Volume 31, Issue 1-2

The tautomerism of phenylpyruvic acid (PPA) and its sodium salt was investigated. The 1 H and 13 C spectra of PPA in aprotic solvents and in methanol show almost complete prevalence of the enol form, whereas the keto form prevails only in water. The Z configuration was assigned to the sole enol tautomer present on the basis of the value of the vicinal coupling constant | 3 J 1 H, 13 COOH |=3.7 Hz. A small amount of the hydrated form of PPA (1-phenyl-2,2-dihydroxyl- propanoic acid) was found in the aqueous solution of its sodium salt and in buffer solution (pD =6) of PPA. By means of infrared spectroscopy one can conclude that crystalline PPA is in the enol whereas its sodium salt is in the keto form. The keto-acid was not obtained in the solid state. The colori­metric method for testing PPA traces in urine depends on the formation of a enol-Fe 3+ complex (2:1) which appears stable in dimethylsulfoxide (DMSO).

The effects of varying concentrations of Mn 2+ on the absorption of divalent cations, viz., Ca 2+ and Zn 2+ from 2 concentrations were studied. Mn 2+ at all concentrations tested were inhibitory to Ca 2+ uptake from 0.1 and 5 mᴍ CaCl 2 . However, when Ca 2+ uptake was measured at concentrations ranging from 0.05 to 10 mᴍ CaCl 2 , MnCl 2 only at high concentration of 5 mᴍ, inhibited Ca ab­sorption. Zn 2+ uptake from 0.1 mᴍ ZnCl 2 was decreased by all concentrations of Mn 2+ .

Incorporation of doubly labelled tyrosine into stizolobinic acid and stizolobic acid by the etiolated seedlings of Stizolobium hassjoo was studied. The retention of tritium activity in stizolobinic acid was 55%, while that in stizolobic acid was only 4.4% in average. The results strongly suggest that the heterocyclic rings of these two amino acids may be derived from 3,4-dihydroxyphenyl-alanine (DOPA) by extradiol cleavage of the aromatic ring.

Spectroscopic properties of 1,N 6 -ethenoadenosine cyclic 3′,5′-monophosphate were studied and compared with cyclic adenosine 3′,5′-monophosphate. Data of nuclear magnetic resonance*, mass-, ultraviolet-, and fluorescence spectroscopy were given. Additionally biochemical activity was tested in protein kinase system of beef adrenals. No significant biological difference between cyclic adenosine 3′,5′-monophosphate and its fluorescent analog has been found.

The significance of pheromones in olfactory communication in mammals, and the possibility of using synthetic pheromones to influence the behaviour of problem animals, are briefly discussed. The isolation, identification and synthesis of the principal volatile component contained in the pedal gland exudate of the bontebok, Damaliscus dorcas dorcas, viz. (Z)-5-undecen-2-one, are reported. Some of the other major constituents of the secretion have been identified as 2-heptanone, 2 -nonanone, 2 -undecanone and 2,5-undecanedione.

The kinetics of the photodynamic desactivation of lysozyme in presence of acridine orange as the sensitizer have been investigated in detail varying oxygen, protein, dye concentration, ionic strength and pH value. The kinetics can be approximately described as an over all pseudo-first- order rate process. Changing the solvent from water to D 2 O or by quenching experiments in pres­ence of azide ions it could be shown that the desactivation of lysozyme is caused exclusively by singlet oxygen. The excited oxygen occurs via the triplet state of the dye with a rate constant considerably lower than that to be expected for a diffusionally controlled reaction. Singlet oxygen reacts chemically (desactivation, k=2.9 × 10 7 ᴍ -1 sec -1 ) and physically (quenching process, k = 4.1 × 10 8 ᴍ -1 sec -1 ) with the enzyme. The kinetical analysis shows that additional chemical reactions between singlet oxygen and lysozyme would have only little influence on the kinetics of the desactivation as long as their products would be enzymatically active and their kinetical constants would be less than about 1 × 10 8 ᴍ -1 sec -1 .

A universal cell was constructed, which allows measurements of the resistance (d. c.), capacity, optical reflectivity, and spectral absorption of blade lipid films simultaneously. These measurements reflect different properties of the films, supplementary to each other. The purpose is to eliminate differences of experimental conditions especially in recombination experiments with proteins. The cell is totally symmetric, can be completely disassembled, and the parts are exchangeable. It allows application of several methods for film production, and is held together simply by mechanical pressure, without need of any sealing agent. The inner parts are exclusively of glass and teflon. The fluid volume of the cell is 7 -15 ml and the better than 10 14 Ohms.

