Band 31, Heft 7-8

In order to evaluate the distribution of molecular oxygen in biological systems, the oxygen solubility (ml/ml atm) at 37 °C in aqueous solutions of thirty organic substances with different concentrations was measured by the classical Van Slyke principle. The oxygen solubility always (with the exception of hemoglobin) decreases exponentially with increasing concentration of the respective substance. In all cases this behavior is described highly significant by a simple empiric exponential function, when the substance concentration as well as the oxygen solubility of pure water is given. The influence of one substance on oxygen solubility contributes additively to the over-all solubility value of a solution of several dissolved substances. Changes in oxygen solubility and, therefore, oxygen distribution in aqueous solutions may be related to three main phenomena: 1. restriction of the free water volume by the specific partial volume of a dissolved substance (e. g. urea solutions), 2. existence of non-solvent water for oxygen (especially in solutions of amino acids), 3. adsorption of oxygen at the interphase in a heterogenous system (e. g. protein solutions).

Proton Transfer, Pyrazole, Outer-Sphere Coordination Proton transfer in pyrazole systems involving interaction with a paramagnetic transition metal complex has been studied with 1 H and 13 C NMR techniques. Kinetics of the ligand exchange in the pyrazole complex of bis-(acetylacetonato) nickel (II) has been investigated, and hyperfine coupling constants for the interaction of the nickel unpaired electron with the ligand nuclei in the complex have been determined. Catalytic admixtures of bis-(acetylacetonato) nickel (II) are shown to modify the mechanism of the proton transfer in pyrazole. The spectral data are discussed in terms of outer-sphere coordination and cooperative proton transfer effects.

Ultraviolet and infrared absorption spectroscopy, in aqueous and non-aqueous media, have been employed to study the tautomerism of 9-substituted isoguanines, including the nucleoside iso­ guanosine. With the aid of a series of model compounds, it was shown that 9-substituted isogua­ nines, and isoguanosine, in aqueous medium are predominantly in the form N(1)H,2-keto-6-amino. In dioxane solution the tautomeric equilibrium is shifted in the direction of the enol form. The shift towards this form is accentuated for those analogues in which the exocyclic amino group is methylated. With the aid of N 6 ,N 6 ,9-trimethylisoguanine, and its 9-octyl analogue, the tautomeric constant was studied as a function of concentration, temperature, and solvent polarity, and the results applied to evaluate the tautomeric equilibria of 9-methylisoguanine and isoguanosine as a function of these variables. In general the enol form is favoured by a decrease in solvent polarity, by a decrease in concentration in dioxane, or an increase in temperature in chloroform solution. Syntheses are described for several N 6 -amino and methylamino derivatives of 2-methoxy-9-methylpurine, and 3-methyl-5-oxo-7,8-dihydroimidazo (2,1-i) purine, which served as an analogue of the unavailable 1,9-dimethylisoguanine.

The results of MO INDO calculations of some thymine radicals are presented. These include as well as the hydrogen addition radicals the hydroxyl addition radicals to C5 and to C6 of the C5=C6 double bond of the thymine molecule. The radicals resulting from hydrogen abstraction from the methyl group and from the nitrogen N1 are also studied. Except for radicals where the main spin density is localized on nitrogen atoms, the agreement between experimental and theoretical couplings is satisfactory.

Recoillabeling of amino acids can be accomplished in good radiochemical yield in solid state only. Labeling in solution or in mixture with charcoal or silicagel results in formation of a variety of different products.

The structure for α-chitin originally proposed by Carlström is generally considered to be the true structure. However, it fails to account for several remaining problems of chitin structure which include a complete explanation of the infrared spectrum and also the different properties of a-and β-chitins. W e have reexamined the structure by X-ray diffraction using automatic rigid subunit least-squares refinement and also the difference Fourier method. The R-value was reduced from 0.31 to 0.22 by a number of small modifications to the structure proposed by Carlström. Conside­ration of symmetry suggests that two distinct types of statistical modifications could be present in the structure, both of which would allow complete intersheet hydrogen bonding between O 6 H groups within the general framework of Carlström structure. The X-ray results give some support to one of these modifications. However, it is predictable that both would affect the X-ray diffraction only marginally so that a clear proof lies beyond the present sensitivity of the method. The proposed extra hydrogen bonding in a-chitin provides the first clear explanation for the different properties of these kinds of chitin.

