Volume 32, Issue 3-4

A value R is obtained by the ratio of the summed, coincident pulses of both photomultipliers to the coincident pulses of one photomultiplier in the same selected range of the external standard puls height spectrum. This value R together with the external standard channel ratio (ESCR) show the extent of chemical and/or colour quenching in a field described by a set of calibration samples with increasing chemical or colour quenching (Fig. 3). The counting efficiency of samples with different and unknown proportion of chemical and/or colour quenching can be determined much more accurate than by any calibration approach using the ESCR only (Table VI). The method can be used for 3H samples with a counting efficiency ≧10% and 14 C samples of ≧5%, respectively. The new correction procedure is also applicable for 3 H and 14 C double-labelled compounds.

The behaviour of α-benzyl-α-bromo-malononitrile (BBMD) as oxidizing agent was characterized. The dependence of the electrochemical reduction potentials of BBMD on the pH-values of the solutions was examined. BBMD is reduced in a two-electron wave with half-wave potentials between + 230 and + 312 mV vs. Ag/AgCl/Cl - . BBMD is able to oxidize different organic compounds with OH-, SH-, NH- and CH- groups as well as a series of biological reducing agents as ubihydroquinone, plastohydroquinone, dihydro-lipoic acid, FADH 2 , NADPH and NADH. In all these cases BBMD is reduced to benzylmalononitrile. Kinetic investigations of this reduction process using NADH as reducing agent and also NMR-investigations using deuterated NADH-models showed that BBMD is directly reduced to benzylmalononitrile without producing benzylidenemalononitrile as intermediate. The reduction of BBMD with reduced pyridine nucleotides as well as that of benzylidene­ malononitrile were accelerated by an enzyme which was concentrated from human erythrocytes, using 2-chloro-benzylidenemalononitrile (CS) as substrate.

The C 18 juvenile hormone of insects can be dissolved in aqueous solutions up to a concentration of 2.5 - 3.0 × 10 -5 M; changes in pH, buffer composition and ionic strength hardly affect this solu­bility. The hormone is salted out gradually by increasing ammonium sulphate concentrations. The juvenile hormone is bound to proteins such as bovine serum albumin or goat immunoglobulin G and can be kept in solution by these proteins up to 10 -3 M. The hormone is strongly absorbed by many commonly used plastic materials but only to a lesser extent by glass and teflon.

Electrochromic spectra of monolayers of carotenoids (lutein and β-carotene) in contact with monolayers of chlorophylls and of pheophytin a are measured in thin capacitors. A specific inter­ action of one of the OH-groups of lutein with the Mg-atom of chlorophyll is found. The formation of this oriented complex accounts for the fact that a part of the electrochromic absorption-change of lutein depends linearly on the electric field strength, whereas for lutein alone only a smaller, quadratic electrochromism is found. In the preparation with chlorophyll a, the maximum of this linear electrochromism is located at shorter wavelengths (512 nm) than in the preparation with chlorophyll b (517 nm). The permanent field that has been postulated in photosynthetic membranes (to explain the linear dependence of the field-indicating absorption-changes of the carotenoids) may also be at­ tributed to such a complex formation with chlorophylls. Especially, the field-indicating absorption-change at 520 nm can now be attributed mainly to a lutein-chlorophyll b complex. The absorption-change at 520 nm, calculated according to this model from the present experiments in vitro, is of the same order of magnitude as observed in vivo. Furthermore, this model agrees with the hitherto unexplained observation that in chlorophyll-b-lacking mutants the absorption-change at 520 nm is smaller than in normal plants, and the maximum is located at shorter wavelengths. Besites, it is concluded that lutein is mainly located in the regions of photosystem II. The contributions of the other carotenoids (especially of neoxanthin) to the spectrum of the field-indicating absorption-changes are also discussed. From the above model, some conclusions are drawn on the asymmetrical arrangement of the different pigments in the membrane of photosynthesis: The bulk chlorophyll molecules that serve as complex partners for the carotenoids should be located near to the inner surface of the thylakoid membrane, and the carotenoids attached to these chlorophylls should be located more to the out­ side. The phytol chain of a chlorophyll molecule should form an acute angle with the plane of the porphyrin ring.

