins by A. D. Nikulin (-).
Chirality and Handedness of Protein Structures by A. V. Efimov (-).
The Molten Globule Concept: 45 Years Later by V. E. Bychkova; G. V. Semisotnov; V. A. Balobanov; A. V. Finkelstein (-).
Thirty Years of Studies of Qβ Replicase: What Have We Learned and What Is Yet to Be Learned? by A. B. Chetverin (-).
50+ Years of Protein Folding by A. V. Finkelstein (-).
Three-Dimensional Organization of Polyribosomes–A Modern Approach by Z. A. Afonina; V. A. Shirokov (-).
The Institute of Protein Research of the Russian Academy of Sciences Is 50 Years Old by E. S. Nadezhdina (-).
Methods for Screening Live Cells by A. A. Gordeev; A. B. Chetverin (-).
Studies of the Process of Amyloid Formation by Aβ Peptide by O. V. Galzitskaya; E. I. Galushko; O. M. Selivanova (-).
Archaeal Flagella as Biotemplates for Nanomaterials with New Properties by S. N. Beznosov; M. G. Pyatibratov; O. V. Fedorov (-).
Recombinant monoclonal antibodies for rabies post-exposure prophylaxis by E. N. Ilina; M. V. Larina; T. K. Aliev; D. A. Dolgikh; M. P. Kirpichnikov (1-12).
Rabies virus is a prototypical neurotropic virus that causes one of the most dangerous zoonotic diseases in humans. Humanized or fully human monoclonal antibodies (mAb) that neutralize rabies virus would be the basis for powerful post-exposure prophylaxis of rabies in humans, having several significant benefits in comparison with human or equine rabies polyclonal immunoglobulins. The most advanced antibodies should broadly neutralize natural rabies virus isolates, bind with conserved antigenic determinants of the rabies virus glycoprotein, and show high neutralizing potency in assays in vivo. The antibodies should recognize nonoverlapping epitopes if they are used in combination. This review focuses on basic requirements for anti-rabies therapeutic antibodies. The urgency in the search for novel rabies post-exposure prophylaxis and methods of development of anti-rabies human mAb cocktail are discussed. The rabies virus structure and pathways of its penetration into the nervous system are also briefly described.
The role of interleukin-33 in pathogenesis of bronchial asthma. New experimental data by M. R. Khaitov; A. R. Gaisina; I. P. Shilovskiy; V. V. Smirnov; G. V. Ramenskaia; A. A. Nikonova; R. M. Khaitov (13-25).
Interleukin-33 (IL-33) belongs to the IL-1 cytokine family and plays an important role in modulating immune system by inducing Th2 immune response via the ST2 membrane receptor. Epithelial cells are the major producers of IL-33. However, IL-33 is also secreted by other cells, e.g., bone marrow cells, dendritic cells, macrophages, and mast cells. IL-33 targets a broad range of cell types bearing the ST2 surface receptor. Many ST2-positive cells, such as Th2 cells, mast cells, basophils, and eosinophils, are involved in the development of allergic bronchial asthma (BA). This suggests that IL-33 directly participates in BA pathogenesis. Currently, the role of IL-33 in pathogenesis of inflammatory disorders, including BA, has been extensively investigated using clinical samples collected from patients, as well as asthma animal models. In particular, numerous studies on blocking IL-33 and its receptor by monoclonal antibodies in asthma mouse model have been performed over the last several years; IL-33-and ST2-deficient transgenic mice have also been generated. In this review, we summarized and analyzed the data on the role of IL-33 in BA pathogenesis and the prospects for creating new treatments for BA.
Biotransformation of progesterone by the ascomycete Aspergillus niger N402 by O. S. Savinova; P. N. Solyev; D. V. Vasina; T. V. Tyazhelova; T. V. Fedorova; T. S. Savinova (26-31).
The ability of the ascomyceteAspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6β,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6β-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.
Modulation of δ-aminolevulinic acid dehydratase activity by the sorbitol-induced osmotic stress in maize leaf segments by M. Jain; S. Tiwary; R. Gadre (32-36).
