[Direct incorporation of 18O isotopes into peptides and proteins for quantative analysis by mass spectrometry method].

The method of direct introduction 18O isotopes in peptides and proteins carboxylic groups through the exchange with H218O in the presence of TFA is shown. The isotope label is steady enough in awide range of pH. Because the labeled compounds retain their physical and chemical characteristics, they can be used as an internal standard in quantitative determination of authentic compounds in the analyzed sites by mass spectrometry methods. The technique may be applicable for quantitative analysis of peptides and proteins in biological environments, for quantitative study of the kinetics of metabolism and enzymatic activity. For polypeptides and proteins the quantitative analysis is combined with trypsinolysis. If necessary, the isotope label may be introduced simultaneously in all peptides and proteins control bioassays, making it suitable for use as a standard for the comparative analysis of experimental bioassays.

[Detection and identification of the tumor-associated antigens in the fraction enriched with plasma membranes of the Zajdela hepatoma cells].

Tumor-associated antigens 45, 57, 80 and 130 kDa were detected using tumor-specific rabbit immune serum in the fraction enriched with plasma membrane of the rat Zajdela hepatoma cells and isolated on the immunosorbent. Revealed proteins were identified as integrin beta-1, ecto-nucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3), basigin, epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP) and protein chaperones--glucose-regulated protein 78 (GRP78) and protein disulfide-isomerase (PDI) A1 by mass spectrometry technique. Functions and characteristics of these proteins in tumor cells and some aspects of the Zajdela hepatoma cells origin are discussed.

[Comparative investigation of antigens associated with plasmatic membranes of rat hepatoma and myogenic cells using anti-kidney serum].

The investigation of antigenic diversion of tumor cells resulting from the expression of heteroorganic antigens has been continued. Tumor-associated heteroorganic antigens with mol. weight 200-210 kDa (identified before as laminin), 105-130, 75-80 and 43 kDa were detected by anti-kidney serum in fractions of plasmatic membranes of cells of rat ascitic Zajdela hepatoma and cultured HTC hepatoma; the antigen 43 kDa was isolated on immunosorbent and identified by mass spectrometry as beta-actin. Anti-kidney serum revealed laminin in fractions of plasmatic membranes of cultured L8 and L6J1 myoblasts, and L6J1 myotubes; apparently, synthesis of laminin by hepatoma and myogenic cells is not connected with their proliferative activity. Besides, anti-kidney serum detected components 38, 42, 44, 48, 62, 78 and 120 kDa, expression of which on myogenic cells surface might be consequence of active cell proliferation and (or) differentiation.

[Revelation and identification of laminin in the structure of plasma membrane of ascitic Zajdela hepatoma cells].

Cell dysdifferentiation during neoplastic transformation is a crucial problem of cell biology and oncology. Antigenic diversion of cancer cells is a typical characteristic of dysdifferentiation. It involves the appearance of antigens which are unusual for normal tissue of this type. Components organospecific for membrane proteins of normal kidney were previously found among plasma membrane proteins of hepatocellular rat tumors, rat hepatocytes after carcinogen treatment, and regenerating liver, respectively. In the present work we showed that a protein with mol. weight about 200 kDa reacting with laminin-1 immunoserum is the basic component of plasma membranes of the rat Zajdela hepatoma cells, which is responsive for organospecific anti-kidney immunoserum in Western blot. A mass-spectrometer analysis of trypsin proteolysis fragments was carried out in SDS-PAGE slices containing the investigated component. The analysis showed the presence of beta1, beta2 and alpha4 laminin chains peptides. The component with mol. weight about 180 kDa, found in the Western blot with laminin-1 immunoserum, was also subjected to the mass spectrometer analysis. As a result, a gamma1 laminin chain was found. An increased amount of laminin was revealed in the ascitic liquid and sera of rat with developed Zajdela hepatoma, in comparison with sera of normal rats. In addition, we found the appearance of laminin on the hepatocyte surface on the 4th day after hepatocarcinogen injection (N-diethylnitrosamine, DENA). Thus, for the first time tumor associated antigens were revealed and identified in the structure of plasma membranes of Zajdela hepatoma cells, being specific to rat kidneys. Our results allow to conclude that in the process of carcinogenesis in rat liver laminin synthesis occurs, which is also characteristic of the rat hepatoma Zajdela cells.

