Twin plastocyanin dimorphism in tobacco.

Two iso-plastocyanin fractions, oxidized b-plastocyanin, PCb(II) and reduced a-plastocyanin, PCa(I), have been isolated from whole tobacco leaves by conventional chromatography on DEAE-cellulose. The isoelectric points of PCa and PCb at 10 degrees C were found to be 3.99 and 3.97, respectively. When the primary structures were analysed, a microheterogeneity within both PCa and PCb was observed. By appropriate peptide arrangements the amino-acid sequences of two PCa (PCa' and PCa") and two PCb (PCb' and PCb") have been differentiated. All four sequences contain 99 amino-acid residues. PCa' and PCa" differ in one position, where Ser-58 in PCa' is replaced by Pro in PCa".PCb' and PCb" differ in three positions, where Gly-65, Thr-81 and Ala-85 in PCb' are replaced by Ala, Ser and Ser in PCb", respectively. PCa (PCa'/PCa") generally differs from PCb (PCb'/PCb") in three positions, where Val-52, Glu-61 and Tyr-62 in PCa'/PCa" are replaced by Ala, Asp and Leu in PCb'/PCb", respectively. Fluorescence spectra of oxidized tobacco PCa and PCb have been characterized with an emission-maximum position at around 340 nm. The presence of one extra tyrosyl (Tyr-62) in PCa results in a weak increase of the maximal intensity in conjunction with a slight blue-shift of the maximum position.

Microheterogeneity of parsley plastocyanin.

A procedure for isolation of two iso-plastocyanins from parsley has been described here. Three consecutive chromatographic steps on DE-52-Whatman cellulose were applied for isolation of two total plastocyanin (PC) fractions, oxidized [PC(II)] and reduced [PC(I)]. By chromatofocusing of PC(II) on Polybuffer exchanger 74 two different plastocyanins, designated as plastocyanin a (PCa) and plastocyanin b (PCb), were obtained. The isoelectric points (pI) of PCa and PCb at 10 degrees C are 4.16 and 4.14, respectively. The complete amino acid sequences of PCa and PCb were determined. The two iso-proteins consist of 97 amino acid residues and differ only at sequence position 53, where Glu in PCa is replaced by Asp in PCb.

Complete amino acid sequence of the protease inhibitor from buckwheat seeds.

The complete amino acid sequence of protease inhibitor BWI-1 from buckwheat (Fagopyrum esculentum Moench) seeds has been established by automatic Edman degradation and mass spectrometry. The molecule of the inhibitor consists of 69 amino acid residues, with a molecular mass calculated as 7743.8 Da. The active site of the inhibitor contains an Arg45-Asp46 bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the proteinase inhibitor I family.

Characterisation of high Mr wheat glutenin polymers by agarose gel electrophoresis and dynamic light scattering.

Agarose gel electrophoresis has been used to separate the complex mixture of wheat gluten polymers into fractions ranging in Mr, determined by dynamic light scattering, from about 500,000 to over 5x10(6). The separation is reliable and reproducible and well suited to the routine analysis of multiple samples.

Structure of microcin C51, a new antibiotic with a broad spectrum of activity.

The structure of microcin C51, a new antibiotic produced by E. coli, has been determined. This antibiotic was shown to be a 1.18 kDa nucleotide peptide. It consists of a heptapeptide with formylmethionine as the N-terminus and a C-terminal asparagine linked with nebularin-5'-monophosphate through the three-methylene bridge. The OH-group of threonine is substituted. The peptide chain of microcin C51 synthesized on ribosomes is the longest among the known biologically active nucleotide peptides.

Primary structure of three cationic peptides from porcine neutrophils. Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation.

The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.

Carbohydrates in mammalian tryptophanyl-tRNA synthetase.

Homogeneous preparations of bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) contain monosaccharides (mannose, fucose, galactose, N-acetylglucosamine) as revealed by liquid chromatography. Their content comprises 2.5-3.0% (w/w) of the enzyme composed of two subunits (60 kDa x 2). The same set of sugars was detected in elastase and CNBr-generated fragments (with molecular masses of approx. 40 kDa and 30 kDa, respectively). It is concluded that bovine tryptophanyl-tRNA synthetase, in addition to being a metallo- and phosphoprotein, is also a glycoprotein.

