A new antioxidative vitamin B6 analogue modulates pathophysiological cell proliferation and damage.

The new large scale synthesis of the yellow colored vitamin B6 analogue 5'-O-phosphono-pyridoxylidenerhodanine (2) (B6PR) leads to oligohydrates of its monosodium salt (4). The light-red hemiheptadecahydrate (8 1/2 hydrate) (4a) was crystallized and its three-dimensional structure determined by X-ray crystallography. Special nucleotide and protein interaction properties together with scavenging antioxidative function are combined in this simple water-soluble vitamin B6 analogues B6PR. High (mM) concentrations were untoxic to 'healthy' not affected cells and primary tissues. Complexation of ions (e.g. Ca2+, Fe2+, and Zn2+), modulation of nitric oxide synthases (NOS I-III), nitric oxide (NO) metabolism, and reactive oxygen species (ROS) was found. Special cytoprotecting, immunomodulating, stimulating and inhibiting activities were observed in vitro, not in comparison with some natural and synthetic pyridoxines. Low B6PR suppressed proliferation, high induced selective cell death of some cancer cell lines. Low B6PR protected HIV-1-infected CD4+ HUT 78 cells against HIV-1-mediated destruction (complete inhibition of HIV-1-induced syncytia formation and cell death) and reduced p24 level. Autoreactive S100beta-specific T cells of Lewis rat, a model of multiple sclerosis, could be influenced. Oxidative damage and age, acquired and inherited disease related pathophysiological disorders can be treated by this new cytopathology-selective versatile acting B6PR.

Limited proteolysis of alpha 1-proteinase inhibitor (alpha 1-PI) in acute leukemia: studies on the resulting fragments and implication for the structure of the inactivated inhibitor.

In patients with acute myeloid leukemia, a 41 kDa glycoprotein appears in the urine during remission induction chemotherapy. We have recently reported on the isolation and preliminary characterization of this protein and the generation of specific monoclonal antibodies which showed that it is a proteolytically modified form of alpha 1-proteinase inhibitor (Dengler et al., 1992, Biol. Chem. Hoppe-Seyler 373, 581-588). In the paper presented here, results from further characterization experiments as well as from studies on the effects of proteolysis on the conformation and the resulting functional properties of the truncated inhibitor are reported. N-terminal amino acid sequence analysis showed that proteolysis has occurred in the N-terminal part as well as in the reactive site loop of alpha 1-PI. The resulting core protein of 41 kDa is composed of approximately 324 amino acid residues with the C-terminus located close to Lys343 of alpha 1-PI.A 4 kDa peptide remaining bound to this fragment throughout the entire purification procedure could be separated by SDS treatment. N- and C-terminal sequence analysis of this peptide after isolation by gel filtration showed that it is comprised of residues Ile359 up to Lys394, thus representing the peptide located C-terminal to the reactive site loop of alpha 1-PI. Transverse urea gradient gel electrophoresis indicates that the proteolyzed inhibitor is in the thermodynamically stable, relaxed (R)-conformation known for proteinase-complexed and cleaved serpins. The truncated inhibitor exhibits no chemotactic activity towards neutrophils when tested in a standard assay.

The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site.

Like many proteins with a single transmembrane domain the IL-6R exists in a membrane-associated and soluble form. The soluble IL-6R is generated by limited proteolysis of the membranous receptor. This process, also called shedding, is drastically enhanced by PMA, an activator of protein kinase C. The soluble receptor protein was purified to homogeneity from supernatants of COS-7 cells transfected with a cDNA coding for the transmembrane IL-6R. The COOH-terminus of the shed receptor protein was analyzed by carboxypeptidase treatment and subsequent amino acid analysis. The established cleavage site Gln357/Asp358 was extensively altered by point mutations and small deletions to define the structural requirements for cleavage. Although point mutations around the cleavage site reduced shedding of the IL-6R up to fivefold, deletions of 5 or 10 amino acids almost completely abolished shedding. Deletion of the cytoplasmic domain of the receptor had no influence on shedding of the protein. It turned out that a potential N-glycosylation site close to the proteolytic cleavage site of the IL-6R is used. However this N-glycosylation does not affect the efficiency of the shedding process. Furthermore, we demonstrate for the first time that the human IL-6R is constitutively phosphorylated and that this phosphorylation can be stimulated by PMA but is not correlated with shedding of the receptor protein. The knowledge of the mechanism by which the soluble IL-6R is generated will help to identify the processing enzyme involved and to analyze its regulation.

