Band 31, Heft 5-6

Proton transfer in aqueous solutions of 3-hydroxypyridin (3HP) has been studied by tempera­ture jump relaxation and ultrasound absorption techniques. Two chemical relaxations have been observed. In the range pK 1 <pH<pK 2 the slower of the two processes corresponds to proton transfer between the N and O atoms. A mechanism is proposed which describes the pH and con­centration dependence of the relaxation time measured. Proton transfer involves two types of pro­cesses which operate in parallel: I. pseudomonomolecular, by consecutive diffusion-controlled protonation and deprotonation steps, and 2. bimolecular by diffusion-controlled proton transfer between the neutral 3HP molecule and its corresponding cation or anion. There is no evidence for direct monomolecular proton transfer within the hydration shell of the molecule (k → ~ k ← < 2 × 1O 3 sec -1 ).

The formation of H-aduition radicals in monocrystals of 9-methyl adenine, deoxyadenosine monohydrate, and adenosine hydrochloride by irradiation with X-rays has been studied using ESR spectroscopy. In 9-methyl adenine and adenosine-HCl, hydrogen atoms add exclusively to position C8 of the imidazole part of the purine ring. On the other hand, in deoxyadenosine · H 2 O crystals, H-addition radicals at position C2 of the pyrimidine part of the purine ring occur together with H-addition radicals at position C8. Both radicals could be isolated by using their differential stability under warming or illumination with light. The C8-addition radical is characterized by two equivalent β-protons of 38.0 ± 0.5 G and the C2-addition radical by two non-equivalent β-protons of 34.0 ±1.0 and 50.0 ± 1.0 G. The nitrogen splittings perpendicular to the purine ring are 27.0 and 6.6 G for the C2-addition radical and 20.5 and 9.2 G for the C2-addition radical. The coupling constants of both radicals are in agreement with INDO calculations. It is further shown that the added hydrogen atom comes partly from the hydrogen bonding scheme and partly from the non­exchangeable hydrogens for the C8-addition radical. Together with additional experiments on polycrystalline samples, these results lead to the conclusion that hydrogen atoms add non-selectively to C2 and C8 of the neutral molecules, whereas protons add predominantly to C8 of anion radicals. This is supported by Hückel molecular orbital calculations.

Undecapeptide of Ferricytochrome c, Electron Spin Resonance, Mössbauer Spectroscopy, Visible Spectroscopy The ESR, Mössbauer, and visible spectra of the undecapeptide of ferricytochrome c (H P p) were measured for both the solid peptide and solutions of the peptide at pH 1.5, 7, and 10. At the low pH, the iron exists in the H Pp in the high-spin species. At neutral and alkaline pHs, the low-spin ferric species predominate. The results of these measurements support the previous sug­gestions concerning the ligands in the fifth and sixth position of HPp at the neutral and alkaline pHs. The spectra of lyophilized preparations of HPp show the presence of both high and low-spin ferric species. The study of the Mössbauer spectra of the lyohilized preparations as function of temerature shows that there exists a spin-spin equilibrium between the two spin states. The relative amounts of the two components vary with the preparation. In these preparations there exist two components, one of which exists in the equilibrium mixture and the second in the high-spin form.

The α -haemocyanin molecules of Helix pomatia were decomposed into halves and studied in solution by small-angle X-ray scattering. The following parameters of the molecule could be ob­tained : radius of gyration, volume, molecular weight, overall shape and dimensions of the molecule. With small-angle X-ray scattering fluctuations of the electron density within the protein cause parasitic scattering at larger angles. According to Stuhrmann and Kirste it is possible to eliminate it mathematically by varying the electron density of the buffer. For this purpose different quan­tities of glycerol respectively saccharose were added to the solvent to study the scattering of α -haemocyanin halves in solvents of varied electron density. The change of the isopotential specific volume of haemocyanin and the strong increase of the statistical errors of its scattering by de­creasing of the excess scattering of solution over solvent per unit volume did not allow an ap­plication of the method of Stuhrmann and Kirste. The data obtained for α -haemocanin halves in different solvents are given. Besides also the sedimentation of the α -haemocyanin halves were studied in solutions containing varied amounts of glycerol and saccharose. An attempt was made to calculate the change of the partial specific volume of haemocyanin by adding glycerol or saccharose.