The photochemically activatable heterobifunctional reagent 5-azido-3-nitro-ω-bromo-acetophenone (3) was synthesized by condensation of 3,5-dinitrobenzoyl chloride with diethyl malonate, acid-catalyzed decarboxylation of the formed malonester derivative 1 to 3,5-dinitro-acetophenone (2a), selective reduction of one nitro group in 2a to 5-amino-3-nitro-acetophenone (2b) and diazotization to 2c. Nucleophilic displacement of the diazonium group in 2c by sodium azide forms 5-azido-3-nitro-acetophenone (2d) which gives 3 after bromination. Compound 3 is photochemically labile and forms an highly reactive nitrene during ultraviolet irradiation. Since compound 3 is able to alkylate amino-or mercapto-groups of amino acids or nucleosides via its bromoacetyl residue, the bifunctional reagent 3 should cross link proteins or nucleic acids after nitrene generation.

Pb 2+ Uptake Short-term experiments were carried out to understand the mechanism of absorption and transport of Pb 2+ in young pea seedlings. It was found that considerable fraction of Pb 2+ absorbed could be exchanged with Ca 2+ in the medium. Furthermore, increasing concentrations of Ca 2+ , Mg 2+ , or K+ in the absorption medium significantly reduced the absorption of Pb 2+ . Uptake of Pb 2+ was largely non-metabolic, as shown by its insensitivity to temperature changes and the presence of a metabolic inhibitor. Presence of Pb 2+ in the medium was highly inihibitory to the absorption of micronutrients, viz., Fe2+, Mn 2+ , and Zn 2+ . The inhibition appeared to be of a physical nature, like blocking the entry or binding of the ions to the ion-carrier.

A new cyanogenic glycoside was isolated from Sorbaria arborea L. (Rosaceae) and identified as 2-β-ᴅ-glucopyranosyloxy-4-p-hydroxybenzoyloxy-3-methylenbutyronitrile (cardiospermin-p-hydroxybenzoate) mainly by 1 H-NMR-spectroscopy. The new glycoside is atypical for this family known for its mandelonitrileglycosides.

Sesquiterpene hydrocarbons of the hexane extract from Dumortiera hirsuta were analyzed by a combining method of gas chromatography and mass spectrometry, and ten sesquiterpene hydrocarbons, (δ-elemene, α-copaene, β-bourbonene, β-elemene, γ-elemene, β-pompene, γ-muurolene, α-muurolene, bazzanene and cuparene, were identified.

Twentyone anthraquinones have been found in the roots of Digitalis schischkinii (Ivan.) of which the following are new natural pigments: 4,5-dihydroxydigitolutein, 5-hydroxy-and 4,5-dihydroxy-nordigitolutein.

[Ring B-U- 14 C]-5,7,3′,4′-tetrahydroxyflavanone-7-O-glucoside could be synthesized and a new way of flavanone-degradation is demonstrated. The B-ring is split off under formation of 5,7-dihydroxychromone- 7-O-glucoside and [ring-U- 14 C]T,2,4-trihydroxybenzene.

Two reaction products of thallium-III-trifluoracetate catalyzed degradation of 5,7,3′,4′-tetra-hydroxyflavanone could be isolated and identified as 5,7-dihydroxychromone and 1,2,4-trihydroxy-benzene.

The common A-dihydrobiliverdin chromophor proposed to be present in both phycocyanin and phytochrome P r has been characterized by using octaethylbiliverdin and its A-dihydro derivative as model compounds. The UV-vis spectra for the free bases, cations, anions and zinc complexes are reported, and the related protonation-deprotonation equilibria have been studied. The data support the postulated structures for the biliproteid chromophor, and at the same time they indicate A-dihydrooctaethylbiliverdin to be a useful model.

The synthesis of the protected pentapeptide tert-butyloxycarbonyl-Gln-Ile-Met-Lys (X) -Gly-p-nitrobenzylester (X = benzyloxycarbonyl, 3-chlor-benzyloxycarbonyl) which is part of the carboxylend of a partial sequence of the active center of the streptococcal proteinase is described. Side reactions are observed if the tert-butyloxycarbonyl-protective group is cleaved by trifluoroacetic acid, not with HCl/dioxane. Obviously the presence of methionine is responsible for the formation of by products. Formation of methioninesulfoxide or tert-butylsulfoniummethionine could not be proved. tert-butyloxycarbonyl-Gln-1-hydroxybenzotriazolylester was obtained in a crystalline state from the reaction of ferf-butyloxycarbonyl-Gln, dicyclohexylcarbodiimid and 1-hydroxybenzotriazol.