[Side reactions in peptide synthesis. V. O-sulfonation of serine and threonine during removal of pmc- and mtr-protecting groups from arginine residues in fmoc-solid phase synthesis]. / Nebenreaktionen bei Peptidsynthesen, V. O-Sulfonierung von Serin und Threonin während der Abspaltung der Pmc- und Mtr-Schutzgruppen von Argininresten bei Fmoc-Festphasen-Synthesen.

A novel side reaction in Fmoc-solid-phase synthesis, which occurs during removal of protecting groups and detachment from the resin, was elucidated by investigations on model peptides: During the cleavage of Pmc- or Mtr-protecting groups from arginine residues by trifluoroacetic acid in peptides with O-tert-butyl-protected aliphatic hydroxyamino acids, peptides containing O3-sulfo-serine and O3-sulfo-threonine are formed as side-products in high yields, if suitable scavengers are absent. Subsequent to their isolation and purification, the structures of these peptide sulfuric acid mono-esters could unequivocally be proven by chemical and spectroscopic (MS, NMR, IR) methods.

Application of high resolution two-dimensional polyacrylamide gel electrophoresis of polypeptides from cultured neonatal rat cardiomyocytes: regulation of protein synthesis by catecholamines.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to cultured neonatal rat heart muscle cells, incubated for 72 h at 37 degrees C in serum-free medium, either in the absence or in the presence of 0.1 microM norepinephrine. After silver staining, about 340 and 550 protein spots could be seen in cardiomyocytes, cultured either in the absence or presence of norepinephrine. Of these spots, 141 could be further characterized according to isoelectric point and molecular weight, with 71 protein spots being present under both conditions. In cells cultivated in presence of norepinephrine, 58 new protein spots appeared, whereas 12 spots disappeared, and 22 spots increased (whereas 3 spots decreased) in intensity. In comparison with 2-D PAGE of rat cardiomyocytes, the protein pattern of the intact heart of neonatal rats is incongruent. 2-D PAGE of polypeptides of cultured neonatal rat cardiomyocytes may be a suitable tool to study the regulation of protein synthesis by various stimuli with relevance to cardiac growth adaptation, inotropy and heart failure.

High resolution two-dimensional mapping of tissue-specific polypeptides in the desert locust, Schistocerca gregaria.

High resolution two-dimensional gel electrophoresis (2-DE) using immobilized pH gradients was used to map the tissue-specific polypeptides of the desert locust, Schistocerca gregaria. Highly specific comprehensive 2-DE reference maps ("master gels") were developed for the brain, corpus cardiacum, subesophageal ganglion, and hemolymph. The polypeptides were well resolved within the pH 4-7 range in the first dimension and within the 14-94 kDa molecular mass range in the second dimension.

Photoaffinity labeling of functional states of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor was subjected to photoaffinity labeling in different conformational and functional states. The photolabel used was the ion-channel blocker [3H]-TPMP+. A procedure is described for isolating labeled delta-polypeptide chains from the receptor complex by preparative SDS-polyacrylamide gel electrophoresis. The photolabel was localized in the primary structure of the delta-chain. The site of labeling was found to be identical when photoaffinity labeling was performed in the resting, desensitized, or antagonist state, respectively.

Homeothermic fish and hemoglobin: primary structure of the hemoglobin from bluefin tuna (Thunnus thynnus, Scromboidei).

Some fish are warm-bodied, e.g. the bluefin tuna (Thunnus thynnus), which has a muscle temperature 12-17 degrees C higher than its environment. This endothermy is achieved by aerobic metabolism and conserved by means of a heat-exchanger system. The hemoglobins of bluefin tuna are adapted to these conditions by their endothermic oxygenation, thus contributing to the preservation of the body energy. This is a new and so far unique property of tuna hemoglobin. The primary structure of the alpha and beta chains of bluefin tuna hemoglobins is presented. The sequence was determined after enzymatic and chemical cleavages of the chains and sequencing of the peptides in gas- and liquid-phase sequencers. The alpha chains consists of 143 residues and are N-terminally acetylated. The beta chains have 146 amino acids and show two ambiguities at positions 140 and 142. The alpha chains differ from the human alpha chains in 65 amino-acid residues, the beta chains in 76. The hemoglobins of bluefin tuna, carp and man are compared and their different physiological properties are discussed in relation to the sequence data. From the primary structure of tuna hemoglobins, it is possible to propose a molecular basis for their peculiar endothermic transition from the T to the R structure.

