Volume 32, Issue 1-2

The formation of H-addition radicals in monocrystals of adenosine, adenosine ·HCl, adenine ·HCl· ½H 2 O, and adenine·2 HCl by X-irradiation has been studied by using electron spin resonance spectroscopy at 9.5 GHz and at 35 GHz. In all crystals, both H-addition radicals at position C2 and at position C8 were observed. The coupling constants of these two H-addition radicals are different and depend strongly on the protonation state of the adenine base. INDO calculations reproduce well the observed trends of the coupling constants. It is shown that the C2- addition radical is transformed into the C8-addition radical by heat and vice versa the C8-addition into the C2-addition by light of λ>360 nm.

A fast freezing technique using liquid propane was used to obtain frozen solutions of methemoglobin at pH 7. With this method it was possible to eliminate largely the presence of a low spin component which is usually found in slowly frozen solutions but not present in the sample at room temperature. The magnetic susceptibility and Mossbauer spectra of Hb(H 2 O) in the temperature range 4.2 K ≦ 7 ≦ 250 K have been measured. The data of the high spin compound of Hb (H 2 O) were evaluated with a Hamiltonian containing the Coulomb repulsion of the five 3d-electrons of the Fe 3+ ion, a crystal electric field of C 2v symmetry and the spin orbit coupling. The term describing the crystal electric field depends on five energy parameters ε 1 , ε 2 , ε 3 , D, and E which are determined by least squares fits to the experimental data. The most relevant parameters ε 2 and ε 3 which equal the energies of the antibonding single 3d-electron orbitals 3d z 2 and 3d x 2 -y 2 with respect to the 3d sy orbital are compared with earlier results of these energies ε 2 and ε 3 of the high spin compound of Mb(H 2 O). From this comparison conclusions regarding the different spatial ar­rangements of Fe 3+ in Hb(H 2 O) and Mb(H 2 O) are drawn.

Polarized infrared ATR spectra of dry oriented multilayers of dipalmitoylphosphatidylethanol- amine, sheep brain phosphatidylethanolamine, dipalmitoylphosphatidyl-N-methylethanolamine, dipalmitoylphosphatidyl-N-N-dimethylethanolamine, dipalmitoylphosphatidylcholine and egg phos­phatidylcholine are reported. Structural features of hydrocarbon chains and polar headgroups are discussed. The average deviation of hydrocarbon chains from the normal to the plane of the bi­layer was found to be 20-30°. However it was not possible to decide whether the chains are oriented parallel to each other. The fatty acid ester groups in β- and γ-position have different conformations. The phosphate group of dipalmitoylphosphatidylethanolamine exists probably in the I protonated and not in the ionized (>PO 2 - ) state. However, the latter state is ex­pected for all other phospholipids of this series. The deviation of the bisector of ∢ (OPO) of the >PO 2 - group from the normal to the bilayer is less than 45° and the mean orientation of all polar head groups is rather parallel than perpendicular to the plane of the bilayer. The polar headgroup of phosphatidylcholine assumes at least two different conformations of the O - C - C - N moiety, i. e. gauche and trans. A variety of conformers has to be expected also for the polar head groups of most of the other phospholipids investigated in this work.

From sporophores of Chamonixia caespitosa the 2,5-diarylcyclopentane-1,3-diones gyrocyanin (1), chamonixin (2) and gyroporin (5) have been isolated, the first two being responsible for the blueing of the fruiting bodies. This result suggests a close relation of Chamonixia to the order Boletales.

The identification of four further major constituents of the pedal gland exudate of the bontebok, Damaliscus dorcas dorcas, viz. α-terpineol, 2-n-heptylpyridine, m-cresol and (Z)-6-dodecen-4-olide and the investigation of the stereochemistry of the double bond in (Z) -6-dodecen-4-olide by means of iterative computer analysis are described. An improved synthesis of this compound is outlined.

The defensive secretion of Peripatopsis moseleyi (Onychophora) consists of 84% water and 16% protein and free amino acids. The secretion’s defensive effectiveness is an anti-predator “sticking” action. The secretion is flung out of the oral papillae in liquid state. It is then denaturized by the air and develops increasingly sticky white threads, probably through the devel­opment of disulfide bridges from the protein content. The elastic properties of the secretion threads indicate a micellar structure. The defensive secretion contains no volatile organic components or carbohydrates. This was confirmed by gas- liquid chromatography and thin-layer chromatography. After acidic hydrolysis of the secretion the following amino acids were determined quantita­tively: aspartic acid, threonine, serine, proline, glutamic acid, glycine, alanine, valine, cysteine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine. A “rare” amino acid was not identified. Tryptophane was not present (basic secretion hydrolysis). The quantita­tive determination of free amino acids, based on the total content, showed the following results: glycine (40.9%), glutamic acid (10.8%), aspartic acid (2.65%), lysine (1.3%). This result shows, that the secretion is stored in a watery glycine/glutaminic acid buffer in the oral papillae of Peripatopsis moseleyi. High voltage paper electrophoreses and gel filtration experiments with dextran and agarose gels showed, that the secretion protein consists of, at least, two fractions with different molecular weight.

