Die Proteinstruktur der Defektmutante PM 2 des Tabakmosaikvirus

Summary The protein structure of the TMV mutant PM2 has been studied. PM2 produces a non-functioning protein unable to aggregate into rodlike particles (Siegel et al., 1962). The two amino acid replacements in PM2-protein are localized in position 28 (threonineisoleucine), and position 95 (glutamic acidaspartic acid). This finding is discussed with relation to the spatial structure of the virus.

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Herrn ProfessorG. Melchers zum 60. Geburtstag gewidmet.     

Induction,purification and characterisation of arabinases produced by Aspergillus niger

The induction of arabinases in Aspergillus niger N400 was studied on different simple and complex carbon sources. Sugar beet pulp was found to be an inducer of three arabinan degrading enzymes (-l-arabinofuranosidase A, -l-arabinofuranosidase B and endoarabinase). These enzymes were purified from A. niger culture fluid after growth of the fungus in medium employing sugar beet pulp as the carbon source and were characterised both physico-chemically (Mw 83 000, 67 000, 43 000 Da and, pI 3.3, 3.5 and 3.0 for -l-arabinofuranosidases A and B and endo-arabinase, respectively) and kinetically (K _m on p-nitrophenyl--l-arabinofuranoside 0.68 and 0.52 mM for -l-arabinofuranosidases A and B, resp.; K _m on sugar beet arabinan 0.24 and 3.7 g/l for -l-arabinofuranosidase B and endoarabinase, resp.). The amino acid compositions of the three enzymes were determined also. The enzymic properties were compared with those of arabinases purified from a commerical A. niger enzyme preparation. Differences were found though the kinetic data suggest considerable similarity between the enzymes from the different sources. Antibodies raised in mice against the three enzymes were found to be highly specific and no crossreactivity with other proteins present in culture filtrates was observed. A mixture of these antibodies has been used to analyze specific induction of these individual enzymes on simple and complex substrates by Western blotting.Abbreviation PNA p-nitrophenyl--l-arabinofuranoside     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Reversion of l-lysine inhibition of penicillin G biosynthesis by 6-oxopiperidine-2-carboxylic acid in Penicillium chrysogenum PQ-96

Summary 6-Oxopiperidine-2-carboxylic acid (OCA; cyclic -aminoadipic acid) reverses the l-lysine inhibition of penicillin G production by Penicillium chrysogenum PQ-96. The reaction probably depends on the recovery of l--aminoadipic acid for penicillin G production from OCA. Offprint requests to: W. Kurzkowski     

Fungal thermostable α-dialkylamino acid aminotransferase: occurrence,purification and characterization

-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K _m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K _i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyrate - PLP pyridoxal 5-phosphate     

Assay of 6-oxopiperidine-2-carboxylic acid in fermentations of Penicillium chrysogenum PQ-96

Summary 6-Oxopiperidine-2-carboxylic acid (OCA; cyclic -aminoadipic acid) was assayed in fermentations of Penicillium chrysogenum PQ-96 by isotachophoresis and HPLC. From 0.36 mg ml^–1 to 2.41 mg ml^–1 OCA was accumulated in fermentations during penicillin G biosynthesis. Offprint requests to: W. Kurzkowski     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Amino acid activation and polymerization at modular multienzymes in nonribosomal peptide biosynthesis

Summary The biosynthesis of microbial bioactive peptides is accomplished nonribosomally by large multifunctional enzymes consisting of linearly arranged building blocks of 1,000–1,500 amino acid residues. Each of these units acts as an independent enzyme which catalyzes the selection, activation, and in some cases modification (epimerization, N-methylation) of its cognate amino acid, as well as the elongation of the peptide product. The specific linkage of amino acid activating modules upon such polyenzymes defines the sequence of the peptide product. A series of functional domains could be identified upon an amino acid activating module which are involved in the sequential reactions in nonribosomal peptide biosynthesis.Abbrevations aaRS aminoacyl tRNA synthetase - GS1 gramicidin S synthetase 1 (phenylalanine racemase) - GS2 gramicidin S synthetase 2 - TY1 and 2 tyrocidine synthetase 1 and 2 - ACV [-(l--aminoadipyl)-l-cysteinyl-d-valine] - FITC fluorescein 5-isothiocyanate - FAB-, ESI-MS fast atom bombardment-, electrospray ionization-mass spectrometry - Pan 4-phosphopantetheine - NMR nuclear magnetic resonance - ACP acyl carrier protein - SAM S-adenosyl-l-methionine - CM carboxy-methyl - NES Nethylsuccinimido     

Mutationsvorgänge bei der Entstehung von Hämoglobinvarianten

Using recent information on the DNA code, the exact base replacements were ascertained for 46 amino acid substitutions in haemoglobine polypeptide chains. Transitions (replacements purine purine and pyrimidine pyrimidine) turned out to be significantly more frequent than expected under the assumption of random substitution, whereas transversions (replacements purine pyrimidine and pyrimidine purine) were less frequent. In 12 codons, 7 of the -chain, 3 of the -chain, and 2 of the -chain, two triplets each were excluded.

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Direktor: Prof. Dr. F. Vogel

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Die Untersuchungen der Verfasser zum Mutationsproblem werden von der Deutschen Forschungsgemeinschaft unterstützt.     