The synergistic effects of four sucrose substitutes, polysorbate and five artificial sweeteners were studied in vitro on growth pattern and acid production of seven glucose-grown Streptococcus mutans strains, representing the five serological groups after Bratthall. Four distinct growth pat­ terns during glucose fermentation were observed: high rate of growth with low acid production, moderate growth rate with moderate acid production, moderate growth rate with high acid pro­duction, and slow rate of growth with moderate acid production. Depending on the strain used, the final OD at 546 nm ranged from 0.55 to 0.99 and the final pH of the medium varied between 4.65 and 4.15. While added sucrose substitutes, with exceptions, usually enhanced growth rate, most artificial sweeteners suppressed or, at higher concentrations, even inhibited growth of S. mutans-, addition of polysorbate to the medium always increased growth rate of S. mutans significantly. The presence of sucrose substitutes during glucose fermentation had no effect on final pH of the medium, but addition of artificial sweeteners, especially sodium saccharin, elevated final pH up to 1.8 units. The observed physiological patterns and differences within the several strains of S. mutans during glucose fermentation in vitro do not necessarily relate to the five serological groups of the species.

d (+) and 1(-) Methyl-2-chloro-3-(4-chlorophenyl)-propionate (common name chlorfenprop-methyl; trade mark Bidisin ® ) were prepared and their herbicidal activities studied on Avena fatua L. and Avena sativa L. (“Flämingskrone”). The 1(-) enantiomer was found to be twice as active as the racemate; the d (+) form almost inactive.

Vitamin B 12 , Methylcorrinoids, Coα/Coβ-isomerism, E. coli Activity In 2-methyladenyl-(Coβ-methyI) -cobamide and in adenyl-(Coβ-methyl)-cobamide the nucleotide base is coordinated to the cobalt atom in neutral and weak acidic aqueous solutions (in the cor­ responding adenosylcorrinoids the nucleotide base is not coordinated). 2-Methylthioadenyl-(Coβ-methyl)-cobamide resembles, with regard to the coordination of the nucleotide base, the benz-imidazole-corrinoids. The partial synthesis via cobalt (I) corrinoids results in a variable proportion: (Coα-methyl) iso­ mer/(Coβ-methyl) isomer, e .g . 7/93 (cobalamin) and 50/50 (p-cresylcobamide). This proportion is generally low, if in the corresponding cyanocorrinoid the nucleotide base is firmly coordinated; it is high, if the coordination in the corresponding cyanocorrinoid is weak or absent. These results are compatible with the assumption that in the cobalt (I) corrinoids the nucleotide base is to a certain extent coordinated. In Escherichia coli 113-3 the examined (Coα-methyl)-corrinoids show a weaker bioactivity than the corresponding (Coβ-methyl) -corrinoids.

The preparation of a new photochemically-activatable polymer was accomplished by condensation of aminohexyl-6-yl-agarose with the N-hydroxysuccinimide ester of 5-azido-2-nitro-benzoic acid. The latter compound was obtained by diazotization of 5-amino-2-nitro-benzoic acid, exchange of the diazonium group through azide and condensation with N-hydroxysuccinimide. As shown for the photolysis on the monomer level, irradiation of the polymer led to the intermediate formation of a highly reactive nitrene, which is able to immobilize ligands e. g. ʟ-phenylalanine to the polymer.

The influence of Mg 2+ ions and temperature on the structure of the enzyme ribulose-1,5-bisphosphate carboxylase was investigated using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). The binding of ANS to the enzyme molecule caused a significant increase of fluorescence emission which was further enhanced by the addition of Mg 2+ . The temperature dependence of the fluorescence emission indicated a conformational change of the enzyme between 12 and 24 0 C. The Mg 2+ and the temperature effects were additive. ANS itself did not change the conformation of the enzyme. The influence of the substrates carbon dioxide and ribulose-1,5-bisphosphate, and the effect of the pH of the medium and of a sulfhydryl reducing reagent on fluorescence emission were analysed.

When photosynthetic membranes from Rhodospirillum rubrum, devoid of loosely bound small molecules and proteins, were passed through a French-pressure cell, the enzyme adenosine-5′-triphosphatase (EC 3.6.1.3.) (ATPase) was released into the soluble fraction. The solubilized ATPase was purified to homogeneity. In many respects it behaved like the enzyme purified by other workers, but it also hydrolyzed Mg-ATP with a small, but significant rate. Furthermore, it was much more stable. Maximal restoration of photophosphorylation in ATPase-depleted membranes was achieved by addition of about 1 mg purified ATPase per mg bacteriochlorophyll. For reconstitution of NAD + - photoreduction, about half of this amount was saturating.