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits.

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.

The reaction site of a non-competitive antagonist in the delta-subunit of the nicotinic acetylcholine receptor.

A site in the primary structure of the nicotinic acetylcholine receptor from Torpedo marmorata covalently labeled with the non-competitive antagonist [3H]triphenylmethylphosphonium (TPMP+) was localized. The label was found in position 262 of the delta-polypeptide chain. This site is specifically labeled in the presence of the agonist carbamoylcholine. Labeling is prevented by the non-competitive antagonist histrionicotoxin. Position 262, probably a serine, is located in the highly conserved membrane-spanning helix M2 (according to the predicted folding scheme of Finer-Moore and Stroud (1984). The relationship of this site to the receptor's ion channel and its regulation is discussed.

The expression of alpha D-chains in the hemoglobin of adult ostrich (Struthio camelus) and American rhea (Rhea americana). The different evolution of adult bird alpha A-, alpha D- and beta-chains.

The hemoglobin of adult American rhea (Rhea americana) and ostrich (Struthio camelus) contains two components identified to be HbA (alpha 2A beta 2) and HbD (alpha 2D beta 2). The amino-acid sequence of alpha D-chains from HbD of adult American rhea and ostrich has been determined. The sequence was studied by Edman degradation of tryptic peptides and chemical cleavage products in a liquid phase sequencer. By homologous comparison with pheasant HbD (Phasianus colchicus colchicus), the alpha D-chains of American rhea differ by 28 amino-acid exchanges, the alpha D-chains of ostrich by 23 residues. These differences are higher than those observed for alpha A- as well as for beta-chains of HbA from the same species. The ratio of amino-acid exchanges for beta:alpha A:alpha D for American rhea and ostrich is found to be 1:5.5:6.5. At present the reason for the differences in evolution rates for the beta-, alpha A- and alpha D-chains of bird hemoglobins is still unclear.

[The hemoglobin of adult Andean condors (Vultur gryphus, Cathartiformes). The amino acid sequence of the major (HbA) and minor component (HbD)]. / Das Hämoglobin des adulten Andenkondors (Vultur gryphus, Cathartiformes). Die Aminosäuresequenz der Haupt-(HbA) und Nebenkomponente (HbD).

The complete amino-acid sequence of the alpha A- and the beta-chains of the major component (HbA) and the alpha D- and the beta-chains of the minor component (HbD) of Andean Condor (Vultur gryphus) is presented. The minor component with the alpha D-chains is present in smaller amounts (17%) than in other birds (25%). The comparison with the corresponding chains of Greylag Goose (Anser anser) shows 17 different amino acids (17 nucleotides, only one-point mutations) in the alpha A-chains and 8 (8 nucleotides) in the beta-chains. The alpha D-chains differ from those of the pheasant (Phasanius cholchicus cholchicus) in 24 amino acids (27 nucl., 3 two-point mutations). Seven alpha 1 beta 1-, one alpha 1 beta 2-, three alpha 1 alpha 1-contacts and one beta 1 beta 1-contact are exchanged. The systematy of Cathartiformes, Ciconiiformes and Phoenicopteriformes is discussed, based on the amino-acid exchanges of all known adult hemoglobins of birds.

[Hemoglobin of tree sparrows (Passer montanus, Passeriformes): Sequence of the major (Hb A) and minor (Hb D) components]. / Die Hämoglobine des Feldsperlings (Passer montanus, Passeriformes). Die Sequenz der Haupt- (Hb A) und Nebenkomponente (Hb D).