In a series of 190 samples, higher fungi, especially from the genus Agaricus, were analyzed by atomic absorption spectroscopy on their content of the trace metals cadmium, zinc, and copper. Cd was found to be present in high concentrations in some Agaricus species belonging to special taxonomic groups. In relation to soil concentrations, Cd is highly enriched in some Agaricus spec., the enrichment being a taxonomic criterion of special value. Among the remaining genera of higher fungi, only Leucoagaricus and Amanita showed similiar enrichment properties for cadmium. The chemically related metals Zn and Cu were found to be relative uniformely distributed in all analyzed fungi. No synergistic or antagonistic relationship between Cd and the other two metals could be detected in the mushrooms.

Lichens, Lipids, Phenols The three lichen species Alectoria ochroleuca, Stereocaulon vesuvianum var. pulvinatum and Icmadophila ericetorum have been chemically investigated with regard to their content of aliphatic and aromatic compounds. Tetrahydroxy fatty acids were isolated in all three species. A mono-acetylated pentol, higher fatty acids and an unsaturated triglyceride were detected as new con­ stituents of A. ochroleuca and I. ericetorum. S. vesuvianum and I. ericetorum both contained phenolic components which not previously have been discussed in literature. Chemical and spectro­ scopic evidences are presented in these studies.

Two homologues of spermidine, N-(3-aminopropyl)-1,3-diaminopropane (1) and N-(4-amino-butyl)-1,4-diaminobutane (2), were identified in Scenedesmus acutus 276-3a. For identification, extracts of algae were trifluoroacetylated and investigated by combined gas chromatography/mass spectrometry.

Young leaves from three plants which accumulate hexadecatrienoic acid rather specifically in the sn-2-position of monogalactosyl diglyceride (MGD) were incubated with radioactive CO 2 or acetate to investigate the origin of this specificity. Labelled glycerolipids were extracted and analyzed for time-dependent changes of radioactivity in their fatty acids. The investigation of labelled MGD and digalactosyl diglyceride (DGD) included determination of radioactivity in hydrolysis products, separation of molecular species by argentation chromatography and analysis of the positional distribution of fatty acids. The results agree with previous observations on the accumulation of radioactive oleic acid in phosphatidyl choline (PC) and formally with the possibility of a PC-coupled desaturation to linoleic acid. They do not support the proposed function of PC as donator of polyenoic acids. Instead the radioactivity patterns may be interpreted as pointing to a relation between fatty acid desaturation and many if not all glycerolipids, although a different interpretation is also possible. Fatty acid patterns in lipids and their labelling indicate the existence of several pools for 1) MGD, from which only that without C 16 -unsaturated fatty acids is accessible for galactosylation to DGD ; 2) palmitic acid, from which one part is accessible to desaturation via C 16:0 and C 16:2 to C 16:3 . Since these acids are found labelled in the sn-2-position of MGD, the specific positioning may be related to this separation of C 16:0 pools. Desaturation of C 16:0 seems to be the major source of C 16:3 ; 3) linolenic acid, from which those parts present in the sn-2-position of galactolipids or in PC are characterized by a strikingly slow labelling.

Eleven biflavones have been isolated from the leaves of Araucaria rulei and identified by NMR spectroscopy and via the permethylated and peracetylated products. The biflavones found until now in the Araucaria genus are summarized in a table.

A single procedure for the preparation of lactate dehydrogenase (EC 1.1.1.27), the mitochondrial and cytoplasmic forms of malate dehydrogenase (EC 1.1.1.37), adenylate kinase (EC 2.7.4.3) and pyruvate kinase (EC 2.7.1.40) from pig heart is described. The five enzymes are obtained in preparative amounts in homogenous form with specific activities equal to or higher than those pre­ viously reported. Some molecular properties of pig heart pyruvate kinase are determined.

A protein has been isolated from spinach leaves which catalyzes the following isotopic exchange reaction: Cys-SH+H 2 35 S ↔ Cys- 35 SH + H 2 S . This enzyme has an pH-optimum above 9.0; its molecular weight has been estimated on a Sephadex-G-100 column to be around 64 000 daltons. During purification this exchange reaction activity follows cysteine synthase activity on DEAE-cellulose and Sephadex-G-100 column chromato­ graphy; however this enzyme fraction has not been purified to homogeneity to prove that both activities are catalyzed by the same protein. The apparent K m for a) H 2 S has been determined to be 0.86 mM using cysteine as substrate and 0.6 mM using O-acetylserine as substrate; b) for cysteine was found to be 3.3 mM; and c) for O-acetylserine to be 3.3 mM. For catalysis no metal ion is required and the reaction proceeds without addition of pyridoxalphosphat. This exchange reaction might prove to be a simple method to prepare labelled cysteine from non-labelled cysteine and labelled H 2 S. The exchange reaction was found too in Chlorella pyrenoidosa and in Rhodospirillum rubrum.