Lokalisierung von Aminos?ureaustauschen bei Nitritmutanten des Tabakmosaikvirus

Summary The positions of amino acid exchanges within eleven mutants of tobacco mosaic virus (TMV) isolated after treatment with nitrous acid have been localized. The protein chemical results are given in Tables 1–11 in detail and summarized in Table 12.In the discussion the localized amino acid exchanges of 36 mutants which arose spontaneously or were isolated after nitrite treatment or fluorouracil incorporation and whose primary protein structure have been described in this and earlier papers were compared with the current codon dictionary. All of the amino acid exchanges can be explained by the postulation that alteration of only one of the three nucleotides within a codon (Table 13) has occurred. Of the 26 amino acid exchanges localized in mutants isolated after treatment with nitrous acid all but two (isoleucinmethionine and glutamic acidaspartic acid) are compatible with the nucleotide alterations CU and AG postulated for the action of nitrous acid on TMV-RNA.The distribution of the localized amino acid exchanges along the protein chain of TMV is shown in Fig. 1.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

The effects of reduced nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot

The addition of l-glutamine, -alanine or l-glutamic acid strongly stimulates somatic embryo formation in carrot, not only in the number of somatic embryos formed but also with respect to their development. The effects of the amino acids on somatic embryogenesis were stronger than that of ammonium ion. In particular, l-glutamine strongly stimulated the development of somatic embryos. To clarify the different effects of amino acids and ammonium ion, the activity of glutamine synthetase (GS; EC 6.3.1.2), a key enzyme involved in nitrogen assimilation, was measured. Its activity decreased during the later stages of embryo development.Abbreviations -Ala -alanine - Glu l-glutamic acid - Gln l-glutamine - 2,4-D 2, 4-dichlorophenoxyacetic acid - -GHA l-glutamic acid -monohydroxamate - GS glutamine synthetase - MS medium Murashige & Skoog (1962) medium - MS-NH₄ medium MS medium without NH₄NO₃ - MS+NH₄ medium MS-NH₄ medium with 10 mM NH₄Cl - MS+ala medium MS-NH₄ medium with 10 mM -alanine - MS+GLU medium MS-NH₄ medium with 10 mM l-glutamic acid - MS+GLN medium MS-NH₄ medium with 10 mM l-glutamine - NIR nitrite reductase - NR nitrate reductase     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

Cation effects on the conformations of muscle and non-muscle α-actinins

We examined the effects of changing KCl concentration on the secondary structures of -actinins using circular dichroism (CD), 1,1-bis(4-anilino) naphthalene-5,5-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH₂PO₄, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in -helix conformation for brain, platelet and heart -actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet -actinin, but had no effect on heart -actinin. bisANS fluorescence of all three -actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca²⁺ or Mg²⁺ did not have significant effects on the bisANS fluorescence of any -actinin. Digestion of brain, platelet and heart -actinins with -chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca²⁺ or Mg²⁺ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that -actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different -actinins.     

Nikolaj Ivanovič Vavilov (26. 11. 1887–2. 8. 1942)

Ohne ZusammenfassungGekürzte Fassung eines Vortrages, gehalten auf der Sitzung der Sektion Genetik der Moskauer Gesellschaft der Naturforscher am 26. 11. 1957, auf der gemeinsamen Sitzung der Geographischen Gesellschaft der UdSSR und der Allunionsbotanischen Gesellschaft am 6. 12. 1957 in Leningrad und am 11. 2. 1958 in Moskau vor Studenten der Timirjasev-Akademie. Aus dem Russischen übersetzt von I.Grebenikov undF. Scholz, Gatersleben. F. Kh. Bakhteyev     

Properties of Aspergillus japonicus β-fructofuranosidase immobilized on porous silica

-Fructofuranosidase from Aspergillus japonicus MU-2, which produces fructo-oligosaccharides (1-kestose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside); and nystose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside) from sucrose, was immobilized, covalently with glutaraldehyde onto alkylamine porous silica, at high efficiency (64%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. After immobilization, the enzyme's stabilities to temperature, metal ions and proteolysis were improved, while its optimum pH and temperature were unchanged. The highest efficiency of continuous production of fructo-oligosaccharides (more than 60%), using a column packed with the immobilized enzyme, was obtained at 40% to 50% (w/v) sucrose. The half-life of the column during long-term continuous operation at 55°C was 29 days.     

Effects of α-galactosidase digestion on lectin staining in human pancreas

Summary Effects of -galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B₄(GSAI-B₄) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B₄ positive acinar cells. -Galactosidase digestion following -galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B₄ positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple -galactosidase digestion as well as sequential digestion with - and -galactosidase. However, when -l-fucosidase digestion procedure was inserted between - and -galactosidase digestion, UEA-I staining imparted by -galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B₄ positive acinar cells. Furthermore, after sequential digestion with -galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B₄ positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion. These results confirmed that the -galactosidase induced GSA-II reactivity and the fucosidase induced PNA reactivity are due to precursors of different kinds of blood-group determinants and suggest that at least two kinds of B antigen determinants, i.e. Gal(1-3)[Fuc(1-2)]Gal(1-3,4)GlcNac and Gal(1-3)-[Fuc(1-2)]Gal(1-3)GalNAc are produced in GSAI-B₄ positive acinar cells. The synthesis of the latter type of B antigen is assumed to be controlled under the secretory gene in human pancreas.Abbreviation GalNAc N-acetyl-d-galactosamine - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - NeuNAc N-acetylneuraminic acid (sialic acid)     

Oligomerization of 3′-amino-3′-deoxyguanosine-5′-phosphorimidazolidate on a d(CpCpCpCpC) template

Summary 3-Amino-3-deoxyguanosine-5-phosphorimidazolidate (ImpGnh ₂) oligomerizes more rapidly and regiospecifically than related nucleotide derivatives on a d(CpCpCpCpC) template. The greater nucleophilicity of the amino group leads to efficient oligomerization even when the structure of the double-helical complex formed by the template and the substrate is not optimal for reaction. The use of amine-containing analogues should permit us to develop models of potentially prebiotic polymerization reactions that cannot be studied easily using natural nucleotides.