6,7-Dihydroxycoumarin (Aesculetin) was found to be a substrate of rat liver Catechol-O-methylfransferase (COMT) (EC 2.1.1.6). Incubation of this substrate with S-Adenosyl-ʟ-[methyl- 14 C] methionine and/or S-Adenosyl- methionin-hydrogensulfate in the presence of COMT yields the highly fluorescent compounds 7-hydroxy-6-methoxycoumarin (Scopoletin) and 6-hydroxy-7-methoxycoumarin (Isoscopoletin) in the ration of about 2:1. The O-methylated products obtained from Aesculetin were identified after separation by thin layer chromatographpy mainly by the reversed isotope dilution technique. The fluorescence of the isolated methylethers was proportional to concentration within the range from 10 -5 - 10 -7 M. A reciprocal plot of activity versus substrate concentration gives a K m of 1 × 10 -6 M.

Aerobacter aerogenes mutant 62-1 AC requires high concentrations of 4-aminobenzoate for growth. The mutant accumulates N-glucosyl-4-aminobenzoate and has an intact 4-aminobenzoate synthetase (Bacher, Gilch, Rappold, and Lingens, Z. Naturforsch. 28c, 614 - 617 [1973]). On the other hand the ability of the mutant to synthesize dihydropteroate is markedly reduced. The dihydropteroate synthetase level of mutant 62-1 AC is 1% as compared to the parent strain. Spontaneous revertants of mutant 62-1 AC show wild type levels of dihydropteroate synthetase. We conclude that the requirement for 4-aminobenzoate in mutant 62-1 AC is due to poor utilization of 4-aminobenzoate as a consequence of the low level of dihydropteroate synthetase activity.

Antiserum against crystallized 20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was used for different immunodiffusion and immunoprecipitation tests to quantify the bacterial enzyme in cell-free supernatants of the microorganism. After immunoprecipitation and gel electrophoresis the molecular weight of the subunits of 20β-hydroxysteroid dehydrogenase was calculated to be 27 300 ± 700.

N-Methyl-N-β-chloroethyl-hydrazine-HCl and its benzaldehydhydrazone inhibit growth and multi­plication of yeast cells. The DNA-synthesis is partially blocked. After removal of the substances the rate of this synthesis is much more increased than that of RNA and protein. The both sub­ stances we tested are more effective than simple alkylhydrazines.

N-methyl-N-β-chloroethylhydrazine and its benzaldehydhydrazone inhibit the influx of [6- 3 H]- uracil and ʟ- [U- 14 C] leucin in yeast cells as well as the incorporation in RNA and protein. Experi­ments with synchronously and asynchronously growing cells showed the N-methyl-N-β-chloroethyl- hydrazine essentially influences the dividing phase while the benzaldehydhydrazone is more ef­fective in the growthphase. The effect of these two substances on respiration, glycolysis and the concentration of fructose-1,6-diphosphate, triose phosphate, and adenosine triphosphate is small.

A polypeptide fraction with the apparent molecular weight 11000 was isolated from stroma-freed chloroplasts from Anthirrhinum majus. An antiserum to this polypeptide fraction inhibits photosynthetic electron transport in chloroplasts from Nicotiana tabacum. The relative degree of inhibition is pH dependent and has its maximum at pH 7.4. The maximal inhibition observed was 93%. The dependence of the inhibition on the amount of antiserum yields a sigmoidal curve which hints at a cooperative effect. A calculation of the Hill interaction coefficient gave the value of 10. The inhibition occurs on the water splitting side of photosystem II between the sites of electron donation of tetramethyl benzidine and diphenylcarbazide. Tetramethyl benzidine donates its electrons before the site where diphenylcarbazide feeds in its electrons. Analysis of the steady state level of the variable fluorescence also indicates that the inhibition site is on the water splitting side of photosystem II. Tris-washed chloroplasts are equally inhibited by the antiserum and the inhibition is also observed in the presence of an inhibitor of photophosphorylation like dicyclo-hexyl carbodiimide and in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydra-zone (CCCP) which means that the inhibitory action is directed towards the electron transport chain. Valinomycin which is supposed to affect the cation permeability of the thylakoid membrane has no influence on the inhibitory action of the antiserum. The same is valid for gramicidin. Methylamine on the other hand can induce a state in the thylakoids in which the antiserum is not effective. If the antibodies are already adsorbed prior to the methylamine addition then the high inhibitory effect by the antiserum remains unchanged upon addition of methylamine. From the experiments it follows that a component from the vicinity of photosystem II is ac­cessible to antibodies that is, the component is located in the outer surface of the thylakoid mem­ brane. It appears that the inhibitory effect is produced in the course of the light reaction.