Mixed monomolecular films of cytochrome (Cyt) and chlorophyll (Chl) were spread at a nitrogen-water interface. A large interaction is observed between reduced Cyt f and Chl a in a mixed film. Oxidized Cyt f and Chl a in a mixed film show little evidence for interaction. Mixed monomolecular films of Chl a with either reduced or oxidized Cyt c appears to result in denaturation of the protein at the surface. A light reaction is observed only in mixed films of reduced Cyt f and Chl a.

The reactions of chloroplast thylakoid lamellae with the chemical probe ρ-(diazonium-) benzene­ sulfonic acid (DABS) in the light have been reinvestigated. In contrast to a previous report, electron transport from a photosystem I electron donor to methylviologen was found to be inhibited by this treatment. During the incubation of chloroplasts with DABS in the light, the probe is altered with high rates. Under aerobic conditions, a concomitant oxygen uptake is observed, which is stoichiometric to the amount of DABS altered. Under anaerobic conditions, the binding of the 35S-labeled probe to the membranes in the light is stimulated 2 - 3 fold as compared to the binding under aerobic conditions. The data are taken as evidence that the photoreduction of the probe rather than a conformational change of the membrane may be at least partially responsible for the increased reagent binding observed in the light.

A previously published working model for the regulation of chlorophyll formation has been tested studying early steps of chlorophyll and porphyrin biosynthesis in developing cotyledons of Helianthus annuus. The activities of δ-aminolevulinate synthetase (ALAS), δ-aminolevulinate dehydratase (ALAD), and the porphobilinogenase complex (PBGase) at any given time have been found to be strongly associated with endogenous developmental processes. Highest activities in dark­ness have been observed at times when maximum chlorophyll formation would have occurred had the plants been exposed to light. Only in the case of ALAS was the maximum activity in light much greater than that observed in the dark. Density labeling experiments and other data suggest that enzyme synthesis is mediated both by development and by illumination. Moreover, ALAS activity appears to be subject to inhibition, presumably by products of the porphyrin biosynthesis, as indicated by halflife experiments. Rapid enzyme degradation in the absence of light seems to be less probable. Slight ALAS activity in darkness is present as long as the plastids are not fully developed. In contrast to findings with cell cultures of tobacco, in Helianthus cotyledons ALAS certainly plays the main role in the regulation of chlorophyll biosynthesis. Nevertheless, increasing activities of the succeeding enzymes, located in the plastids, ensure that increased concentrations of δ-aminolevulinate (ALA) are drawn into the chlorophyll biosynthetic pathway. The experiments corroborate the suggestion that chlorophyll biosynthesis is controlled by different but interdependent mechanisms. The dominant regulatory mechanism is dependent on the stage of development.

In the heavy fraction of deoxycholate-treated spinach chloroplasts the chlorophyll an activity is high and the chlorophyll a I activity is low when no artificial electron donor is added. The addition of the photosystem I donor system N-methyl-phenazonium sulphate plus sodium ascorbate (PMS + Asc) leads to a complete reactivation of the chlorophyll a I reaction. The addition of the photo­system II donor system p-benzohydroquinone plus sodium ascorbate (HQ + Asc) leads to an inhibition of the chlorophyll a II activity. From these results we conclude: 1. Besides an interruption of the linear electron flow between the two photosystems deoxycholate-treatment leads to a block of the electron flow from water to photosystem II. 2. In deoxycholate-treated chloroplasts the linear electron flow in photosystem II just like in Triswashed, heat-treated or aged chloroplasts, is replaced by a cyclic one.

Photosystem I fragments were prepared by digitonin treatment of spinach chloroplasts. The mid­point potential of cytochrome b 6 in the fragments is close to 0 V, showing a one electron transition. No cytochrome b 559 was detectable, neither in difference absorption spectra nor in light-induced absorbance changes. In the absence of added cofactors only cytochrome b 6 photoreduction can be observed. This photoreduction is stimulated by ferredoxin. Ferredoxin-NADP + reductase appears not to be involved in cytochrome b6 reduction. Photooxidation of cytochrome b 6 is dependent on plastocyanin addition and inhibited by DBMIB, a plastoquinone antagonist. Addition of plastocyanin restores cytochrome f photooxidation as well, reacting quite specifically in about equimolar concentrations to bound cytochrome f. The stimulation of cytochrome f oxidation is abolished by an antibody prepared against plastocyanin, indicating a surface location of plastocyanin in digitonin treated membranes. Biphasic kinetics of dark-reduction of cytochrome f by ascorbate indicate that part of this cyto­chrome f is relatively inaccessible in the membrane. After preillumination a monophasic reduction is observed and the slowly oxidized component is absent. Illumination in the presence of plastocyanin causes a fast and complete reduction of cytochrome f, suggesting equilibration of cyto­chrome f with added plastocyanin, residing in the membrane surface. It appears that actinic light causes conformation and/or structural changes in the membrane of these digitonin fragments, influencing cytochrome f accessibility.

