Volume 33, Issue 3-4

Dual-wavelength spectroscopy allows the precise and fast measurement of small absorbance changes in highly absorbing and scattering samples (≦ 10 -3 absorbance). The sensitivity of this method makes it possible to monitor directly redox changes of electron carriers in complex particulate biological systems. In this article, a description of the measuring principle of dual­wavelength spectroscopy is given with special reference to its application in photosynthesis.

The title compound crystallizes in space group P 4 s 2 1 2 with a-b=9.558 Å, c = 36.649 Å. The structure has been determined by direct methods on the basis of 1624 X-ray counter data and refined by least squares methods to R = 6.7%. The acidic proton resides at the carboxyl group and not at adenine N(1) and the acetal methyl group is exo while the bulkier carboxyethyl group is endo relative to the ribose. The nucleoside occurs in the syn conformation, the conformation about C(4′)-C(5′) is trans, gauche, thus prohibiting an intramolecular O (5′) - H ... N (3) hydrogen bond. The ribose is in a slightly puckered C(3′)-exo envelope conformation, the carboxyethyl group is all-lrans rendering the molecule in an extended form with a distance 11 Å between the carboxyl group and amino group N(6). The acetal methyl group in exo displays a pmr signal at 1.35 ppm and can serve to distinguish the two methyl signals of O-2′,3′-isopropylideneadenosine at 1.38 ppm and at 1.65 ppm.

Light scattered at a planar lipid membrane is modulated by an electric a. c. voltage across the membrane. This effect is studied by a schlieren optic method. It shows a large contribution from the bilayer/torus border. From the inner part of the membrane no modulation of scattered light was detectable. Membranes were made from egg lecithin + cholesterol/decane in 0.1 n KCl aqueous solution.

Quantitative ultratrace analysis (10 nmol to 10 /cmol/1) of cesium in biological samples such as human body fluids and animal tissues is performed without any prior purification or concentration steps. The normal level of cesium ions in heart cells was determined. After poisoning these cells with high concentrations of the alkali cation much higher levels were found inside the cells then had been suggested previously. It is demonstrated that field desorption mass spectrometry is a unique tool for the qualitative and quantitative investigations of metal cations in biological material.

Recent investigations point to the presence of paracrystalline lattice disorder in the nerve myelin in the swollen, fixed and to some extent also in the native state. This leads to the distortion of the Q-function of the membrane stack and the Q 0 -function of the unit cell, which can be ex­perimentally isolated through swelling. Representing the distance statistics between neighbouring membranes and the electron density distribution of the single membrane with Gaussian functions, the Q-function can be expressed analytically as a convolution polynomial of the electron density, its mirror image and the distance statistics functions. Fitting this model (^-function to the experimental Q-function obtained by the inverse Fourier transformation of the scattered intensity, one can determine the optimal parameters of the electron density distribution and the distance statistics functions. The elimination of the distance fluctuations between neighbouring membranes permits the calculation of the undistorted Q-function or the Q 0 -function. The Fourier Analytical Deconvolution of the undistorted Q 0 -function enables a unique determination of the double membrane profile, thus a unique phase determination.

When myoglobin is irradiated in the presence of amino acids, the most radiation-reactive species, like the aromatic and sulfur-containing amino acids, will bind preferentially to the protein. The radiation-induced binding is strongly dependent on the concentration of protein and amino acid. Subsequent to irradiation of myoglobin in the presence of radioactively labelled tryptophan followed by tryptic hydrolysis, only a single radioactive spot was detected on the fingerprint. The binding of amino acids is thus not randomly distributed over the protein molecule but occurs at specific reactive sites.

The energy dependence of Zn 2+ accumulation by human erythrocytes was studied by preparing resealed ghosts loaded with ATP or AMP. Uptake of Zn 2+ was followed by radioactivity or atomic absorption spectroscopy measurements. Zn 2+ taken up is not adsorbed by the mem­brane but transported into the ghosts. Ghosts containing ATP took up more Zn 2+ than those con­taining AMP. The effect is not specific for ATP, however, GTP and CTP showing the same in­creased uptake. Zn 2+ uptake by ATP ghosts can be further drastically enhanced by addition of Cu 2t . Therefore, in spite of the enhancement of Zn 2+ uptake by ATP, Zn 2+ seems to be accumulated by a passive mechanism even when the ghosts contain ATP. The accumulation is probably brought about by binding of Zn 2+ to substances inside the ghosts.