Osmotic stress induced with 1 M sorbitol inhibited δ-aminolevulinic acid dehydratase (ALAD) and aminolevulinic acid (ALA) synthesizing activities in etiolated maize leaf segments during greening; the ALAD activity was inhibited to a greater extent than the ALA synthesis. When the leaves were exposed to light, the ALAD activity increased for the first 8 h, followed by a decrease observed at 16 and 24 h in both sorbitol-treated and untreated leaf tissues. The maximum inhibition of the enzyme activity was observed in the leaf segments incubated with sorbitol for 4 to 8 h. Glutamate increased the ALAD activity in the in vitro enzymatic preparations obtained from the sorbitol-treated leaf segments; sorbitol inhibited the ALAD activity in the preparations from both sorbitol-treated and untreated leaves. It was suggested that sorbitol-induced osmotic stress inhibits the enzyme activity by affecting the ALAD induction during greening and regulating the ALAD steady-state level of ALAD in leaf cells. The protective effect of glutamate on ALAD in the preparations from the sorbitol-treated leaves might be due to its stimulatory effect on the enzyme.
Characterization and mutational analysis of two UDP-galactose 4-epimerases in Streptococcus pneumoniae TIGR4 by L. L. Chen; D. L. Han; Y. F. Zhai; J. H. Wang; Y. F. Wang; M. Chen (37-44).
Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galE sp1 and galE sp2 ) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalESp1 and GalESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalESp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalESp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalESp1. The biochemical properties of GalESp2 were studied. GalESp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalESp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalESp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalESp2.
Adenylyl cyclase-associated protein 1: Structure, regulation, and participation in cellular processes by G. V. Kakurina; E. S. Kolegova; I. V. Kondakova (45-53).
This review summarizes information available to date about the structural organization, regulation of functional activity of adenylyl cyclase-associated protein 1 (CAP1), and its participation in cellular processes. Numerous data are generalized on the role of CAP1 in the regulation of actin cytoskeleton and its interactions with many actin-binding proteins. Attention is drawn to the similarity of the structure of CAP1 and its contribution to the remodeling of actin filaments in prokaryotes and eukaryotes, as well as to the difference in the interaction of CAP1 with adenylyl cyclase in these cells. In addition, we discuss the participation of CAP1 in various pathological processes.
MicroRNA sponge knockdowns miR-483-5p and upregulates serum ALT/AST in transgenic mice by Yanfen Zhang; Dantong Wu; Xuemei Zhang; Ning Ma; Yanhong Liu (54-59).
MicroRNAs are involved in many biological processes. Studying microRNA function requires genetic strategies generating loss-of-function phenotypes, especially in vivo. However, few microRNA loss-of-function models have been reported in mice. Here, we generated several transgenic mouse lines to stably and specifically knockdown miR-483-5p by overexpressing microRNA sponges from CAG promoters. The different levels of expression of microRNA sponges resulted in different levels of mature miR-483-5p, which upregulated serum ALT/AST in these transgenic lines. These results indicate microRNA sponges are effective in mice in vivo, and they can be used in microRNA loss-of-function research.
Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations by Xue-Jiao Ma; Yin-Juan Zhang; Cheng-Ming Zeng (60-68).
Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC50 of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.
Interaction between RAD51 and MCM complex is essential for RAD51 foci forming in colon cancer HCT116 cells by Jun Huang; Hong-Liang Luo; Hua Pan; Cheng Qiu; Teng-Fei Hao; Zheng-Ming Zhu (69-75).
Colon cancer remains one of the most common digestive system malignancies in the World. This study investigated the possible interaction between RAD51 and minichromosome maintenance proteins (MCMs) in HCT116 cells, which can serve as a model system for forming colon cancer foci. The interaction between RAD51 and MCMs was detected by mass spectrometry. Silenced MCM vectors were transfected into HTC116 cells. The expressions of RAD51 and MCMs were detected using Western blotting. Foci forming and chromatin fraction of RAD51 in HCT116 cells were also analyzed. The results showed that RAD51 directly interacted with MCM2, MCM3, MCM5, and MCM6 in colon cancer HTC116 cells. Suppression of MCM2 or MCM6 by shRNA decreased the chromatin localization of RAD51 in HTC116 cells. Moreover, silenced MCM2 or MCM6 decreased the foci forming of RAD51 in HTC116 cells. Our study suggests that the interaction between MCMs and RAD51 is essential for the chromatin localization and foci forming of RAD51 in HCT116 cell DNA damage recovery, and it may be a theoretical basis for analysis of RAD51 in tumor samples of colon cancer patients.
Isolation of large amounts of highly pure mitochondria for “omics” studies by M. A. Afanasyeva; A. S. Ustiugova; S. A. Golyshev; A. T. Kopylov; A. V. Bogolyubova; D. E. Demin; P. V. Belousov; A. M. Schwartz (76-85).
Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.

References: V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V. 
 V.