Two multiple forms of α-glucan phosphorylase which migrate about half way in polyacryl-amide-gel electrophoresis (named “slow“ and “fast” isozyme), were isolated by combined chromatography and preparative electrophoresis after freezing the tissue from freshly harvested and from sprouting potato tubers respectively. Depending on the primer used for the synthesis reaction their pH optimum varied between 5.2 and 6.0 and the optimum temperature was 30 and 35 °C. The isoelectric point for the slow isozyme was at pH 5.0±0.1 and for the fast isozyme, pH 5.5±0.1, the molecular weights were 209 000±10 000 and 165 000±5000 and for their subunits 104 000±4000 and 40 000±2000 respectively. Both isozymes were inhibited by Hg 2+ , Ag + and p-chloromercuro-benzoate (p-CMB). Fe 2+ ions inhibited them partially. Mg 2+ , Mn 2+ and sulfhydryl compounds activated both. K m values for the slow and fast isozymes with glucose-l-phosphate in presence of soluble starch was 6.7 and 8.0 mM, of amylose 14.3 and 20.0 mм and of glycogen 22.2 and 40.0 mм respectively. The affinity of the primer for the slow and the fast isozymes were as follows: soluble starch 0.5 and 1.0 mм, amylose 2.6 and 3.8 mм, glycogen 6.2 and 7.7 mм respectively. K m values of phosphorolysis with soluble starch was 0.8 and 0.5 mм, with amylose was 3.1 and 1.1 mм, and with glycogen was 6.5 and 1.3 mм respectively. As substrate and primer the soluble starch was superior and the glycogen was inferior. Amylose was in between. Kinetic parameters suggested the existence of α-glucan phosphorylase isozymes with different specificities: the slow one being more active in the direction of starch synthesis and the fast isozyme degrading faster the polyglucans. These observations suggest that the polyglucan synthesis and degradation in potato tubers may be regulated by the change in the proportion of slow and fast isozymes.

The blue-green alga Anacystis nidulans (strain L 1402-1) was grown at + 37 °C in air (0.03 vol.% CO 2 and in air enriched with 3.0 vol.% CO 2 . The effects of several inhibitors on the activity of aminotransferases, 14 CO 2 fixation and radioactive photosynthetic products of Anacystis were studied. No serine-pyruvate aminotransferase activity could be found in 10 -2 м isonicotinyl hydrazide (INH) ; under the influence of this inhibitor aspartate and alanine aminotransferase were decreased about 49% respectively 17.6%. Serine-pyruvate and alanine aminotransferase activity decreased to more than 50% in 10 -3 м glyoxalbisulfite. The obtained inhibitory effect of 10 -4 м HPMS on serine-piruvate aminotransferase (35%) was stronger than on the other aminotransferases. DCMU (5 × 10 -6 м) inhibition on alanine aminotransferase activity was 83.7%. Under the influence of 10 -3 м glyoxalbisulfite no 14 C-labelled amino acids could be detected after 5 min photosynthesis; 14C-labelling of phosphoenolpyruvate, malate, phosphoglycolate and glycolic acid increased. Isonicotinyl hydrazide (10 -2 м) caused in comparison to the control experiment a lower radioactivity in aspartate, glutamate and phosphoenolpyruvate. The results are discussed with reference to the operation of the glycolate pathway and a carboxylation reaction of phosphoenolpyruvate in the blue-green alga Anacystis nidulans.

Intermolecular interactions between chloroplast lipids and a polypeptide fraction from thylakoids were investigated by far ultraviolet circular dichroism. The polypeptide fraction was isolated from dodecyl sulfate-containing buffers. It exhibits an average molecular weight of 24 000. The circular dichroism of this polypeptide fraction measured as mean residue ellipticity is greater in the presence of sodium dodecyl sulfate than in the absence of this detergent. This effect is reversible. Addition of sulfoquinovosyl diglyceride to the dodecyl sulfate-free solution of the polypeptide also causes an increase of the circular dichroism. This increase was only observed in the pH-range between 6.9 and 7.4. The effect of dodecyl sulfate or sulfolipid on the circular dichroism is interpreted to indicate an increase of α-helix content. Monogalactosyl diglyceride, digalactosyl di­glyceride and phosphatidyl glycerol gave no reaction. The attempt to obtain a conformational analysis of the polypeptide in the different states did not yield an entirely satisfactory result. Antisera to sulfolipid inhibit photosynthetic electron transport of stroma-freed chloroplasts in the region of light reaction I. This inhibition is restricted to the same pH-range as the non-covalent binding of sulfolipid to the polypeptides. It appears that in the cell membrane-bound metabolic processes are regulated by this pH-dependence of the sulfolipid-polypetide interactions.