The isopycnic equilibrium of [ 131 J] albumin digesting particles from mouse liver homogenates occurs, in a sucrose gradient, at a density 1.25, and it does not change with time after albumin injection (5 to 300 minutes).

Previous authors have described that liver cytoplasm and serum from partially hepatectomized rats stimulate the DNA synthesis of isolated nuclei of liver cells. The aim of our investigation was to prove whether such stimulating effects are part of a specific regulation of growth. The sera and the cytoplasm supernatant 105 000Xg) were isolated at several intervals after partial hepatectomy and laparotomy and were tested on isolated nuclei of liver cells from adult rats partially hepatectomized 20 hours before. The supernatants isolated at 8 and 16 hours after both partial hepatectomy and laparotomy have a significant stimulatory effect on the [ 3 H]TTP-uptake by nuclei. The presence of serum (10%) causes a general elevation (75%) of the [ 3 H]TTP-uptake by isolated nuclei. The sera isolated 4 to 12 hours after operation from both partially hepatectomized and laparotomized rats have an additional stimulatory effect on the [ 3 H]TTP-uptake synthes of nuclei. In no case did the sera and liver cytoplasm of partially hepatectomized rats have a significantly different and therefore “specific” effect on the nuclear DNA synthesis. Additional experiments showed that frozen and thawed nuclei of liver cells respond only to the nutritive power but not to the stimulatory effects of the sera. Thus, thawed nuclei cannot be used for growth investigations.

The substrate specificity and the effects of nucleotides and SH-blocking agents on the p-nitro- phenylphosphatase activity of intact Ehrlich ascites tumor cells (EAT) cells were studied, ᴅʟ-β- Glycerophosphate, o-phosphoethanolamine, cholinephosphate, glucose-6-phosphate, o-carboxyphenyl- phosphate,, phosphoenolpyruvate and AMP were not attacked by intact cells. ATP > GTP > UTP > PP i > pNPP were cleaved with decreasing velocity. A stimulation of the cleavage of p-NPP by the following nucleotides was observed with decreasing effectivity: ATP > ADP > GTP > UTP; AMP was ineffective. The phosphatase activity was not affected by malate, tartrate and glutathion disulfide. The SH blocking agents diamide and thimerosal were more effective in­hibitors of the pNPPase than of the ATPase activity, whereas the hydrolysis of ATP is more affected by the ATP analog adenylylimidodiphosphate. The present data are best compatible with a double headed enzyme: Both active sites interact with ATP, only one is active against p-NPP and sensitive against SH-blocking agents.

The period length of the activity rhythm of the cockroach Leucophaea maderae is increased by LiCl solution of 100 mol m -3 offered as drinking water by about 1%.

(1) A procedure has been described for the purification of two glutamine-chorismate-amido- transferase-free anthranilate phosphoribosyltransferases from mutant strains TAX6trpR782 and trpAB1653trpR782 of Salmonella typhimurium. (2) The native enzymes tend to aggregate forming polymers of molecular weights 333,000 in the case of TAXtrpR782 and 220,000 and larger than 1X10 6 in the case of trpABI653trpR782. In the presence of sodium dodecyl sulfate the polymer of trpAB1653trpR782 dissociates into a single component with molecular weight of 72,000. (3) In contrast to anthranilate phosphoribosyltransferase of the wild type component II, the glutamine-chorismate-amidotransferase-free proteins do not complex with component I. They do however show catalytical similarities with the wild type with respect to anthranilate phosphoribosyltransferase activity.

Derepressed free living cells of Rhizobium japonicum strain 61-A-101 with leucine as single nitrogen source develop a maximum nitrogenase activity of 180 nmol C 2 H 4 · mg protein -1 ·h -1 in liquid culture under 2% O 2 in the gas phase. Only 10% of this activity is found with no oxygen in the gas phase during a 90 min incubation period. The maximum activity under 2% oxygen in the gas phase is unaffected by addition of 1 -100 mM NH 4 * and by addition of low concentrations of glutamine (0.36 - 1.44 mᴍ). Specific activities of alanine dehydrogenase (E.C. 1.4.1.1.) aspartate aminotransferase (E.C. 2.6.1.1.) and, with much lower activities, of GOGAT (E.C. 1.4.1.13) in nitrogenase active free living cells are more similar to bacteroids than to nitrogenase repressed free living cells from liquid culture. The activities in nitrogenase repressed cells were about 50% lower. Glutamine synthetase (E.C. 6.3.1.2.) activity in bacteroids and in nitrogenase active cells were also similar, but only about 25 - 30% of that found in nitrogenase repressed Rhizobium japonicum cells.