The influence of temperature on the structure and enzyme activity of ribulose-1,5-bisphosphate carboxylase, isolated from Euglena gracilis cells, was studied. Freezing of the purified ribulose-1,5-bisphosphate carboxylase preparation causes a severe loss of enzyme activity, which can be restored again by incubation of the enzyme molecules at higher temperatures (50 °C). The titration of both enzyme samples with the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) re­vealed an increase of the fluorescence emission of the low temperature form of the enzyme. Two different enzyme conformations can be assumed which differ in the number of binding sites for ANS and V max values for the carboxylase reaction but show similar binding constants for ANS and the apparent K m values for C0 2 .

Chromatin, RNA Polymerase, Potato Tuber Tissue, Aging Phenomenon The synthesis of RNA by chromatin-bound RNA polymerase (E.C. 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however. Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically. Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue. The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphos­ phates and a divalent metal ion such as Mg 2+ or Mn 2+ and totally inhibited by the presence of pyrophosphate. Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique. The K m of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X10 -5 M, 1.6X10 -5 M, 0.9X10 -5 M and 0.45 X 10 -5 M /1 respectively, α-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase If.

The pyridazinone herbicide San 6706 blocks chlorophyll and carotenoid formation simultaneously in a correlated way in both Raphanus and Hordeum seedlings. With decreasing carotenoid content the carotenoid precursor phytoene appears in San 6706-treated plants. The accumulation of the chloroplast prenylquinones (plastoquinone-9, α-tocopherol, α-tocoquinone and vitamin K 1 ) is much less affected. This indicates that the biosynthesis of C 20 - and C 45 -prenyl chains (geranyl-geraniol, phytol and solanesol) which are incorporated in prenylquinones is not blocked by San 6706. It is assumed that the block of chlorophyll and carotenoid formation is due to one basic inhibition mechanism. The possibility that San 6706 may interfere with chloroplast ribosome formation, is discussed.

The two main non-sedative effects of thalidomide (2-phtalimido-glutarimide) in man are the embryopathy, which finally forced termination of its use as a sedative, and the excellent efficacy in leprosy reactions, alone for whose prevention and treatment the drug is still available. Both effects can be explained on a molecular level by assuming that a non-sedative metabolite of thalidomide mediates inhibition of human prolyl hydroxylase, as suggested by steric considerations and correlation to known data. This metabolite may well be a suitable model compound for drugs designed for selective fibrosuppression and selective immunosuppression.

Distribution of rat liver serine: pyruvate aminotransferase between cytosol and mitochondria varies considerably with the dietary and hormonal state of animals. Feeding a high-protein diet or fasting the animals results in an increase in the enzyme activity of both fractions but more marked in the mitochondrial fraction. A low-protein diet exerts the reverse effect. A single administration of dibutyryl cyclic AMP causes a rapid elevation of the enzyme activity in both fractions, which is effectively prevented by cycloheximide, actinomycin D and cortisone. The activity in mitochondria increases with a lag of 2 h following injection of the nucleotide inducer, in contrast to the cytosol enzyme, which increases without any lag. Gel filtration and DEAE cellulose chromatography of the enzyme from both fractions revealed the similar pattern and some kinetic constants of these two types of the enzyme were not significantly different from each other. These results indicate that rat liver serine: pyruvate aminotransferase is synthesized in the extra-mitochondrial site and transfered to mitochondria.

Type A complete spinach chloroplasts (photosynthetic control ratio ≅ 5) were frozen in liquid nitrogen, in the presence or absence of 50 percent glycerol (v/v). By considering five parameters related to the non cyclic electron transfer in the light (oxidant : K-ferricyanide) it was shown that the uncoupling due to freezing and thawing was partially prevented by glycerol. However glycerol was responsible for a slight uncoupling in fresh chloroplasts. The clearest uncoupling effect of freezing, and chiefly of glycerol, concerned the inhibition of the electron flow which follows the stimulation caused by a limiting quantity of ADP (state 4). The relative decrease of the photosynthetic control and of related parameters due to the use of this method of preservation, has to be balanced with the improved homogeneity and convenience resulting from it.

