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We have conducted a research of mitochindrial internal membrane proteins. This fraction has been received in the form of submitochondrial particles (SMP). SMP have been processed by trypsinum, and the received peptides have been separated from so-called "smoothfaced vesicles". "Smoothfaced vesicles" were blasted, proteinse and peptides were processed by cyanogen bromide and trypsinum. We have received two groups of tryptic peptides and analyzed them separately with the help of proteomic methods, such as chromatography, mass spectrometry and protein identification in different databases. To identify more proteins and find minor components of mitochindrial proteome we have considered possible non-specific fragmentation of proteins. 298 proteins have been identified, we also have conducted the analisys of their functions and cell localization.

A fraction of the so-called mitochondrial soluble proteins was obtained after the destruction of purified mitochondria by sonication according to the previously found approach to the identification of protein subsets of the Bos taurus heart proteome. A tryptic destruction of these proteins was achieved. Approximately half of the tryptic hydrolysate was separated into two fractions of cysteine-containing and cysteine-free peptides by covalent chromatography on Thiopropyl Sepharose 4B. The cysteine-containing peptides were modified by iodoacetamide. The peptides were mass-spectrometrically identified in all the three fractions of tryptic hydrolysate, and the proteins were searched for in the amino acid sequence databases. There were 213 unique proteins reliably identified.

This study is part of a large-scale investigation of the proteome of mitochondria from the heart muscle of Bos taurus. We developed a special approach to simplification of the protein mixture by separation of mitochondrial fractions with stable protein compositions. At the first stage of this approach, we isolated and purified internal mitochondrial membranes. The protein composition of this fraction was analyzed by the following proteomic methods: enzymatic or/and chemical cleavage of the proteins, chromatographic fractionation of the complex mixture of the resulting peptides, mass-spectrometric identification of these peptides, and a search for proteins in databases of amino acid sequences. We reliably identified 147 unique proteins with the use of the SwissProt database. The subcellular location and functions of these proteins were analyzed. Approaches to studies of transmembrane domains of integral membrane proteins of the internal mitochondrial membrane were proposed on the basis of proteomic methods of analysis. Considerable coincidence of the experimental data with the results of determination of the 3D structures of the proteins by X-ray analysis was shown.

The subunits of the F0 membrane sector of bovine heart mitochondrial H(+)-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H(+)-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn- glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn- glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero- 3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The cross-linked products were identified by SDS-PAGE and MALDI mass spectrometry.

This review is focused on the import of processable precursor proteins into the mitochondrial matrix; the import of carrier proteins into the inner mitochondrial membrane is also briefly discussed. Post- and cotranslational theories of the import, specific features of the presequence structures, and effects of some cytosolic factors on the import of precursor proteins are reviewed. The data on the structure of the protein translocases of the outer (TOM complex) and the inner (TIM complex) membranes of mitochondria and the current models of the precursor protein import by these translocases are also summarized.

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F1beta subunit from Nicotiana plumbaginifolia (pF1beta) into plant mitochondria. When pF1beta of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF1beta. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF1beta. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca(2+)-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF1beta proteins were strongly associated with the mitochondrial outer membrane.

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1 beta subunit of the ATP synthase (pF1 beta) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondrial than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1 beta with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1 beta into intact mitochondria. Import of pF1 beta through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.

The culture, morphology, and genome of alkalo- and halotolerant bacterial strain Bacillus sp. FTU were characterized; the strain is compared to other representatives of genus Bacillus. The DNA-DNA hybridization data indicate that the strains of Bacillus halodurans DSM 497 and DSM 2513 and Bacillus sp. FTU belong to the same species. Bacillus sp. FTU can be renamed to Bacillus halodurans FTU. The N-terminal amino acid fragments of the subunits I and II of the terminal caa3-type cytochrome c oxidase of B. halodurans FTU were sequenced. The N-terminal fragments of this enzyme and of the caa3-type oxidase of alkalophilic Bacillus firmus OF4 are highly homologous (homology of subunits I and II is over 90 and over 96%, respectively). Such high homology of the terminal oxidases of these bacteria might be due to their alkaline medium.

Protein compositions of venoms of South-American stinging ants, Ectatomma tuberculatum, Paraponera clavata (subfamily Ponerinae), and "tangarana" were analyzed. The venom of E. tuberculatum displayed the most complex protein composition (more than 15 polypeptides). The water-soluble fraction of the venoms of P. clavata and "tangarana" contained acidic proteins (pI < 3.5 to 5.2), whereas the venom of E. tuberculatum contained predominantly basic proteins (pI 8 to > 9.5). N-Terminal residues and N-terminal sequences of a number of polypeptides were determined. High-molecular-mass polypeptides of the P. clavata venom slightly stimulated the ATPase activity of mitochondrial F1-ATPase. Low-molecular-mass nonprotein components of this venom significantly inhibited the ATPase activity of submitochondrial particles and F1-ATPase from bovine heart mitochondria. The venoms of E. tuberculatum and "tangarana" produced no effect on the ATPase activity.