Disulphide structure of a sunflower seed albumin: conserved and variant disulphide bonds in the cereal prolamin superfamily.

Disulphide mapping of a methionine-rich 2S albumin from sunflower seeds showed four intra-chain disulphide bonds which are homologous with those in a related heterodimeric albumin from lupin seeds (conglutin delta). Similar conserved disulphide bonds are also present in alpha-gliadin and gamma-gliadin storage proteins of wheat, but a lower level of conservation is present in a further related group of proteins, the cereal inhibitors of alpha-amylase and trypsin. These differences may relate to the different functions of the proteins.

A direct photo-activated affinity modification of tetracycline transcription repressor protein TetR(D) with tetracycline(1).

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR(D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR(D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.

Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase.

A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 A resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.

De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis.

Antimicrobial peptides (AMPs), named lycocitin 1, 2 and 3, and a peptide with a monoisotopic molecular mass of 3038.70 Da were detected in the venom glands of the wolf spider Lycosa singoriensis. Two of the peptides, lycocitin 1 and 2, are new AMPs whereas lycocitin 3 is highly homologous to lycotoxin II isolated from the venom of spider Lycosa carolinensis. In addition, two other peptides with monoisotopic masses of 2034.20 and 2340.28 Da showing the motif typical for antimicrobial peptides were also identified. These peptides and lycocitin 1, 2 and 3 were de novo sequenced using electron capture dissociation and low-energy collisional tandem mass spectrometry. The amino acid sequence of lycocitin 1 was determined as GKLQAFLAKMKEIAAQTL-NH(2). Lycocitin 2 differs from lycocitin 1 by a replacement of a lysine residue for an arginine residue at the second position. Lycocitin 3 differs from the known lycotoxin II consisting of 27 amino acid residues by a deletion of Gly-26. Both lycocitin 1 and 2 inhibit growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Pseudomonas aeruginosa) at micromolar concentrations.

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.

Antifungal activity of storage 2S albumins from seeds of the invasive weed dandelion Taraxacum officinale Wigg.

In this work, we isolated and characterized novel antifungal proteins from seeds of dandelion (Taraxacum officinale Wigg.). We showed that they are represented by five isoforms, each consisting of two disulphide-bonded large and small subunits. One of them, To-A1 was studied in detail, including N-terminal amino acid sequencing of both subunits, and shown to display sequence homology with the sunflower 2S albumin. Using different assays we demonstrated that dandelion 2S albumins possess inhibitory activity against phytopathogenic fungi and the oomycete Phytophtora infestans at micromolar concentrations with various isoforms differing in their antifungal activity. Thus, 2S albumins of dandelion seeds represent a novel example of storage proteins with defense functions.

Identification of parvalbumin alpha in bovine hypothalamus: a partial primary structure.

In the course of the study of structure-functional properties and molecular mechanisms of neuropeptides and of low molecular weight proteins of the central nervous system we succeeded in isolating from the soluble fraction of bovine hypothalamus a protein having M(r) 11897.3, according to mass spectral analysis. The purification procedure was mainly based on reversed phase HPLC. As the N-terminus of the molecule was found to be blocked, we have subjected it to CNBr degradation. By Edman microsequence analysis of the peptide fragments and by data base searching the isolated substance was identified as parvalbumin alpha (PRVA)-one of the calcium-binding proteins. However, its primary structure was found not to be identical to that of the known PRVAs from other sources. One of the features of PRVA is its stability. Being subjected to an exhausting purification procedure it retains its complete structure. As neuropeptides and low molecular weight proteins are found to be polyfunctional, a central question concerns the biological role of PRVAs in terms of "where and when" they express their action.

Determination of disulfide bonds in gamma-46 gliadin.

The disulfide bonds in gamma-46 gliadin were identified: Cys173--Cys192, Cys212--Cys291, Cys165--Cys199 (or Cys200), Cys283--Cys200 (or Cys199). The disulfide-containing peptides were obtained by limited hydrolysis of the intact protein with chymotrypsin at an enzyme/substrate ratio of 1:1000 at 20 degrees C for 22 h with subsequent digestion of disulfide-containing fragments with trypsin and chymotrypsin. The locations of disulfide bonds were determined by sequencing disulfide-containing fractions and constituent peptides and comparison of the obtained sequences with the partial amino acid sequence of gamma-46 gliadin determined earlier.

