State of oncomarker protein B23/nucleophosmin in HeLa cells.

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38-40 kDa) and oligomers (210-230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210-230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.

[Isolation of the protein B23/nucleophosmin from HeLa cell nuclei].

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35-40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C- and N- terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.

Purification and primary structure of novel lipid transfer proteins from germinated lentil (Lens culinaris) seeds.

A subfamily of eight novel lipid transfer proteins designated as Lc-LTP1-8 was found in the lentil Lens culinaris. Lc-LTP2, Lc-LTP4, Lc-LTP7, and Lc-LTP8 were purified from germinated lentil seeds, and their molecular masses (9268.7, 9282.7, 9121.5, 9135.5 daltons) and complete amino acid sequences were determined. The purified proteins consist of 92-93 amino acid residues, have four disulfide bonds, and inhibit growth of Agrobacterium tumefaciens. Total RNA was isolated from germinated lentil seeds, RT-PCR and cloning were performed, and the cDNAs of six LTPs were sequenced. Precursor 116-118-residue proteins with 24-25-residue signal peptides were found, and two of them are purified proteins Lc-LTP2 and Lc-LTP4.

State of nucleolar proteins B23/nucleophosmin and UBF in HeLa cells during apoptosis induced by tumor necrosis factor.

The structural state of two major nucleolar proteins, UBF and B23/nucleophosmin (both monomeric and oligomeric forms), was for the first time established in HeLa cells treated with apoptosis inducers: tumor necrosis factor (TNF-alpha), emetine, and their combination. The treatment of the cells with either TNF-alpha or emetine did not induce apoptosis and affect the state of UBF and nucleophosmin (both monomers and oligomers). Apoptosis was rather pronounced only if HeLa cells were treated with a mixture of TNF-alpha and emetine. States of the UBF and B23 proteins were analyzed in samples containing 25, 45, and 100% of cells with apoptotic nuclei. It was shown by immunoblotting that TNF-alpha-induced apoptosis of HeLa cells was associated with proteolysis of UBF and production of a 76-kD fragment, the content of which increased in correlation with the fraction of apoptotically changed cells. The N- and C-terminal amino acid sequences of UBF and its 76-kD fragment were characterized, and the site of the apoptosis-induced specific proteolysis was identified. As differentiated from UBF, protein B23 did not undergo proteolytic degradation during the TNF-alpha-induced apoptosis of HeLa cells and its content was unchanged even in the cell fraction with fragmentation of virtually all nuclei. However, the ratio between the monomeric and oligomeric states of B23 protein was changed in apoptotic cells, and apoptosis-specific forms of nucleophosmin were detected.

[Isolation and characterization of fragments of ATP-dependent protease Lon from Escherichia coli obtained by limited proteolysis]. / Vydelenie i kharakteristika fragmentov ATP-zavisimoi Lon-proteinazy Escherichia coli, poluchennykh metodom ogranichennogo proteoliza.

Conditions of limited proteolysis of the protease Lon from Escherichia coli that provided the formation of fragments approximately corresponding to the enzyme domains were found for studying the domain functioning. A method of isolation of the domains was developed, and their functional characteristics were compared. The isolated proteolytic domain (LonP fragment) of the enzyme was shown to exhibit both peptidase and proteolytic activities; however, it cleaved large protein substrates at a significantly lower rate than the full-size protease Lon. On the other hand, the LonAP fragment, containing both the ATPase and the proteolytic domains, retained almost all of the enzymatic properties of the full-size protein. Both LonP and LonAP predominantly form dimers unlike the native protease Lon functioning as a tetramer. These results suggest that the N-terminal domain of protease Lon plays a considerable role in the process of the enzyme oligomerization.

[Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain]. / Strukturnye osobennosti izofermentov Na+,K+-ATP-azy mozga telenka.

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.

Interaction between tubulin and Na+,K+-ATPase in brain stem neurons.