Unmodified and citraconilated OSCP of the pig heart mitochondrial H(+)-ATPase were hydrolysed by proteinase from Staphylococcus aureus V8 and trypsin, respectively. To purify the individual peptides, various types of HPLC and covalent chromatography on SH-Sepharose were used. By the automatic Edman method complete or partial amino acid sequences of the peptides obtained were determined, thus allowing for the reconstruction of the primary structure of pig OSCP. A linear antigenic determinant recognizable by A1 monoclonal antibody against bovine OSCP, was localized. Studies showed Gly43 residue (bovine OSCP) to be replaced by Ala43 (pig OSCP), which is responsible for a decrease of the affinity of the monoclonal antibody A1 to pig OSCP. Comparative analysis of primary structures of bovine and pig OSCP was carried out.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at about 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a = 13.0 nm, b = 25.6 nm and gamma = 86 degrees. Two alternative models for the crystal structural organization were suggested, viz. with one or two protein molecules per unit cell.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at ca. 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a 13.0 nm, b 25.6 nm and gamma 86 degrees. In line with this conclusion, two alternative models for the crystal structural organization are plausible, viz., with one or two protein molecules per unit cell. The first model suggests an asymmetric incorporation of ATP synthase molecules into the lipid bilayer: extramembranous portions F1 are located on one side of the crystal membrane plane. According to the second model, the incorporation occurs on each side of the lipid bilayer, the unit cell containing the two oppositely oriented protein molecules. Based on the absence of another type of the projected crystal images (a rear view of the membrane), unique to the first model, preference is given to the second model.

Structural analysis of oligomycin sensitivity-conferring protein (OSCP) revealed repeating sequences (residues 1-89, 105-190) suggesting an evolution of the protein by gene duplication. In addition to the reported homology with the delta-subunit of Escherichia coli F1ATPase, OSCP also shows a certain homology with the b-subunit of E. coli F0 and the ADP/ATP carrier of mitochondria.

Oligonucleotides deduced from the amino acid sequence of a hexapeptide Lys-Asp-Phe-Ala-Glu-Asn were synthesized and used as probes to screen a pig kidney cDNA library for a specific DNA sequence coding for the alpha-subunit of Na+, K+-ATPase. It was shown that the mixed oligoprobe, consisting of 64 heptadecamers, could be only suitable for mRNA blot analysis. To identify the clones with specific cDNA inserts, mixed oligoprobes were fractionated by HPLC technique. For the same purpose a new set of oligonucleotides, synthesized as four groups of 16 different heptadecamers each, was used.

Hydrolysis of OSCP of bovine heart mitochondria by proteinase from Staphylococcus aureus V8 was followed by isolation of all individual peptides by means of gel-filtration and HPLC. Structural analysis of the peptides allowed to arrange BrCN-fragments and to reconstruct the complete amino acid sequence of the protein. Comparative structural analysis revealed existence of a certain homology between OSCP and delta- and b-subunits of the E. coli H+-ATPase, which are necessary for interaction of catalytic and proton-conducting parts of the bacterial enzyme.

The F1-type ATPase has been isolated from membrane preparations of marine alkalotolerant bacterium, Vibrio alginolyticus. The enzyme was found to consist of two major subunits of 55 and 58 kDa and at least two minor components (38 and 23 kDa). Amino acid sequences of N-terminal regions of the major subunits revealed close homology with those of E. coli H+-ATPase and of Propionigenium modestum Na+-ATPase.

The complete amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria is reported. The protein contains 190 amino acids and has a molecular mass of 20 967. Its structure is characterized by a concentration of charged amino acids in the two terminal segments (N 1-77 and C 128-190) of the protein, whereas its central region is more hydrophobic. The earlier reported homology of the protein with the delta-subunit of E. coli F1, based on the terminal amino acid sequences of OSCP, is further substantiated.

The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the beta and beta' subunits of Escherichia coli DNA-dependent RNA polymerase and of their structural genes, yielding a continuous nucleotide sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the beta subunit, comprising 1342 residues, and the N-terminal sequence of the beta' subunit (176 residues).

Trypsin and cyanogen bromide were used for cleavage of the OSCP preparations. The peptide mixtures thus formed were separated into individual components by a combination of various chromatographic procedures: gel filtration, ion exchange and paper chromatography, as well as reversed-phase HPLC. As a result, 31 tryptic peptides and 9 out of 10 possible cyanogen bromide peptides were isolated. Determination of the amino acid sequences of these peptide allowed the alignment of cyanogen bromide fragments in the polypeptide chain that shed light on the "architecture" of the protein molecule as a whole. It also afforded the overlappings for tryptic peptides, 16 in the N-terminal and 8 in the C-terminal portions of the molecule.