A rapid and specific method for isolation of thiol-containing peptides from large proteins by thiol-disulfide exchange on a solid support.

Activated thiol-Sepharose [agarose-(glutathione-2-pyridyl disulfide) conjugate] has been used to immobilize proteins with a single or a few thiol groups via disulfide bridges. The immobilized proteins were subsequently proteolytically degraded. After washing, the thiol-containing peptides were eluted with a reducing agent. A single preparative paper electrophoresis, occasionally after a modification such as oxidation, was sufficient to obtain pure peptides in good yields. The method was applied to the major parvalbumin from hake muscle (a protein with 108 amino acid residues and one cysteine residue), to mercaptalbumin from bovine serum (565 residues and one cysteine), and to human serum ferroxidase [EC 1.16.3.1; iron (II):oxygen oxidoreductase] (ceruloplasmin) (1065 residues and three cysteines). The use of the technique, e.g., as a simple means of obtaining homologous peptides in related proteins, is discussed.

Complete amino acid sequence of the protease inhibitor BWI-4a from buckwheat seeds.

The complete amino acid sequence of the protease inhibitor BWI-4a from buckwheat (Fagopyrum esculentum Moench) seeds has been established by automated Edman degradation in combination with MALDI-TOF mass spectrometry. The inhibitor molecule consists of 67 amino acid residues with a single disulfide bond. Its N-terminus is blocked by a pyroglutamic acid residue. The reactive site of the inhibitor contains an Arg43-Asp44 bond. Mass spectrometry revealed that inhibitor BWI-4a is present in buckwheat seeds in two isoforms differing by a single amino acid substitution of Gly40 for Ala40. Analysis of the amino acid sequence of the BWI-4a inhibitor indicates that this inhibitor is a member of the potato proteinase inhibitor I family.

Amino acid sequence of the protease inhibitor BWI-4a from buckwheat seeds.

The complete amino acid sequence of protease inhibitor BWI-4a from buckwheat (Fagopyrum esculentum Moench) seeds, consisting of 67 amino acid residues with a single disulfide bond, has been established by Edman degradation in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Its N terminus is blocked by a pyroglutamic acid residue. Mass spectrometric analysis revealed that inhibitor BWI-4a is present in buckwheat seeds in two isoforms with a single amino acid substitution of Ala40 for Gly40. The reactive site of the inhibitor contains an Arg43-Asp44 bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the potato proteinase inhibitor I family.

The complete amino acid sequence and disulphide bond arrangement of oat alcohol-soluble avenin-3.

We have elucidated the complete amino acid sequence of one of the avenin components, avenin-3, isolated from oat (Avena sativa L.), variety Narymsky 943. The sequence of the protein was determined by sequencing of CNBr and trypsin-generated peptides in combination with mass spectrometry. The protein is a single polypeptide chain, consisting of 201 amino acid residues with M(r) 23,252.8. The N-terminal amino acid residue of the protein is blocked with 5-oxoproline (pyroglutamic acid). All eight cysteine residues in avenin-3 are involved in disulphide bonds. The positions of these bonds were established by identification of a CNBr cleavage product of the intact avenin containing all the disulfide bonds (S-S core). Subsequent subdigestion of this S-S core allowed isolation of disulphide bonded peptides detected by differential reverse-phase HPLC before and after reduction. As a result, all four disulphides Cys50 Cys183, Cys58 Cys77, Cys84 Cys85 and Cys97 Cys191 were identified. Comparison of avenins with other prolamins demonstrates a high degree of similarity, which is especially pronounced around the cysteine residues. Avenins differ slightly from other prolamins in having unique N-terminal sequences and some differences in the repeated sequence motifs.

Identification of an allelic variant of isoform MLC1-V/sB (human myosin light chain).

A study of 250 specimens of human myocardium by two-dimensional gel electrophoresis revealed an allelic variant of isoform MLC1-V/sB, which was identified by immunoblotting with monoclonal antibody against MLC1-V/sB and peptide mapping after in situ tryptic digestion of electroblotted proteins. The substitution Asn-144 for His-144 was found in this new allelic variant of MLC1-V/sB.