Photosystem I activity of isolated chloroplasts has been coupled to photosynthetic hydrogen evolution via methylviologen and a Clostridium hydrogenase. Rates of 125 μmol hydrogen evolved per mg chlorophyll and hour have been obtained. The conditions for such optimal activity suggest that a closed thylakoid vesicle is required in order to prevent side-and backreactions of reduced methylviologen, which compete with and reduce hydrogen evolution.

A kinetic model for the chloroplast translocators (mediating equal and opposite exchange fluxes between the external medium and the stroma) is derived. This model is a modification of the classical Widdas model corresponding to the exchange of an arbitrary number of compounds with no contribution to net transport. It describes the rate of transport of each compound in simple terms and with a minimum number of kinetic constants. Predictions from the model agree with experimental data as recorded in the literature. From the experimental apparent kinetic constants V MAX and K m those in terms of the model are calculated. These constants complete the model and make it applicable to the study of metabo­lism of photosynthesis by means of computer simulation. The set of differential equations describing the operation of the phosphate translocator is solved numerically for some illustrative examples.

A monospecific antiserum to cytochrome f agglutinates stroma-free swellable chloroplasts from tobacco and Antirrhinum. Consequently, antigenic determinants towards which the antiserum is directed are located in the outer surface of the thylakoid membrane. The antiserum inhibits linear photosynthetic electron transport. Just as described earlier for the antiserum to polypeptide 11000 this inhibition develops in the course of the light reaction. Ultrasonication in the presence of anti­ serum abolishes the light requirement and the maximal inhibition of the electron transport reaction is immediately observed. Electron transport in chloroplasts from a tobacco mutant which ex­ hibits only photosystem I-reactions is also inhibited by the antiserum. No time lag in the light for the onset of inhibition is observed with these chloroplasts. As chloroplasts of this mutant have only single unfolded thylakoids it appears that light might preponderantly open up partitions. If the light effect is interpreted in this way, cytochrome f should be located in the partition regions but nevertheless in the outer surface of the thylakoid membrane. However, a rearrangement of molecules in the membrane in the light by which the accessibility of cytochrome f is changed can­ not be excluded. The inhibition of linear electron transport by the antiserum is approximately 50 per cent and can only be increased to 75% upon the addition of antibodies to plastocyanin. The inhibition by the antiserum to cytochrome f as well as the combined inhibition by the antisera to cytochrome f and plastocyanin can be by-passed by DCPiP. It appears that cytochrome f and plastocyanin cannot be connected in series in the electron transport chain but are both closely associated in the thylakoid membrane. PMS-mediated cyclic photophosphorylation in chloroplasts from wild type tobacco and the tobacco mutant NC95 is only inhibited if the chloroplasts are sonicated in the presence of anti­ serum. If one disregards, that ultrasonication might cause reaction artifacts, it is thinkable that the cytochrome f, involved in the PMS-mediated cyclic photophosphorylation reaction, might be located inside the membrane.

Sarcoplasmic Calcium Uptake, Oxalate and Phosphate Uptake, Oxalate-Phosphate Competition, Calcium Turnover 1. During ATP supported active calcium uptake oxalate as well as phosphate are accumulated with calcium. The uptake of calcium exceeds that of both anions by a small quantity - accounting for calcium binding to vesicular proteins and lipids. 2. From assay media containing phosphate and oxalate - nearly exclusively either oxalate or phosphate are taken up together with calcium by the sarcoplasmic reticulum vesicles. The mutual exclusion occurs in a very narrow concentration range of the anions. 3. In solutions containing phosphate and oxalate, calcium phosphate or calcium oxalate pre­ cipitates are formed according to their solubility properties. 4. When phosphate prevents oxalate from being taken up, calcium transport is inhibited. In­ hibition occurs, because the concentration of ionized calcium inside the vesicles rises approximately 100-fold when oxalate is replaced by phosphate. The activity of the calcium dependent ATPase parallels the calcium uptake activity. 5. It is excluded that the inhibition of calcium uptake produced by phosphate is caused by an enhanced permeability of the sarcoplasmic reticulum membranes for calcium in the presence of phosphate.