Blood of the adult Tree Sparrow (Passer montanus) contains two hemoglobin components, Hb A (ca. 85%), Hb D (ca. 15%). They differ in their alpha-chains (alpha A, alpha D), the beta-chains are identical. The complete primary structures of alpha A-, alpha D- and beta-chains are presented. Comparison with the Greylag Goose (Anser anser) hemoglobin (Hb A) showed that the alpha A-chains differ by 22 amino-acid exchanges, the beta-chains by 16. Comparison with the minor component of the Pheasant (Phasianus colchicus colchicus) hemoglobin (Hb D) showed that the alpha D-chains differ by 34 amino-acid exchanges. Proline is found incorporated in an internal position of an alpha-helix (pos. 124, H7). In comparison to that of the Starling (Sturnus vulgaris) the ratio of amino-acid exchanges for beta: alpha A: alpha D chains is 1 : 7 : 4; in comparison to other birds this ratio is found to be 1 : 2 (1.4-2.2):3 (2.2-4).

Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus.

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined. This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine. The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography. The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides. It consists of 274 amino-acid residues, like that of subtilisin Carlsberg. By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively. It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor. The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently.

[Hemoglobins of thecommon starling (Sturnus vulgaris, Passeriformes). The primary structures of the alphaA, alphaD and beta chains]. / Hämoglobine vom Gemeinen Star (Sturnus vulgaris, Passeriformes), Die Primärstrukturen der alpha A-, alpha D- und beta-Ketten.

The primary structures of the major hemoglobin component HbA (alpha A- and beta-chains) and the minor component HbD (alpha D-chains, beta-chains are identical with those of HbA) of the Starling (Sturnus vulgaris) are given. The order Passeriformes, to which this species belongs, represents 5/8 of all species of birds. Comparing Starling and Greylag Goose (Anser anser) hemoglobins, HbA alpha A-chains differ by 20 amino acids or 23 nucleotides (3 two point mutations), beta-chains by 14 amino-acid and nucleotide exchanges. Eight substitutions modify alpha 1-beta 1 contacts and one phosphate contact (beta 2(NA2)His----Gln). In both chains some histidine residues are substituted by neutral amino acids. Comparing Starling and Pheasant (Phasianus colchicus colchicus) hemoglobins, HbD alpha D-chains differ by 33 amino acids or 39 nucleotides (6 two point mutations). Five alpha 1-beta 1 contacts, one alpha 1-beta 2-contact and one heme contact are changed. Valin was found in the N-terminal position of Starling alpha D-chains instead of methionin which is the N-terminus in all other avian alpha D-chains so far investigated. Not only alpha A- and beta-chains, but also alpha A- and alpha D-chains differ significantly from each other in the mutation rates. Substitutions are discussed in relation to evolution, function and physiology.

[Primary structure of subtilisin DY]. / Die Primärstruktur von Subtilisin DY.

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis, strain DY, is presented. The enzyme's primary structure was elucidated using peptides obtained by tryptic hydrolysis and peptides released from BrCN, tryptophan and Asn-Gly cleavage (using hydroxylamine). The peptides were isolated by gelfiltration and by reversed phase high performance liquid chromatography and were degradated automatically in the sequenator. The complete sequence has been verified by peptide overlapping. The subtilisin DY polypeptide chain, like that of subtilisin Carlsberg, consists of 274 amino-acid residues. 32 Amino-acid replacements were found between these two molecules (37 nucleotide mutations, 5 of them two-point mutations). Between the subtilisins DY and Novo 82 amino-acid residue replacements (106 nucleotide mutations, 24 two-point mutations) and one deletion were found. The polypeptide chains of the three subtilisins mentioned were compared and some differences discussed.

[Hemoglobin of the golden eagle (Aquila chrysaetos, Accipitriformes): amino acid sequence of the alpha A- and beta chains of the principal component]. / Hämoglobine des Steinadlers (Aquila chrysaetos, Accipitriformes): Die Aminosäure-Sequenz der alpha A- und beta-Ketten der Hauptkomponente.

The primary structures of the alpha A- and beta-chains from the major hemoglobin component of Golden Eagle (Aquila chrysaetos) are given. By homologous comparison, Greylag Goose (Anser anser) hemoglobin and Golden Eagle alpha A-chains differ by 17 amino acids or 19 nucleotides (2 two-point mutations), beta-chains by 9 exchanges. Five substitutions have modified alpha 1 beta 1-contacts, one substitution, one alpha 1 beta 2-contact and one alpha 1 alpha 1-contact. Differences by homologous comparison to other hemoglobins of birds are discussed.