Functionally active Na+,K+-ATPase isozymes containing three types of the catalytic subunits (alpha1, alpha2, and alpha3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (beta3 isoform) in the high-molecular-weight complex of Na+,K+-ATPase alpha3beta1 isozyme from brain stem axolemma and the junction between Na+,K+-ATPase alpha3 subunit and tubulin beta3 subunit are shown for the first time.

Na+,K(+)-ATPase isozymes in the bovine brain grey matter and brain stem.

Active preparations of Na+,K(+)-ATPase containing three types of catalytic isoforms were isolated from the bovine brain to study the structure and function of the sodium pump. Na+,K(+)-ATPase from the brain grey matter was found to have a biphasic kinetics with respect to ouabain inhibition and to consist of a set of isozymes with subunit composition of alpha 1 beta 1, alpha 2 beta m and alpha 3 beta m (where m = 1 and/or 2). The alpha 1 beta 1 form clearly dominated. For the first time, glycosylation of the beta 1-subunit of the alpha 1 beta 1-type isozymes isolated from the kidney and brain was shown to be different. Na+,K(+)-ATPase from the brain stem and axolemma consisted mainly of a mixture of alpha 2 beta 1 and alpha 3 beta 1 isozymes having identical ouabain inhibition constants. In epithelial and arterial smooth muscle cells, where the plasma membrane is divided into functionally and biochemically distinct domains, the polarized distribution of Na+,K(+)-ATPase is maintained through interactions with the membrane cytoskeleton proteins ankyrin and spectrin (Nelson and Hammerton, 1989; Lee et al., 1996). We were the first to show the presence of the cytoskeleton protein tubulin (beta 5-isoform) and glyceraldehyde-3-phosphate dehydrogenase in a high-molecular-weight complex with Na+,K(+)-ATPase in brain stem neuron cells containing alpha 2 beta 1 and alpha 3 beta 1 isozymes. Consequently, the influence of not only subunit composition, but also of glycan and cytoskeleton structures and other plasma membrane-associated proteins on the functional properties of Na+,K(+)-ATPase isozymes is evident.

[Structural analysis of isoforms of Na+,K+-ATPase from calf brain]. / Strukturnyi analiz izoform Na+,K+-ATP-azy mozga telenka.

The isoform composition and types of functioning of Na+,K(+)-ATPase complexes, as well as their ouabain-inhibition constants, were studied for calf brain membranes. The catalytic subunit alpha 3 within the native enzyme complex was found to exhibit an increased sensitivity to endogenous proteolysis. The site of specific proteolysis was localized in the region of the polypeptide chain that is unique for all alpha 3 type isoforms: PNDNR492 decreases (Y493) (according to the numeration of human alpha 3-subunit). It was shown for the first time that in all enzyme preparations containing the alpha 2 and alpha 3 isoforms isolated by both Jorgensen's and Esmann's method two other proteins were present: the beta 5 chain of tubulin and glyceraldehyde-3-phosphate dehydrogenase; the biological meaning of their association is still unclear.

Determination of the sidedness of the carboxy-terminus of the Na+/K(+)ATPase alpha-subunit using lactoperoxidase iodination.

The orientation of the carboxy-terminal pair of tyrosines of the Na+/K(+)-ATPase alpha-subunit with respect to the plane of the plasma membrane was determined. The approach was based on lactoperoxidase-catalysed radioiodination of the tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line and those accessible in a broken plasma membrane fraction and in isolated membrane-bound Na+/K(+)-ATPase. The labeled alpha-subunit was isolated by SDS gel electrophoresis followed by electroblotting. Then the COOH-terminal amino acids were hydrolyzed by carboxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the COOH-terminal tyrosine residues of the alpha-subunit were only accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the COOH-terminus of the Na+/K(+)-ATPase alpha-subunit is located on the cytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.

Na(+)-K(+)-ATPase isoforms in different areas of calf brain.