A gaschromatographic study has been made of the alkane-and alcoholic fractions from the leaf waxes of some Coffea species. The n-alkanes have the odd-carbon-numbered, the primary alcohols the even-carbon-numbered members pre­ dominating. The major homologues of the alkane fractions are C 29 and C 31 and of the alcoholic fractions C 30 and C 32 . The sub-divisions Erythrocoffea and Pachycoffea could be distinguished by the alkane patterns. Im Rahmen einer chemotaxonomischen Studie der Gattung Coffea wurden die Alkan-und Alkohol­ fraktionen der Blattwachse untersucht.

The pterocarpan pisatin has been identified as a major phytoalexin in fifteen species of Lathyrus. Thus, the genus is closer in its phytoalexin response to Pisum than to Vicia, which is known to produce furanoacetylenes on fungal in­ vasion.

X-Ray An X-ray analysis of two compounds isolated from Piper sanctum confirmed the trans configuration for 5-(methoxy) -cinnamyliden-4-methoxy-but-3-enolide (= piperolide [1]) and for 5-[trans-(2) -β-γ epoxy-α-methoxy-cinnamyliden]-4-methoxy-2 (5H) -furanon (= epoxypiperolide [2]). The stereochemistry of the semicyclic double bond is in favour of the Z-isomeres.

Pulvinic acid lactams have been synthesized by reaction of the lichen compound vulpinic acid and 2-hydroxyethoxy-acetamide. The structure were established by consideration of infrared, mass and nuclear magnetic resonance spectra.

RNA with a sedimentation constant of 28S and 18S can easily be separated from 4S and 5S RNA. The method depends on the different solubulities of nucleic acids in solutions of the cationic detergent N-cetyl-N,N,N-trimethyl-ammoniumbromide at various ionic strengths. The separation can be achieved with high efficiency in one step.

This work describes an enzyme which degrades adenosine 3′:5′-cyclic phosphate (3′ :5′-cyclic-AMP) and adenosine 2′:3′-cyclic phosphate (2′:3′-cyclic-AMP). The reaction pro­ ducts are 3′-AMP and 5′-AMP in case of 3′:5′-cyclic-AMP, 2′-AMP and 3′-AMP when 2′:3′-cyclic-AMP is given as substrate. Inorganic phosphate acts as a strong inhibitor, whereas theophyllin does not act up to a concentration of 10 -3 m . Probably the adenosine cyclic phosphates degrading enzyme has no function in a ‘second messenger’ system with 3′:5′-cyclic-AMP.

A precipitating antigen of bovine leukemia virus was isolated by isoelectric focusing and Sephadex gel filtration. In SDS-polyacrylamide gel electrophoresis it was found to be a homogeneous protein with a relative molecular weight of 69 000 daltons. Because of its relative molecular weight and staining characteristics it was designated as BLV gp69. A protein with the same molecular weight could also be demonstrated in BLV particles. In 34 out of 35 sera from cattle affected by enzootic bovine leukosis antibodies against gp69 were detected, whereas the sera from 197 animals, free of bovine leukosis, did not react in immunodiffusion test.

Young Oocytes of the Loach, Noemacheilus barbatulus (L.) (Teleostei, Cobitidae) Young oocytes of the loach, Noemacheilus barbatulus (L.), were studied by electron microscopy. Fistular paracristalline bodies are found in the oocytes during state I. They consist of many osmiophilic particles with a diameter of 150 - 300 Å. Ultrahistochemical experiments have demonstrated that the paracristalline bodies are mainly composed of pro­ teins. The paracristalline bodies are yolk precursors.

The regio olfactoria of Ophicephalus was studied by transmission electron microscopy. Two types of receptor cells were found: ciliary receptor cells and microvillous receptor cells. Both of these types represent primary sensory cells. So one can regard the light microscopical findings of Kapoor and Ojha as disproved.

The summed action potential (SAP) of the receptor axons in the tympanic nerve is a very regular, often nearly harmonic oscillation around an average level. The oscillation amplitude depends on the slope of the stimulus ramp, whereas the average level of the response is determined by the plateau of the ramp. Earlier hypotheses upon the tympanic SAP do not explain the above findings. Yet the transient solutions of a simple oscillator model reproduce the tympanic oscillations.

The mating behaviour of a spider belonging to the Liphi­ stiomorphae (Mesothelae) is described. Mating is introduced by palpal signals of the male. The copulation takes place just in front of the opened trap-door of the female’s burrow. The male inserts only one palpal organ. After the copulation the couple separates quickly but peacefully.