[Interaction of laminin with the plasma membrane components of ascitic Zajdela hepatoma cells].

The laminin affinity chromatography was used for isolating laminin-binding proteins from the plasma membrane of Zajdela hepatoma cells synthesizing laminin. These were components with mol. weights about 80, 67, 60, 55, 52, 48 and 43 kDa. The isolation of laminin integrin receptors from plasma membranes of Zajdela hepatoma cells in the presence of MnCl2 detected only a protein with mol. weight about 80 kDa in EDTA-elution conditions. This protein was identified by mass spectrometry method as the 78 kDa glucose-regulated protein precursor (GRP78). It belongs to the family of 70 kDa heat shock proteins, recently GRP78 was reported to be localized on the surface of different cell types, including hepatocytes.

[Detection and identification of a tumor-associated heteroorganic antigen of Zajdela hepatoma in non-histone proteins of chromatin].

By polyacrylamide gel electrophoresis, a phosphoprotein with mol. weight of 42 kDa was detected in non-histone proteins (NHP) of chromatin of Zajdela ascitic hepatoma cells eluted from phosphocellulose with 0.4-0.5 M NaCl. A protein of the same mol. weight is present in narrow fractions of rat kidney chromatin, but is absent in rat liver. It is suggested that the revealed protein corresponds to the tumor-associated heteroorganic NHP antigen detected earlier in NHP chromatin of rat tumor cells. By MALDI mass spectrometry, this phosphoprotein was identified as ERK2/mitogen-activated protein kinase.

[Study of dimerization of polysynthetic derivatives of the antibiotic eremomycin by ESI MS and its role in elucidating antibacterial activity]. / Izuchenie dimerizatsii polusinteticheskikh proizvodnykh antibiotika éremomitsina metodom ESU MS i ee rol' v proiavlenii antibakterial'noi aktivnosti.

The dimerization constants for glycopeptide antibiotics vancomycin, ristocetin, and eremomycin and nine semisynthetic eremomycin derivatives were determined by the electrospray ionization mass spectrometry; the constants for natural antibiotics turned out to be close to those previously determined by NMR. No correlation between these dimerization constants and antibacterial activities of all the compounds toward the clinical strains of Gram-positive bacteria was found.

[MALD-MS in the quantitative analysis of peptides and proteins]. / Ispol'zovanie MALDI-MS dlia kolichestvennogo analiza peptidov i belkov.

A modified method of isotope dilution was applied to the quantitative determination of peptides and proteins by MALDI MS at subpicomolar level. The essence of the method consists in the quantitative analysis of the enzymic hydrolysis products rather than the starting compounds. This allows the measurements to be performed at a higher resolution and makes the method independent of the molecular mass of oligopeptides and proteins examined. Fragments obtained by hydrolysis of the same oligopeptide or protein in a known concentration by the same enzyme and labeled with the stable 18O isotope are used as internal standards. The label is introduced by carrying out the hydrolysis in H(2)18O, and the oligopeptide concentration is calculated from the isotope distribution between the labeled and unlabeled hydrolysis products in the mass spectrum. This method was tested in the determination of concentrations of the angiotensinogen (1-14) fragment (oligopeptide), extracellular RNAase from Bacillus amyloliquefaciens (protein) and its protein inhibitor, barstar M. Usefulness of this method in kinetic studies was also demonstrated.

[Duodenase--a potential activator of cascade of digestive proteases]. / Duodenaza--potentsial'nyi aktivator kaskada pishchevaritel'nykh proteinaz.

The substrate specificity of duodenase from bovine duodenum mucosa to synthetic and natural polypeptides was studied. Amino acid residues preferential for duodenase in the P1 and P2 positions of the substrate were determined. It was shown that the enzyme is synthesized in epithelial secretory cells of duodenal (Brunner's) glands and enters, as part of the secreta, into the lumen of the duodenum. The possible role of duodenase as an activator of proenteropeptidase is discussed.

[Fungal aspartic proteinase from Trichoderma viride. Specificity during oligopeptide hydrolysis]. / Gribnaia aspartatnaia proteinaza iz Trichoderma viride. Spetsifichnost' pri gidrolize oligopeptidov.