Substituted p-benzoquinones (= quinones) and substituted hydroquinones [bis-trimethylsilyl- ethers or bis-trifluoroacetates] can be separated by gas-liquid chromatography using 50 meter long thinfilm capillaries (inner diameter: 0.25 mm) coated with silicone OV 17 (50% phenyl, methyl) or silicone DC 550 (25% phenyl, methyl). Total retention times of various derivatives of p-benzoquinone and hydroquinone were deter­mined. Complex model mixtures of these derivatives can be separated by gas-liquid chromatography with temperature programs and the individual components can be clearly identified by their mass spectra in the sub microgram range by using combined gas-liquid chromatography/mass-spectrometry. This microanalytical technique was applied to identify the defensive secretion components from members of the diplopod order Julida (Arthropods). Julus nitidus produces a defensive secretion that consists exclusively of 2-methyl-3-methoxyquinone. The expelled defensive secretions of Unciger foetidus, Cylindroiulus coeruleocinctus (Iondinensis), Cylindroiulus punctatus, Cylindroiulus luridus and Ophyiulus psilosus consist of a 2-methylquinone and 2-methyl-3-methoxyquinone mixture. The presence of the corresponding hydroquinones-2-methyl-hydroquinone and 2-methyl-3-methoxyhydroquinone-was also determined in all cases.

The paper describes a method for the large-scale isolation of protein III (protein E, major intrinsic protein) from the human erythrocyte membrane with little expenditure of time. By treat­ment of the erythrocyte ghosts with deluted HCl at pH 2.3 and 0 °C some membrane proteins can be extracted. From the remaining “rest”- membranes, whose major constituents, besides phospholipides, are protein PAS-1, other carbohydrate containing proteins and protein III, the latter can be separated in pure form by means of gel filtration. The paper reports on the amount, purity and possible structural modifications of the protein III obtained by this isolation method.

The arginine-rich histone F3 has been purified by covalent affinity chromatography. By the use of activated Thiol-Sepharose 4B for the purification of cysteine containing histone F3 a highly pure protein was obtained. The simple purification procedure offers the opportunity to get larger amounts of pure histone F3 within short time.

Cytochrome b-559, Algae, Spinach Cytochrome b-559, an integral protein of diloroplast thylakoids, was prepared in a homogeneous form from the alga Bumilleriopsis filiformis. The protein is easily denatured and can be solubilized by treatment of isolated thylakoids with high concentrations of urea and detergents in addition to weak sonification. The reduced form exhibits absorption maxima at 559, 530 and 429 nm. By comparative determination, a molecular weight of 17 000 was found for the protein from Bumilleriopsis, whereas that for spinach has 37 000 daltons. Both proteins have a low, but variable lipid content which is not a necessary part of the enzymatically active cytochrome b-559. The purified cytochrome exhibits a low midpoint the preparation a transient “high-potential” form (reducible by hydroquinone) was also solubilized.

Starting with well known β-diketoesters the corresponding y-enolethers are synthesized and described. Both classes of compounds inhibited the growth of some microorganisms. The inhibition is abolishable by addition of certain amino acids to the definite medium. A possible mechanism of inhibition is discussed and compared with the mechanism of some β,γ-unsaturated α-amino acids. The known unsaturated a-amino acids, their origin and their biological activity are summarized in two tables.

The NAD analogue [3-(3-acetylpyridinio)-propyl] adenosine pyrophosphate forms enzymically inactive complexes with glyceraldehyde-3-phosphate dehydrogenase from yeast and rabbit skeletal muscle. In the latter enzyme four mol of the analogue are bound with equal affinity inhibiting the enzyme in a competitive way: K I = 0.3 mM as compared to the dissociation constant K D =O.6 mм. The brominated derivative [3- (3-bromoacetylpyridinio) -propyl] adenosine pyrophosphate is co­valently bound to both enzymes causing irreversible loss of enzymic activity. Complete inactivation of the enzyme from muscle requires two moles of the analogue per mol of tetramer. The remaining two sites are still able to bind two mol of NAD + without regain of enzymic activity. In the case of the yeast enzyme four mol of the analogue are bound. Inactivation of the rabbit muscle enzyme is accompanied by the disappearance of two out of four highly reactive sulfhydryl groups; in the yeast enzyme the four active site cysteine residues are still able to react with DTNB 1 the reactivity being diminished significantly. Hybrid formation between the native enzymes from yeast and skeletal muscle is not affected by the modification of the enzyme. Similarly the sedimentation properties of the covalently modi­fied enzyme are indistinguishable from those of the native molecule. This indicates that both the native and the irreversibly inhibited enzyme are identical regarding their quaternary structure.