[Primary structures of the alpha and beta chains from the major hemoglobin component of the ostrich (Struthio camelus) and American rhea (Rhea americana) (Struthioformes). Aspects of respiratory physiology and taxonomy]. / Die Primärstruktur der alpha- und beta-Ketten der Hauptkomponenten der Hämoglobine des Strausses (Struthio camelus) und des Nandus (Rhea americana) (Struthioformes). Aspekte zur Atmungsphysiologie und Systematik.

The primary structures of the alpha A- und beta-chains from the major hemoglobin component of the Ostrich (Struthio camelus) and American Rhea (Rhea americana) are given. The minor component with the alpha D-chains was detected in Ostrich in several concentrations, in American Rhea as in chicken and pheasant (about 40%). By homologous comparison, Greylag Goose (Anser anser) hemoglobin and Ostrich alpha A-chains differ by 15 amino acids or 16 nucleotide (1 two-point mutation) exchanges, beta-chains by 4 exchanges. Four substitutions modify alpha 1 beta 1-contacts and one phosphate contact. American Rhea and Greylag Goose hemoglobin alpha A-chains differ by 20 amino acids or 23 nucleotides (3 two-point mutations), beta-chains by 4 exchanges. Two substitutions modify alpha 1 beta 1-contacts, one phosphate contact and one heme contact. Oxygen affinity of three hemoglobin components of Ostrich are measured and the results are discussed. Systematic and evolution of Ostrich and American Rhea are discussed.

[Primary structure of alpha and beta chains from the major hemoglobin component of the magpie goose (Anseranas semipalmata, Anatidae)]. / Die Primärstruktur der alpha- und beta-Ketten der Hauptkomponente der Hämoglobine der Spaltfussgans (Anseranas semipalmata, Anatidae).

The amino acid sequence of the alpha and beta chains from the major hemoglobin component (HbA) of Australian Magpie Goose (Anseranas semipalmata) is given. The minor component with the alpha D chains was detected, but only found in low concentrations. By homologous comparison, Greylag Goose hemoglobin (Anser anser) and Australian Magpie Goose alpha chains differ by 13 amino acids or 17 nucleotide (4 two point mutations) exchanges, beta chains by 6 exchanges. Seven alpha 1 beta 1 contacts are modified by substitutions in positions alpha 30-(B11)Glu leads to Gln, alpha 34(B15)Thr leads to Gln, alpha 35(B16)-Ala leads to Thr, alpha 36(B17)Tyr leads to Phe, beta 55(D6)Leu leads to Ile, beta 119(GH2)Ala leads to Ser and beta 125(H3)Glu leads to Asp. Further, one alpha 1 beta 2 contact point was changed in beta 39(C5)Gln leads to Glu. Mutation in this position, except in two abnormal human hemoglobins, was not found in any species. Amino acid exchanges between hemoglobin of Australian Magpie Goose and other birds are discussed.

The amino acid sequence of Canada goose (Branta canadensis) and mute swan (Cygnus olor) hemoglobins. Two different species with identical beta-chains.

The amino acid sequences of the alpha- and beta-chains from the major hemoglobin component (HbA) of Canada goose (Branta canadensis) and mute swan (Cygnus olor) are given. The alpha-chains are of the alpha A-type, since alpha D-type was expressed but only found in low concentrations. By homologous comparison, greylag goose hemoglobin (Anser anser) and Canada goose hemoglobin alpha-chains differ by two exchanges, and beta-chains by three exchanges. A valine substitution for threonine was found at position alpha 34 (B15). This exchange is a result of a two point mutation. Thus, there are three nucleotide mutations in alpha-chains, as in beta-chains. Substitutions in positions alpha 34 (B15) and beta 125 (H3) have modified intersubunit contacts (alpha 1 beta 1-contacts). A comparison of mute swan hemoglobin with greylag goose hemoglobin shows four exchanges in alpha-chains and three in beta-chains. Canada goose and mute swan have identical beta-chains, while alpha-chains differ in two amino acids. One of these exchanges is implicated in one of the alpha 1 beta 1-contact points (alpha 34) where isoleucine substitution for valine was found. Comparison of hemoglobins from different species in the same tribe (Anserini) shows a high homology between Canada goose and mute swan hemoglobins.