We isolated, purified, and characterized an aspartic protease from fungus Trichoderma viride. The pH-dependence of the enzyme functioning was determined, and its specificity in the limited proteolysis of insulin and melittin was compared to the specificities of pepsin A and gastricsin. The kinetics of melittin hydrolysis by these enzymes was studied by mass spectrometry.

[Digestion of Luliberine analogues containing D-alanine residue by human gastric juice]. / Rasshcheplenie zheludochnym sokom cheloveka analogov liuliberina, soderzhashchich ostatok D-alanina.

The digestion of surphagone and other luliberine analogues containing residues of D-amino acids by human gastric juice was studied. By means of chromatography and mass spectrometry, these peptides were shown to undergo hydrolysis of the bond formed by D-Ala at the P-1 position, and this hydrolysis was shown to be catalyzed by gastricsin rather than pepsin A. Gastricsin was assumed to be a highly specific protease. The results are in a good agreement with the concept of conformational specificity of proteases towards the natural oligopeptides.

[Proteolysis of human proinsulin catalysed by native, modified, and immobilized trypsin]. / Proteoliz proinsulina cheloveka, kataliziruemyi nativnym, modifitsirovannym i immobilizirovannym tripsinom.

Proteolysis of recombinant human proinsulin by the native trypsin, by trypsin modified with a copolymer of vinylpyrrolidone and acrolein, and by the same modified trypsin immobilized on Silochrom 1.5 was studied by RP HPLC and mass spectrometry. Rate constants of the main stages of proinsulin hydrolysis by the native trypsin were estimated. The values of rate constants of the digestions of the most easily hydrolyzable bonds (those formed by the pairs of the basic amino acid residues) in proinsulin were found to be of the same order as those formed by the separate lysine residues (Lys7) and those formed by the four basic amino acid residues of the C-terminal cluster of melittin. It was established that covalent trypsin binding to the copolymer did not change the ratio of the rate constants of the individual stages of proinsulin hydrolysis, whereas after the immobilization of modified trypsin on the Silochrome, the formation of diarginyl insulin-ArgArg, intermediate forms of hydrolyzed insulin, and desThr-insulin proceeds with comparable rates.

[Recombinant proteins containing oxytocin oligomeric sequences]. / Rekombinantnye belki, soderzhashchie oligomernye posledovatel'nosti oksitotsina.

Expression plasmids were constructed with genes encoding the ILOX3, ILOX6, and ILOX9 recombinant proteins, which contain the C-terminal fragments of trimer, hexamer, or nonamer of oxytocinoyl-Lys. Upon expression in E. coli, all three genes yielded inclusion bodies containing protein products of similar length and heterogeneous in the C-terminal region. It is likely that in the case of the ilox3 gene, the obtained protein mixture includes the full-length product of translation with the C-terminal lysine. In the case of the ilox6 and ilox9 genes, the protein products are formed as the result of a site-specific proteolysis in the regions between the second and the fourth oxytocin units.

[ECP 32 proteinase: characteristics of the enzyme, study of specificity]. / Proteinaza ECP 32: kharakteristika fermenta, issledovanie spetsifichnosti.

Hydrolysis of the C-peptide from recombinant human proinsulin, porcine insulin, and melittin by the E. coli actin-degrading proteinase ECP 32 was studied by reverse phase high performance liquid chromatography and mass spectrometry with electrospray ion source. Proteinase ECP 32 hydrolyzed only melittin at the Ala15-Leu16 or Leu16-Ile17 bonds (KM = 2.4 x 10(-6) M). The effects of pH and buffer composition on the rate of enzymatic hydrolysis were studied. The pH optimum of melittin hydrolysis was 7. Phosphates inhibited, whereas ATP stimulated the hydrolysis of melittin. Melittin was suggested as a substrate for determining the activity of proteinase ECP 32.

[Functionally important tyrosine residues in Saccharomyces cerevisiae pyrophosphatase. I. Chemical modification and localization in the primary structure]. / Funktsional'no vazhnyi ostatok tirozina v pirofosfataze S. cerevisiae. I. Khimicheskaia modifikatsiia i lokalizatsiia v pervichnoi strukure.