Free and ethidium bromide (EB) complexed alkali denatured T7 DNA molecules were re-natured at 58 and 62 °C respectively for 1 -3 h. The structures of the renaturation products of the free candidates were as usual showing native like, branched and unrenatured DNA whereas the structures of the ethidium bromide complexed one were somewhat different, showing non-renatured loop-like and entangled regions present inbetween the renatured segments. On the basis of the linear base sequence of T7 DNA, these non-renatured parts are indicative of the inhibition of renaturation by the complexed EB molecules. Mapping of the non-renatured regions showed that they were present at some specific sites, which inturn suggested that the EB-binding has some base sequence specificity.

Acetyl-CoA carboxylase (E.C. 6.4.1.2) was isolated from rat liver. The purified enzyme contains phospholipids with a rather large amount of phosphatidylinositol (26%). Incubation of the purified acetyl-CoA carboxylase with phospholipase A 2 (E.C. 3.1.1.4) or with phospholipase D (E.C. 3.1.1.4) diminishes the phospholipid content by 70%, this treatment leading to a complete inactivation of the enzyme. After removal of the phospholipases, the lipid-depleted enzyme can be reactivated to a certain degree by incubation with a phospholipid extract from rat liver, with phosphatidylinositol alone, or with serum albumin.

1. Kinetic studies on the binding of 125 I-Streptolysin-O exhibited immediate fixation of activated toxin to natural and artificial membranes. Once fixed to the membrane no release of Streptolysin-O or Streptolysin-O-lipid-complexes has been observed. 2. In contrast to activated toxin (free SH-groups!), oxidized Streptolysin-O was shown to become also fixed to membranes, however, with different binding kinetics. The binding of oxidized material was clearly dependent on temperature and time. When the toxin was oxidized twice the amount of labelled material was bound as compared with the hemolytically active Streptolysin-O. This suggests that oxidized Streptolysin-O, too, possesses a “binding site” within the molecule, though free SH-groups were expected to be essential for toxin fixation at the membrane. It has been shown that oxidized (inactive) and reduced (active) Streptolysin-O forms stable “complexes” with liposomes in aqueous solution, which could be separated by chromatography on Sepharose gels. 3. The binding of 125 I-toxin to membranes and liposomes was specific since specific antisera against Streptolysin-O inhibited fixation of toxin completely. 4. Hydrolysis of phospholipids and release of lysophosphatides by Streptolysin-O esterase (EC 2.1.1.2) has not been observed, thus providing no evidence for an enzymatic concept of membrane damage.

Continuous light-dependent H 2 production was studied in a reconstituted in vitro system using Spinacea oleracea chloroplasts, Clostridium pasteurianum hydrogenase and Spirulina maxima ferredoxin. PhotosystemII dependent production at 30 0 C is 60 -70 μmol H 2 /mg chlorophyll. At 15 0 C, this reaction proceeds for up to 20 h producing 1O μmol H 2 /mg chlorophyll. O 2 (glucose, glucose oxidase) and peroxide (ethanol, catalase) traps do not extend the lifetime but enhance the rate of H 2 production. Stoichiometry of the coupled system for H 2 production in this system is 2 H 2 formed : 1 glucose consumed. A conversion efficiency of water photolysis for H 2 evolution of 70% was determined from the O 2 produced, measured as the amount of glucose consumed, during the first 2 h of continuous illumination. Cessation of H 2 production by the reconstituted system involves inactivation of photosystem II and a limitation in the coupling of low potential electrons to hydrogenase. Increasing ferredoxin leads to more rapid H 2 evolution but longevity of the system remains unchanged. When H 2 evolution ceases due to inactivation of water-splitting activity of photosystem II, about 40% of the hydro­genase and 25% of photosystem I activity are still present; inactivation is unclear when photo­system I is used to drive H 2 production since when H 2 production ceases, hydrogenase and photo­system I still retain activity. This may suggest that coupling between low potential reducing equivalents from photosystem I to hydrogenase is impaired.