1. A method for the reproducible preparation of CF 1 -depleted thylakoid membranes was established. By repeated washes with pyrophosphate solution and subsequent treatment with sucrose media, containing micromolar concentrations of Ca 2+ or Mg 2+ , different degrees of CF 1 depletion and uncoupling were adjusted. 2. The membranes were completely uncoupled when about 50% CF 1 was removed. Re-coupling with isolated CF 1 was maximal at this point. It decreased with progressive CF 1 extraction. 3. The re-attached CF 1 particles regained their ability of energy-dependent adenine nucleotide exchange, an essential reaction in photophosphorylation. 4. CF 1 -depleted membranes showed two main morphological alterations, a. unstacknig of the grana, and b. a perforation of the thylakoid membranes in those chloroplasts which showed a poor recoupling activity. Whereas CF 1 -depletion and unstacking were found to be reversible, the formation of holes was an irreversible effect. 5. The decreasing re-coupling activity was discussed in context to the irreversible perforation of the thylakoid membranes.

Isolated spinach chloroplasts (type A complete) were used to study the changes in the photo-chemical activities upon storage in order to establish optimum conditions for prolonged storage. Chloroplasts stored at - 5 °C were found to retain over 70% of their photosynthetic electron transport from H 2 O even after 5 days. Increases in the level of state 2 electron transport (due to uncoupling) with concomitant loss of state 3 phosphorylation activity was observed during the initial period of ageing. Addition of 1% bovine serum albumin decreased the level of uncoupling and maintained the phosphorylation activity for a longer period. Chloroplasts stored at 77 °K maintained their phosphorylation capacity for a period of 10 days even after repeated freezing and thawing. Photosystem I activity was found to be more stable over the whole ageing period of 15 days. Loss of plastoquinone may be responsible for the decrease in electron transport between photosystems II and I.

1. Development and fine structure of the chromoplasts in hips of Rosa rugosa have been in­ vestigated by electron and polarizing microscopy. 2. The chromoplasts develop from chloroplasts. During disorganization of the thylakoid system characteristic strutures become visible: ‘oblique’ grana, U-shaped thylakoids, and occassionally ‘thylakoid teeth’ as well as thylakoid anastomoses. - In this early stage, tubules appear in the plastid matrix. They are sometimes connected with membranes, especially with thylakoids. 3. In ripe hips the chromoplasts are packed with tubules. These chromoplast tubules, which attain lengths of up to at least 1.5 μm, are non-ramified and lie parallel to each other. Their diameter is variable, with a mean of 18 nm. They are polygonal in cross-section with electron-dense walls and electron-transparent cores. Association of ellipsoidal osmiophilic globules with the tubules can often be seen. Irregularly swollen remnants of former thylakoids are found in connection with these tubules. In spindle-shaped chromoplasts, the tubules are oriented parallel to the longitudinal axis. Positive birefringence and positive dichroism are observed. 4. The chemical composition of tubule fractions has been analyzed. The tubules contain appreciable amounts of carotenoids, phospho- and glycolipids, and proteins. 5. The origin and development of the tubules is discussed. It appears likely that reorganization of part of the thylakoid system and synthesis of carotenoids are involved in the formation of tubules. 6. The plastids in the yellow autumn leaves of Rosa rugosa contain globules but not tubules.

It is suggested that turgor pressure sensing in plant cells occurs via compression of the cell membranes; either at the plasmalemma where a pressure gradient exists, or the tonoplast due to the pressure developed inside the cell. Considerations of electro-mechanical forces in electrical breakdown of cells suggests that significant changes in the thickness of some regions of the membrane can indeed occur. It is readily envisaged how such changes in membrane thickness can be coupled to changes in active transport processes.

Ribonucleoprotein Particles Experiments indicate that eggs and embryos of the North Sea sea urchin Echinus esculentus are excellent material to investigate the regulation of protein synthesis before and after fertilization. The dormant egg contains several proteins which are absent from embryos. Such additional proteins have been detected on the small ribosomal subunits, in the salt wash of whole ribosomes, and as­ sociated with cytoplasmic messenger ribonucleoprotein particles.