Extension growth of 1-cm segments of Avena coleoptiles (studied in liquid medium by means of a flow chamber) was anomalously increased after short anaerobic treatment. The normal growth rate in air-saturated medium without indole-3-acetic acid (IAA) was about 5 µm/min; the rate decreased under anaerobiosis to about 1 µm/min, followed by an increase up to 30 µm/min when aerobic conditions were restored. This “growth burst” continued for about 10 min; then the rate decreased to the initial value of 5 /tm/min. In the same segment growth enhancement could be induced repeatedly by anaerobic treatments. The phenomenon of growth burst after anaerobiosis may be named “anaerobiosis-aerobiosis-transition effect” (ANA-effect). The ANA-effect was also observed under conditions of acid-induced growth at pH 4. Further­ more it appeared in coleoptile segments whose growth rate had already been enhanced by IA A . Anaerobic pre-treatment not only stimulated extension growth but when applied before IAA addition also overcame the lag phase of IAA-induced growth enhancement. It is supposed that a compound essential for coleoptile growth is accumulated during anaero biosis; such compound together with oxygen may cause the observed growth burst. Possible candidates for this compound are discussed.

Tumor induction by Agrobacterium tumefaciens on Kalanchoe leaves is suppressed by charcoal mixed with the inoculum. The inhibition works best with a certain mass relation between bacteria and charcoal. The bacteria become adsorbed to the charcoal particles and in this state they are unable to induce tumors. If charcoal is added to bacteria-infected wounds only 15 min after infection it is not interfering with tumor formation. These results are supporting the hypothesis of sites in the wound to which Agrobacterium has to be attached to incite tumors.

Neighbouring pyramidal cells in the mouse cortex sometimes have different densities of dentritic spines. This was shown by a quantitative analysis of 10 neurons. For this, a method was worked out which corrects for the spines hidden behind (and in front of) the dendrite. The main result is that there is a positive correlation between the spine densities on different parts of the dendritic tree of one neuron. The possible functional meaning of these findings is briefly discussed.

Lowering of extracellular Ca 2+ -concentration from 14 × 10 -3 м to ca. 10 -6 м (using 1 mм ethyleneglycol bis β-Laminoethylether) -N,N′-tetraacetic acid (EGTA)) results in weaker light adap­tation by a 2 s light exposure and faster dark adaptation (half time 45 ±1%) of crayfish visual cells.

Three stable free radicals have been prepared which are akin to 5-aziridino-2,4-dinitrobenzamide (CB 1954) ; these compounds all contain a nitroxide function. The metabolism and excretion of two such compounds in mice has been monitored by electron spin resonance (ESR) spectroscopy and compared with that of the simpler nitioxide, 4-keto-2,2,6,6-tetramethylpiperidino-1-oxyl (tempone).

An isoflavonoid phytoalexin isolated from the fungusinfected leaves of Trijolium hybridum has been identified as 7,4′-dimethoxy-2′-hydroxyisoflavan.

A Cholinesterase deriving from the hemolymph of the mussel Mytilus edulis was partially purified by use of gel-permeation and ion-exchange chromatography; the specifity to different substrates or inhibitors indicates clearly the occurrence of a “true” acetylcholinesterase.

The superposition eye of Cloeon dipterum is a clear zone type of compound eye. The central ommatidia have two rhabdomes, at different levels, a small apical and a main basal one. The number of basal rhabdomes is greater than the number of dioptric apparatuses. This and the role of the retinula cell processes is discussed with regard to super­ position or light-guide theory.

Valine-Isoleucine Interaction, Tobacco Protoplasts ʟ-valine inhibits the division of haploid tobacco mesophyll protoplasts. This inhibition is reversed by simultaneous addition of ʟ-isoleucine to the culture medium.