The protein. RNA and DNA content of wing discs increase exponentially during the last larval instar. Biosynthesis of protein, RNA and DNA was studied by injecting labelled precursors into larvae or white prepupae and measuring the incorporation in the wing discs dissected out after an appropriate time. Protein synthesis is stimulated by ecdysterone, methoprene has no effect. Bio­synthesis of rRNA is increased in wing discs of white prepupae after ecdysterone or methoprene injection. Methoprene inhibits the synthesis of mRNA, while ecdysterone has no clear-cut effect within the limits of our method (gel electrophoretic analysis). Ecdysterone and methoprene have no detectable influence on incorporation of thymidine into DNA, but the incorporation label from uridine into DNA is diminished; this effect may be due to changes in the precursor pool.

Conditions for washed, spinach thylakoid membranes to catalyze cyclic phosphorylation using ferredoxin as the cofactor for electron transfer have been re-examined. It was found necessary to “redox poise” the system; however, the best method to accomplish the poising seemed to be the use of a reductant (glucose-6-phosphate) and not by optimizing the rate of phosphorylation with 3- (3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). Under these conditions, ferredoxin catalyzed cyclic phosphorylation was found to be sensitive to the inhibitors antimycin A and dibromothymo- quinone (DBMIB). The inhibition of ferredoxin catalyzed cyclic phosphorylation by DBMIB, but not by antimycin A, was completely reversed by N, N, N′ N′-tetramethyl-p-phcnylenediamine (TMPD). These data are taken as further support for the function of plastoquinone in ferredoxin catalyzed cyclic phosphorylation. The effect of TMPD in reversing the DBMIB inhibition is inter­preted as the formation of a TMPD bypass on the internal side of the thylakoid membrane around the DBMIB site of inhibition.

Intoxication of class II chloroplasts of spinach with Cu(II) leads to inhibition of millisecond luminescence. The degree of inhibition depends on Cu (II) -concentration. The investgation of the pH dpendence of the inhibition curve of luminescence revealed that (1) there is an inhibition site of copper on the donor side of photosystem II, (2) copper (II) does not act as an uncoupler of photophosphorylation, (3) a protonation equilibrium is involved in the inhibition mechanism, and (4) copper (II) binds to a dissociated residue of a membrane protein.

By measuring ms delayed fluorescence emission, we have investigated the interaction of protons in the reactions of the watersplitting enzyme of photosystem II. (1) In the presence of the electron transport mediators 2,3,5,6-tetramethyl-p-phenylene diamine (DAD) (in its reduced form) and methyl viologen and of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the maximal intensity of the delayed fluorescence measured between 1 and 1.5m s after illumination was greater than the level reached in the presence of DCMU alone. (2) The stimulation of the delayed fluorescence was greater when the suspension was preilluminated by 1 - 8 xenon (or laser) flashes prior to addition of DCMU, and the maximal intensity varied with the number of preilluminating flashes given. (3) The amplitude of the intensity of delayed fluorescence oscillated with a periodicity of 4, with a maximum after 2, and minimum after 4 flashes. A negligible oscillation w r as observed in the absence of the electron transport mediators. (4) When the prompt fluorescence intensity was measured under these conditions, a weak oscillation of period 4 with superimposed periodicity of 2 was observed in F 0, but of insufficient amplitude to account for the observed changes of delayed fluorescence intensity in terms of the bade reaction caused by addition of DCMU. (5) We therefore suggest that the enhancement of the delayed fluorescence is due to the release of protons accompanying transitions of the S-states, and that this release occurs not in synchrony with 0 2 , but in all the transitions of the S-states with the exception of Si → S 2 . We discuss the relative potentials of the transitions at the low internal pH which is generated as a result of cyclic electron transport around photosystem I in the conditions of our experiments.

Three mechanisms of oxygen reduction by chloroplast lamellae in the presence of autoxidizable electron acceptors can be differentiated by product analyis in the presence or absence of either dibromothymoquinone (DBMIB) or superoxide dismutase (SOD) : 1)H2O2 is the product of two-electron oxygen reduction by 2,3-dimethyl-5,6-methylencdioxy-p- benzoquinone, involving only photosystem II. This reaction is not inhibited bv either DBMIB or SOD. 2)Superoxide anion, and H2O2 as the product of its dismutation, are products of monovalent oxygen reduction following autoxidation of certain low potential electron acceptors (herbicides) of photosystem I. These reactions are not inhibited by SOD but are blocked by DBMIB. 3)H2O2 is the product of and “apparent” two-electron photoreduction of oxygen, mediated by certain o-diphenols (caffeic acid). These reactions are inhibited by both DBMIB and SOD in­dicating the involvement of photosystem I and of O2- as an intermediate in the H2O2 producing reaction.