A screening procedure is presented which allows the isolation of yeast mutants (typ tlr) with highly efficient utilization of exogenous deoxythymidine-5′-monophosphate (5′-dTMP) (>50% ). Data are given concerning the phenomenon of 5′-dTMP utilization in general: (i) The ability of S. cerevisiae to incorporate exogenous 5′-dTMP was found to already be a wild type feature of this yeast, i. e. apparently not to be due to any mutation such as typ , tup, tmp per or tum. Consequently these mutations are interpreted as amplifiers of a pre-given wild type potency. So far eight stages of 5′-dTMP utilization were detected as classified by the optimal 5′-dTMP requirement, with 5′-dTMP biosynthesis blocked, of the corresponding mutant strains isolated. All of them fit well into a mathematical series of the type “2 n × 1.5” (n = 0, 1, 2, … , 11), where the product term for n = 11 represents the 5′-dTMP requirement (μg/ml) of the best 5′-dTMP utilizing wild type strain found, (ii) Amplification of the 5′-dTMP utilizing potency obviously is due to any genetically determined alteration of the yeast 5′-dTMP uptaking principle itself or of physiological processes accompanying the monophosphate’s uptake, (iii) The functioning of 5′-dTMP uptake requires acidic (≦ pH 6) conditions in the yeast cell’s outer environment, (iv) Some yeast typ and typ tlr mutants were found to exhibit a more or less pronounced sensitivity towards exogenously offered 5′dTM P. The response of a sensitive strain towards inhibitory concentrations of the nucleotide apparently is co-conditioned by the presence or absence of thymidylate biosynthesis. With 5′-dTMP biosynthesis blocked the 5′-dTMP mediated inhibition is a permanent one and finally leads to the death of a cell. With a functioning thymidylate biosynthesis, in contrast, the inhibition is only temporary, (v) Yeast typ or typ tlr strains were observed to dephosphorylate exogenous 5′-dTMP to thymidine due to a phosphatase activity which cannot be eliminated at pH 7 + 70 mм inorganic phosphate conditions in the growth medium. This 5′-dTMP cleavage obviously occurs outside the cell and does not seem to be correlated both to the monophosphate’s uptake and to the phenomenon of 5′-dTMP sensitivity. The destruction of 5′-dTMP does not disturb (5′-dTMP) DNA-specific labelling.

A correlation has been found between the reactivity in the alkaline hydrolysis reaction of a new series of organophosphates and their ability or disability to produce the clinical manifestation of delayed neurotoxicity in hen. At a dose of 1000 mg/kg only compounds exceeding a lower limit of reactivity (about 6×10 -2 м -1 s -1 ) are active.

After incubation of Saccharomyces cells in [ 3 H] thiopyro­ nine solution the photodynamically high effective dye was detected only to about 1% in the nucleus of the cell. 99% of the dye is distributed in the cytoplasm.

Calf thymus DNA was irradiated in oxygen saturated 0.01 N NaCl solution with 2 μs pulses of 15 MeV electrons. By monitoring the decrease of light scattering intensity after the pulse, two modes of decrease were detected: The fast decrease (τ½ ≈ 0.8 ms) is ascribed to the separation of DNA fragments produced by double strand breaks at positions directly opposite to each other. The slow decrease (τ½ ≈ 8s) is attributed to the detachment of segments generated by single strand breaks at sites on the alternate strands being separated by about 10 nucleotide units.

Levels of the purine nucleoside triphosphates are de­ creasing towards the end of log phase growth of Streptomyces hydrogenans. Induction of 20β-hydroxysteroid dehy-drogenase by addition of 11β,21-dihydroxy-4,17 (20) -pregna-dien-3-one to the growth medium leads to a pronounced drop in purine nucleoside triphosphate levels with is irreversible in contrast to the initial loss and later accumulation of RNA.

Molluscan Embryo, Sulfated Acid Mucopolysaccharide, Sulfated Protein An attempt has been made to partially characterize the substance responsible for the rhythmic incorporation of 35 S (inorganic sulfate) in the course of the cell cycle in early Limnaea embryos. This trichloroacetic acid (TCA) insoluble substance is partly pronase sensitive, and dissociable into two fractions after treatment with NaCl. One of these re­ mains TCA-insoluble while the other is TCA soluble but precipitable by cetyl pyridinium chloride. Thus, unlike in some other higher organisms, the major part of the inorganic sulfate is incorporated here into a fraction which is not a simple mucopolysaccharide, but is more likely to be a protein-mucopolysaccharide complex, rather like the chondro-mucoprotein of chick-embryo cartilage.

Honey Bee, Rectal Pads, Function According to earlier investigations young bees show a rectal fluid hypo-osmotic to the haemolymph. It had been assumed, however, that in certain situations honey bees have to be economical with water and simultaneously form a rectal fluid hyperosmotic to the haemolymph. The latter has been confirmed by the present investigation.