The isoform composition and type of Na(+)-K+ ATPase functional complexes in a number of calf brain membranes were determined. Functionally active enzymes were obtained from microsomes from calf cerebral cortex grey matter, brain stem, and stem axolemma by two different methods involving (1) the selective removal of contaminating proteins according to Jorgensen (1974) and (2) the selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). The protein components of the isolated preparations were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, transferred to an immobilon membrane [poly(vinylidene difluoride) membrane] by electroblotting, and subjected to structural analysis. The N-terminal amino acid sequences of the alpha- and beta-subunits (alpha 1, alpha 2, alpha 3, beta 1, beta 2) and the isoform composition and type of alpha n beta m functional complexes present in the different microsome preparations were determined. Brain grey matter Na(+)-K+ ATPase was characterized by biphasic kinetics with respect to ouabain inhibition (Ki approximately 10(-6) M and -1.5 x 10(-8) M) and comprised a set of isozymes with subunit compositions of alpha 1 beta 1, alpha 2 beta m, and alpha 3 beta m (where m = 1 and/or 2), with the alpha 1 beta 1 form clearly predominating. Na(+)-K+ ATPase from brain stem and axolemma consisted mainly of a mixture of the isozymes alpha 2 beta 1 and alpha 3 beta 1, which had identical ouabain inhibition constants (Ki approximately 10(-7) M), but in the axolemma there was a large quantity of the alpha 3 beta 1 isozyme. The catalytic subunit alpha 3 within the untreated enzyme complex had increased sensitivity towards endogenous proteolysis. It was therefore possible to isolate enzyme containing the alpha 3 catalytic subunit only in the presence of the protease inhibitor diisopropyl fluorophosphate (DIPF). In the absence of this inhibitor there was a specific fragmentation of the polypeptide chain, resulting in the formation of an extremely stable N-terminal fragment of molecular mass 55 kDa.

[Complete amino acid sequence of neurotoxin Os-1 from the venom of the Central Asian black scorpion Orthochirus scrobiculosus]. / Polnaia aminokislotnaia posledovatel'nost' neirotoksina Os-1 iz iada sredneaziatskogo chernogo skorpiona Orthochirus scrobiculosus.

The neurotoxin Os-1 from the venom of the Central Asian scorpion Orthochirus scrobiculosus possesses a high paralytic activity against mice. This neurotoxin was subjected to tryptic, chymotryptic and BrCN cleavages and its total amino acid sequence was established. It was shown that neurotoxin Os-1 consists of 66 amino acid residues an contains four disulfide bonds.

[Neurotoxins from the venom of the Central Asian scorpion Buthus eupeus]. / Izuchenie neirotoksinov iz iada sredneaziatskogo skorpiona Buthus eupeus.

Three main polypeptide neurotoxins M9, M10, M14, possessing paralytic activity in mice, have been isolated from the venom of the Central Asian scorpion Buthus eupeus. The amino acid composition of these toxins was determined. Toxins M9 and M14 were digested with trypsin, chymotrypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The complete amino acid sequences of the toxins M9 and M14 were established.

[Amino acid sequence of 2 neurotoxins from the scorpion Buthus eupeus venom]. / Aminokislotnaia posledovatel'nost' dvukh neirotoksinov iz iada skorpiona Buthus eupeus.

Three polypeptides, M10, M14 and M9, toxic to mammals were isolated from the venom of the Central Asian scorpion Buthus eupeus. All the toxins were shown to be homogeneous according to disc-electrophoresis and N-terminal group analyses. The toxin M9 was digested with trypsin, Staphylococcus aureus proteinase and cleaved with BNPS-skatole. The toxin M14 was subjected to tryptic and chymotryptic hydrolyses. The complete amino acid sequences of the toxins M9 and M14 were established and it was shown that each of them consists of 66 amino acid residues with four intramolecular disulfide bonds.