Four polypeptides which exhibited apparent molecular weights from 40 000 to 48 000 were isolated from the thylakoid membrane. The isolation was achieved by gel filtration of polypeptides, solubilized by means of sodium dodecyl sulfate and, after removal of detergent by anion exchange chromatography in ethanolic solution. The polypeptide of the molecular weight 40000 was iden­tified as the y-component of the coupling factor of photophosphorylation via the effect of its antiserum and by its serological cross reactions. The antiserum not only inhibits photophosphory­lation reactions by 90% but also to the same extent the photoreduction of anthraquinone-2-sulfonate with tetramethyl benzidine as the electron donor, provided ADP, Pi and Mg 2+ are present in the assay. The inhibition of electron transport is accompanied by a corresponding increase in the fluorescence yield. The other three antisera inhibit photosystem I reactions. In addition, the antiserum designated 45 000 PSI-1 inhibits phenazine methosulfate-mediated cyclic photophos­phorylation. This antiserum as well as the two other sera do not contain any detectable anti­coupling factor activity. Antiserum 45 000 PSI-4, in contrast to 45 000 PSI-1, does not inhibit cyclic photophosphorylation. Moreover, it is the only one of the four antisera which does not affect the ratio of the fluorescence yields measured at 735 and 685 nm (F 735/F 685) at 77 °K. Hence, the antigen 45 000 PSI-4 probably plays a role on the acceptor side of photosystem I. On the other hand, the antiserum 48 000 PSI-3 seems to exert its effect on the donor side of photosystem I, because it inhibits the photoreduction of anthraquinone-2-sulfonate with low con­centrations of the electron donor dichlorophenol indophenol. The prominent property of this anti­serum is that the photophosphorylation with fcrricyanide as the electron acceptor is stimulated more than two fold. This, however, it not accompanied by an apparent change of the electron flux between water and ferricyanide. The antiserum causes a decrease of fluorescence yield which is probably due to an increased energy spill-over. All antisera exert their activity only after an il­lumination of the chloroplast preparations for several minutes. The dosis-effect curve is hyperbolic for the antiserum 48 000 PSI-3 and is sigmoidal with the other three antisera.

Olivacine, (-) -apparicine and (-)-vobasine have been isolated from leaves of Pagiantha cerifera Markgraf.

The 13 C resonance spectra of fungal melanins from Aspergillu sniger, Eurotium echinolatum , and Stachybotrys chartarum are reported. The spectra were taken in 5% W/W solution of the substances in 0.1 ɴ NaOD in D 2 O using the Fourier-transform-technique. The spectra and possible as­ signments of the bands are discussed. The similarities of these spectra to those of soil humic acids are unexpectedly small.

Acids A reinvestigation of the velum pigments of Cortinarius armillatus yielded the new anthraquinonecarboxylic acids endocrocin-6,8-dimethyl ether (4) and dermorubin-6-methyl ether (8).

4,5′-dimethylangelicin is a new angular furocoumarin that by irradiation at 365 nm shows a very high photo­ reactivity towards DNA in vivo, without forming any cross-linkage; by photosensitization of Ehrlich ascites tumor cells a strong inhibition of DNA synthesis was observed.

After the inhibition of protein synthesis in Saccharomyces cells with cycloheximid, the content of membrane-bound ribosomes decreases significantly, whereas the content of free ribosomes remains the same as the untreated control for a longer period of time. This is further evidence that in yeast cells protein synthesis takes place only on membrane-bound ribosomes.

Fifteen strains of Fusarium previously shown to degrade flavonoids and isoflavonoids were investigated for pisatin degradation. Fusarium anguioides and Fusarium avenaceum converted the phytoalexin (1) to the nontoxic 3,6a-dihydroxy-8,9-methylenedioxypterocarpan (2).

Cell suspension cultures of soya were incubated with cis-[1- 14 C]octadecenoic acids and trans- [1- 14 C]octadecenoic acids, each of known composition with regard to positional isomers. Each of the positional isomers ranging from ⊿8- to ⊿15-as-octadecenoic acids and ⊿7- to ⊿16-frans-octadecenoic acids was readily incorporated into the acyl lipids of the cells, yet, the ⊿9 cis- and the ⊿9-trans-isomers were the pre­ferred substrates, cis-trans Isomerization did not occur during the incorporation of the fatty acids into the acyl lipids.