In trypsin-treated spinach chloroplasts there is no linear electron flow from water to potassium ferricyanide. The chlorophyll a II reaction, however, is still active but insensitive to 3-(3,4-dichloro-phenyl)-1,1-dimethylurea. From this we conclude that ferricyanide and trypsin together stimulate a mini-cycle in photosystem II.

Ultrastructural changes in in vitro ageing spinach chloroplasts have been studied in detail. Prolonged storage caused swelling of the chloroplasts due to the increase in the thickness and spacing of the thylakoid membranes. The increase in the thickness of the membrane is partly accompanied by the release of lipids. Addition of crystalline bovine serum albumin was found to stabilize the membrane structures. Storage of the chloroplasts at 77 °K even though it resulted in complete breakage of the whole chloroplasts, maintained the thylakoid structures in a highly intact form.

The size of poly(A)-RNA from polysomes and cell nuclei of the yeast Saccharomyces cerevisiae was investigated. Pulslabelled cells ([ 14 C]adenin) were cracked by the French press; polysomes and nuclei were separately isolated and the RNA was finally extracted with phenol. The separation of poly (A)-containing and poly (A)-lacking fractions was achieved by oligo (dT) cellulose. These fractions were characterized by sedimentation analysis. The main portion of polysomal poly (A) -RNA sedimented with a rate of 8 to 14S, whereas the poly (A)-RNA of nuclei exhibited a sedimentation rate of 12 to 17S. Thus nuclear poly (A)-RNA is about 20 -30% larger than polysomal poly (A) -RNA.

After lysis of Pseudomonas testosteroni with lysozyme and non-ionic detergents different DNA-protein complexes can be separated in 5 -25% (w/v) neutral sucrose gradient. The protein to DNA ratio of these complexes varies between 0.5-4.5 to 1, whereby the faster sedimenting forms contain more protein than the slower sedimenting ones. Different initial rates of DNase digestion may indicate various degrees of DNA packing in these complexes. The chromosomal complexes of Pseudomonas testosteroni are relatively stable towards pronase. Treatment with RNase or sodium dodecylsulphate is accompanied by a dramatic increase in viscosity and decrease in relative density. It suggests that DNA in these complexes is maintained in its supercoiled form by RNA molecule (s) in a similar way as in isolated chromosome of E. coli.

By means of paper chromatography, Lactobacillus casei test and the staining method with the Feulgen reaction, the effects of reserpine and amphetamine on riboflavin and melanin were studied in the liver of Triturus cristatus. It was demonstrated that the concentrations of both melanin and riboflavin can be altered by the influence of these drugs. From the results obtained it is suggested that riboflavin shows a correlation with the density of melanin.

Reaction of 6-hydrazinopurine and 2-hydrazinopyrimidine with 2,2,6,6-tetramethylpiperidone-1-oxyl gives 2,2,6,6-tetramethylpiperidone- 1-oxyl-(6-purinyl)hydrazone (3b) and 2,2,6,6- tetramethylpiperidone-1-oxyl- (2-pyrimidinyl) hydrazone (4b) respectively. Compound 3b is inactive even at high dose levels against TLX/5 lymphoma and 3b and 4b are both inactive in tests using the L1210 leukaemia system.

Dibromothymoquinone inhibits photosynthetic NADP reduction by broken chloroplasts. The inhibition of electron flow and coupled ATP formation is effectively reversed by the addition of thiol compounds.

DNA from dormant and germinated embryos exhibit differences in the kinetics of reassociation of thermal denatured nucleic acid. Within 24 h of germination a reduction of the renaturation, expressed as Cot 1/2 of the redundant fraction, is detectable.

The chloroplast population of Acetabularia contains four subpopulations of intact plastids with the buoyant densities at 1.21 g/cm 3 - 1.18 g/cm 3 - 1.14 g/cm 3 - 1.10 g/cm 3 on the discontinuous sucrose gradient. The starch content varies with the buoyant densities. The subpopulations probably represent entities of different biochemical function.