An antiserum to lutein agglutinates thylakoids of Nostoc muscorum and Oscillatoria chalybea. From this it follows that lutein is located in the outer surface of the thylakoid membrane of these blue-green algae. The same result is obtained for an antiserum to neoxanthin. As neoxanthin is supposed not to occur in blue-green algae it follows that in this case the antibody action should be directed towards a carotenoid with allenic structure. The antisera to lutein and neoxanthin inhibit in both investigated algal species photosynthetic electron transport on the oxygen-evolving side of photosystem II. Moreover, the inhibition sites of both antisera are identical in Nostoc muscorum and are located between the sites of electron donation of the artificial electron donors tetramethyl benzidene and diphenylcarbazide. In the case of the blue-green alga Oscillatoria chalybea the inhibition sites of both antisera differ. Whereas the inhibition site of the antiserum to neoxanthin lies again between the sites of electron donation of tetramethyl benzidine and di­phenylcarbazide, the inhibition site of the antiserum to lutein appears to be situated at least partially beyond the site of electron donation of tetramethyl benzidine. The degree of inhibition of electron transport reactions with Nostoc muscorum is for both antisera 50 - 60 per cent and is pH-dependent. The pH-optimum lies at pH 7.2 for the antiserum to neoxanthin and at 7.8 for the antiserum to lutein. In comparison to this data the same antisera inhibit electron transport in chloroplasts from higher plants only by 20%. This low degree of inhibition in higher plants is apparently due to the fact that the surfaces of the thylakoids are not accessible to antibodies within the grana. In contrast to this the thylakoid surfaces of blue-green algae are fully accessible because the thylakoids are unstacked. The thylakoids of Oscillatoria chalybea have the tendency towards aggregation. Therefore, the results concerning the accessibility of the carotenoids to antibodies are not so clear cut as with Nostoc muscorum.

Attempts to substantiate irreversible actions of a variety of magnetic fields on the fruitfly, Drosophila melanogaster, have been successful and unsuccessful in about equal numbers. The most conspicuous mutagenic effects apparently induced by pulsed H F-fields failed to appear under continuous electromagnetic irradiation. This seems to correlate the observed damage with the VLF-components of the pulsed fields. The present investigation is motivated by the occurrence of these components both in the atmosphere and in the vicinity of electrical appliances. A strain of normally viable wild type males and subnormally viable Attached-X γ ω females was used in which the yield, and the sex ratio, of the progeny indicate, respectively, the extent of developmental damage and of sex-linked recessive lethal mutation induced by the exposure to detrimental conditions. Evaluation of 73,800 flies from subsequent generations of a control group and two test groups raised in steady, or rotating, homogeneous 9.6 kHz magnetic fields of about 2.5 G did not reveal any development of hereditare load in the test groups.

To measure pO 2 in gases or fluids and pH in solutions the fluorescence indicators pyrene butyric acid and β-methylumbelliferone, respectively, were nanoencapsulated to obtain nano-probes for measurement in physiological struc­tures of nano-range. For pH the fluorescence changes of β-methylumbelliferone were monitored, for pO 2 the fluorescence quenching of pyrene butyric acid by oxygen were registered. Drawing log(S 1/ S 2 ) versus pH, one obtains a straight line between the pH from 6 to 8. A linear increase in pO 2 between O and 400 torr yields a linear increase in the reciprocal fluorescence signal.

Tetracaine, Fluorescence, Erythrocyte Membrane The fluorescence of tetracaine depends on the micro-environment of the molecule and increases with increasing hydrophobicity. The presence of erythrocyte membranes strongly enhances tetracaine fluorescence. The results support the view that the alkyl chain and aromatic part of tetracaine is embedded in apolar regions of the lipid or protein phase of the membrane.

Oocytes of the pond skater, Gerris najas, display ribosome tetramers that are arranged in the form of sheets in the vicinity of the nucleus. This is the first finding of ribosome crystals in an insect and suggests that ribosome crystallization may be a common phenomenon of cells that are inactive in protein synthesis.

Conformational changes of the thylakoid arrangement during light-dependent etioplast-chloroplast development in cotyledons of Nicotiana clevelandii X N. glutinosa are correlated with a decrease of the iron and phosphorus content in electron-dense stroma inclusions. Parallel to the transformation of the prolamellar body and the stacking process of the thylakoids, both the iron and phosphorus content of the inclusions were found to be reduced. Their elemental composition was analysed by means of the energy-dispersive X-ray microanalysis. Due to their high electron-density these stroma inclusions can be observed by conventional transmission electron microscopy in unstained thin-sections from exclusively glutaraldehyde-fixed material. They seem to be involved in membrane formation processes concomitant with the dispersal of the prolamellar bodies. Thus, the iron and phosphorus containing inclusions were found either closely surrounded by membranes or in the intralamellar space of plastids from plantlets illuminated for 1 - 8 hours. In chloroplasts (illumination period 12 -24 hours) no connections between these inclusions and the thylakoids were noticed.

Treatment of the lysogenic strain M. lysodeikticus 53-40 (N5) with ethyl methylaminosulfonate results in a slight increase in infective center concentration of N5 phages which is only detectable once disturbing cells had been eliminated by lysozyme. After conversion of ethyl methylaminosulfonate with radioactive labelled guanosine at 37 °C, N7-ethyl-guanine could be ascertained. Therefore the biological activity of the drug may be due to an ethylation of DNA.