Inorganic pyrophosphatase (PPase) of S. cerevisiae is effectively inactivated by 7-chloro-4-nitrobenzofuran; the CaPP1 substrate analog has a protective effect. The modified enzyme separated from low molecular weight contaminants has an adsorption maximum at 345 nm. Preliminary modification of PPase SH-groups does not influence the enzyme binding to the inhibitor. The PPase activity is reconstituted by beta-mercapto-ethanol; hence, the inhibiting effect of the reagent is due to modification of tyrosine residues. A single reagent-containing peptide was isolated by specific adsorption from the tryptic hydrolysate of modified PPase. Within the primary structure of PPase, this peptide occupies positions 82-111 and contains two tyrosine residues. Hydrolysis of the isolated peptide by chymotrypsin and determination of the structure of fragments obtained by mass spectrometry and automated sequencing revealed that inactivation of PPase is due to selective modification of Tyr89.

[Determination of nystatin component composition using HPLC and TLC with densitometry]. / Opredelenie komponentnogo sostava nistatina metodami VEZhKh i TSKh s ispol'zovaniem densitometrii.

The component composition of nystatin produced by an improved strain of Streptomyces noursei was determined by HPLC on Milichrom chromatograph (USSR). It was shown that the antibiotic consisted of nystatins A1, A2, A3 and B and admixture substances. The data appeared to be in good agreement with the results of the complex TLC investigation, by using densitometry. The component composition of the samples was evidenced by SIEAP mass spectrometry. Physiochemical and biological characteristics of separate components are presented.

[Study of components of standard samples of polyene macrolide antibiotics by the methods of high performance liquid chromatography and SIEAP mass spectrometry]. / Izuchenie komponentnogo sostava standartnykh obraztsov polienovykh makrolidnykh antibiotikov metodom vysokoéffektivnoi zhidkostnoi khromatografii i mass-spektrometrii ERIAD.

The component composition of reference samples of polyenic macrolide antibiotics such as nystatin, mycoheptin, amphotericin B and levorin was studied by HPLC and chromatographic mass spectrometry in comparison to the WHO standards. It was shown that the samples were close by their component composition to the analogous samples of the WHO standards.

[Study of the structure of anthracycline antibiotics by ERIAD mass spectrometry]. / Izuchenie stroeniia antratsiklinovykh antibiotikov metodom ERIAD mass- spektrometrii.

Fragmentation of antibiotics daunorubicin, carminomycin, doxorubicin and their semisynthetic analogues under conditions of the new mass spectrometry method ERIAD is discussed. Signals of protonated molecular ion (M + H)+ and ions of fragments are present in all the mass spectra. The results are compared with literary data obtained by means of other (EI and FAB MS) mass spectrometry methods.

[The study of degradation of peptides in human plasma and serum by the ERIAD high pressure liquid chromatography-mass spectrometry method]. / Izuchenie degradatsii peptidov v plazme i syvorotke krove cheloveka metodom vysokoéffektivnoi zhidkostnoi khromatografii-mass- spektrometrii ERIAD.

The proteolysis of bradykinin, [Leu5]enkephalin and some of its synthetic analogs. [D-Ala2,Leu5]enkephalin and [D-Ala2,Leu5]enkephalinyl-Arg, by human blood plasma and serum enzymes was investigated. The degradation products were identified. Based on the kinetic data, the principal pathways of degradation and its limiting steps were established.

[Location of amino acid substitutions in human hemoglobin. Mass spectrometric rapid analysis of tryptic peptides]. / Lokalizatsiia aminokislotnykh zamen v gemoglobine cheloveka. Mass- spektrometricheskii ékspress-analiz tripticheskikh peptidov.

Point mutations alpha 58 His----Tyr (Hb M Boston), beta 6 Glu--lys (Hb C) and beta 26 Glu----Lys (Hb E) have been identified in abnormal hemoglobins by means of tryptic hydrolysis of their alpha- and beta-chains followed by mass-spectrometry coupled with direct extraction of ions from solution. The abnormal hemoglobin Hb M Boston alpha 58 (E7) His----Tyr has been for the first time found in the blood of a patient from the USSR. This express-method is generally applicable for the identification of point mutation in proteins. The amount of protein necessary for the analysis is 100-1000 pmole. The stability, proteolytic degradation of the identified abnormal Hb's and Hb Bart's were investigated. The molecular pathogenesis of the hemoglobinopathies are discussed from the point of view of the observed properties.