Vacuolar Na+/H+ antiporter from barley: identification and response to salt stress.

One of the protective mechanisms used by plants to survive under conditions of salt stress caused by high NaCl concentration is the removal of Na+ from the cytoplasm. This mechanism involves a number of Na+/H+-antiporter proteins that are localized in plant plasma and vacuolar membranes. Due to the driving force of the electrochemical H+ gradient created by membrane H+-pumps (H+-ATPases and vacuolar H+-pyrophosphatases), Na+/H+-antiporters extrude sodium ions from the cytoplasm in exchange for protons. In this study, we have identified the gene for the barley vacuolar Na+/H+-antiporter HvNHX2 using the RACE (rapid amplification of cDNA ends)-PCR (polymerase chain reaction) technique. It is shown that the identified gene is expressed in roots, stems, and leaves of barley seedlings and that it presumably encodes a 59.6 kD protein composed of 546 amino acid residues. Antibodies against the C-terminal fragment of HvNHX2 were generated. It is shown that the quantity of HvNHX2 in tonoplast vesicles isolated from roots of barley seedlings remains the same, whereas the rate of Na+/H+ exchange across these membranes increases in response to salt stress. The 14-3-3-binding motif Lys-Lys-Glu-Ser-His-Pro (371-376) was detected in the HvNHX2 amino acid sequence, which is suggestive of possible involvement of the 14-3-3 proteins in the regulation of HvNHX2 function.

(2)H- and(13)C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring.

Biosynthetic preparation of(2)H- and(13)C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing(13)C- or(2)H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of(2)H- and(13)C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.

[Composition and structure of the antibiotic polypeptide 308-I from Actinomadura recticatena]. / Sostav i stroenie antibiotika-polipeptida 308-I iz Actinomadura recticatena.

Antibiotic 308-I isolated from Actinomadura recticatena and the products of its degradation were studied with the methods of electron impact mass spectrometry, chemical ionization with ammonia, field desorption and ion extraction from solution under atmospheric pressure. It was shown that by its composition and structure antibiotic 308-I was identical to antibiotic BBM-928 A.

[Mass-spectrometry with ion extraction from solution at atmospheric pressure for peptide mapping]. / Ispol'zovanie mass-spektrometrii s ékstraktsiei ionov iz rastvora pri atmosfernom davlenii (ERIAD) dlia sostavleniia peptidnykh kart.

SIE AP mass spectra of tryptic peptides from the cyclo-GMP phosphodiesterase gamma-subunit and of chymotryptic peptides from the cyanogen bromide fragments of the same subunit exhibit MH+ ions for all theoretically possible smaller peptides. These facts show that SIE AP mass spectrometry can be successfully applied to peptide mapping using 1-2 nmoles of the compound.

[Isolation and structure of the active component of the antitumor agent blastolysin]. / Vydelenie i struktura aktivnogo komponenta protivoopukholevogo preparata blastolizina.

Active principle of blastolysin, comprising ca. 40% of the total weight of Lactobacillus bulgaricus cell wall preparation, has been isolated and structurally studied by chemical and spectral methods. The substance a glycopeptide with Mr aa. 10,000, consists of two tetrasaccharide moieties connected by oligopeptide bridges; glycerophosphate, glucose, and galactose moieties are also attached to N-acetylmuramyl residue of the peptidoglucan core. Tetrasaccharide-containing muramylpeptides were shown to be responsible for the antitumour activity.

[Mass-spectrometric determination of the structure of glycopeptides from the bacterial cell wall (illustrated by Lactobacillus bulgaricus)]. / Mass-spektrometricheskoe usstanovlenie struktury glikopeptidov kletochnykh stenok bakterii (na primere Lactobacillus bulgaricus).

Mass spectrometry has been applied to the structural analysis of one of the glycopeptides from blastolysin, antitumor bacterial preparation isolated from the Lactobacillus bulgaricus cell wall. The glycopeptide (MW 10,000) was subjected to partial acid hydrolysis (6 N HCl, 100 degrees C) and the resulting products were dansylated or trifluoroacetylated and methylated or deuteromethylated. The mixture of these derivatives was examined by high-performance liquid chromatography or gas chromatography followed by mass spectrometry using electron impact and ammonia chemical ionization techniques.

[Structural-functional characteristics of argiopine--the ion channel blockers from the spider Argiope lobata venom]. / Strukturno-funktsional'naia kharakteristika argiopina--blokatora ionnykh kanalov iz iada pauka Argiope lobata.

Argiopine, a compound capable of blocking the glutamate-activated ion channels in experiments with glutamatergic synapses of blowfly larvae, was isolated from the venom of spider Argiope lobata. Argiopine-receptor complex has KD 6,7 X 10(-7) M. The venom solution was treated with 60% ethanol, centrifuged and supernatant was subjected to reversed-phase HPLC in the acetonitrile concentration gradient. Argiopine structure was established using 1H- and 13C-NMR spectroscopy, mass spectrometry, elemental and amino acid analyses. Argiopine (molecular mass 636) consists of arginine (free alpha-NH2) linke with polyamine--NH (CH2)3NH(CH2)3NH(CH2)5NH--through a peptide bond. The polyamine is connected to the alpha-carboxyl group of asparagine whose alpha-amino group is linked to 2,4-dihydroxyphenylacetic acid.