Volume 36, Issue 7-8

The CD and ORD spectra of the Coar-and Co/β-methyl isomers of cobalamin, 2-methylthio-adenyl cobamide (factor S), cobyric acid and p-kresyl cobamide (factor lb) were recorded in water and acidic solution. In complete and incomplete (nucleotide free) (Coar-methyl)-corrinoids the axial ligands CH3 and H2O are unalterable (fixed) within a wide range of pH (about 1 to 12) at room temperature in the dark. Thus the spectra in this pH range are constant at X > 350 nm; spectral differences at X < 350 nm are effects of the nucleotide bases. The spectra of complete "base-on" (Co/β-methyl)-corrinoids are, like the spectra of (Coa-methyl)-corrinoids, at X > 350 nm not dependent on the nucleotide base. If the nucleotide base is not coordinated ("base-off'), the spectra change strikingly and at X > 350 nm are nearly identical with those of incomplete corrinoids e. g. cobyric acid.

Vitamin B12-factor A (2-Methyladeninyl-cyanocobamide), Vitamin B12, X-Ray Crystal Structure Analysis The crystal and molecular structure of 2-methyladeninyl-cyanocobamide (factor A) has been determined. This compound crystallizes in space group P21212, with a = 2630.6 (15), b = 2210.6 (13) and c = 1592.1 (9) pm. The structure has been solved by the heavy-atom method and refined by least-squares methods on the basis of 3682 X-ray counter data to R = 0.166 and R w = 0.148. As far as we know, this is the first X-ray-investigation of a purine-corrinoid, which differs from cyanocobalamin (vitamin B12) by containing a purine base instead of 5,6-dimethylbenz-imidazole. The structure analysis of 2-methyladeninyl-cyanocobamide shows unambiguously, that the purine base coordinates with cobalt via N B9. The choice of N B9 as coordinative atom can be ascribed partly (or mainly?) to steric influences, since coordination via N B3, which was also discussed, would presumably lead to severe distortion of the nucleotide loop.

The sulfhydryl enzyme malate synthase was shown to undergo an X-ray induced aggregation and inactivation in solution (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980)). Further evidence for the occurrence of aggregation and inactivation and also of fragmen­ tation and partial unfolding of the enzyme upon X-irradiation was obtained by chemical and electrophoretic studies. Irradiation was carried out in a specially designed microcell, experiments were performed on the microlevel. Under conditions of the experiments the formation of H 2 O 2 upon X-irradiation could be proven; therefore the influence of H 2 O 2 on the enzyme was investigated too. Though the quantitative results of the damaged enzyme particles are influenced by many disturbing factors, the findings allow clear statements on the nature of the effects under investigation. 1) Both X-irradiation and treatment with H 2 O 2 caused a decrease of total and an increase of available sulfhydryl groups of the enzyme and led to a loss of enzymic activity. The presence of dithiothreitol turned out to be able to protect the enzyme against X-ray or H 2 O 2 induced inactivation. Moreover, addition of dithiothreitol after X-irradiation or H 2 O 2 treatment allowed a considerable repair of enzymic activity. 2) Polyacrylamide gel disc electrophoreses of X-irradiated enzyme solutions, performed in the presence of sodium dodecyl sulfate, showed the occurrence of covalently cross-linked subunits (preferably dimers and trimers) and of various definite fragments. Electrophoreses in the absence of the denaturant indicated the occurrence of enzyme aggregation. The effects were more pronounced with increasing X-ray doses. The electrophoreses also clearly reflected a radioprotec­ tion by dithiothreitol against cross-linking, but not against fragmentation. Addition of excess of 2-mercaptoethanol or of dithiothreitol to the X-irradiated enzyme clearly demonstrated that part of the covalent cross-links were disulfide bridges; the aggregates themselves, however, were held together primarily by non-covalent bonds. Blocking of exposed enzyme sulfhydryls by means of Ellman's reagent prevented both covalent cross-linking and enzyme aggregation. 3) Similar electrophoretic patterns as found for the X-irradiated enzyme were obtained for the unirradiated enzyme after treatment with H 2 O i . The similarity of the electropherograms, as well as the reversible diminution of enzymic activity and the loss of sulfhydryls in the presence of H 2 O 2 , suggest an involvement of H 2 O 2 in the radiation damage of the enzyme. It seems plausible that oxidation reactions are responsible for the effects caused by X-irradiation or H 2 O 2 treatment.

Enzymes which hydrolyse ATP cause an exchange of 180 of labelled Pi in the presence of ADP. A theory for the evaluation of rate constants from an observation of the time dependence of the concentration of the various Pi species is presented. Application to the 180 exchange catalysed by myosin SI as observed by 31P-NMR shows excellent agreement with values of the rate constants determined earlier.

Enzymes causing an exchange of oxygens from P\ with the surrounding water oxygens are very common. A statistical model for the data evaluation for an observation of this oxygen exchange by isotope methods is presented. It is shown how different cases of inequivalence of the four Pi oxygens may be uncovered. The number of reversals of the oxygen exchange step on the enzyme and the apparent second order rate constant for the binding of Pi to the enzyme are obtained as a result of the data fitting procedure. Cobalt phosphatase, zinc phosphatase, and myosin subfragment 1 are treated as examples.

Alkylating NAD-Analogs, Glyceraldehyde-3 Phosphate Dehydrogenase, Half-of-the-Sites Reactivity co-(3-Bromoacetylpyridinio)alkyldiphosphoadenosines with alkyl chain lengths of 2 -6 me­ thylene groups inactivate glyceraldehyde-3 phosphate dehydrogenase from rabbit muscle. Half-of-the-Sites reactivity is observed in each case: The analogs are covalently bound to highly reactive cysteine residues in two of the four subunits. The remaining two subunits still bind N AD and the reactive SH-groups, although modified by SH-reagents of low molecular weight are not labeled by any of the brominated coenzyme models. This behaviour may be explained by the assumption, that the modification of 2 subunits induces structural changes in the neighboured unoccupied subunits which prevent any attack on reactive cysteine residues caused by fixation and orientation of the bromoketo-coenzyme analog when bound to the active center. Structural similarities of the covalently bound coenzyme analogs in the active center and the native ternary GAPDH-NAD-substrate complex suggest that half-of-the-sites reactivity is a natural characteristic of the enzymes catalytic mechanism.

Extracts of HeLa cell fractions were analyzed by DEAE-and phospho-cellulose chromatog­ raphy for their range of cyclic AMP-dependent and -independent protein kinase activities phosphorylating histone and/or phosvitin; extractions were by phosphate buffered saline (soluble protein kinases) and the non-ionic detergent NP-40 (membrane-bound protein kinases). The soluble fraction contained (i) cyclic AMP-dependent histone kinases type I and II as evidenced by their behaviour on DEAE-cellulose and inhibition by the specific heat-and acid-stable protein kinase inhibitor (PKI) in a dose-related manner; both types I and II as well as their purified catalytic subunit also phosphorylated protamine and — with very low efficiency -casein but not phosvitin; (ii) a histone kinase (H), insensitive to cyclic AMP and PKI, also accepting protamine as substrate but not either casein or phosvitin; (iii) a phosvitin kinase (P), insensitive to cyclic AMP and PKI, which also phosphorylates casein but not histone or protamine. These four enzyme species were also found in NP-40 extracts of 27000x g residues which, however, contained further histone and phosvitin kinase activities as yet unspecified. NP-40 extracts of the microsomal fraction possessed, besides unspecified histone and phosvitin kinase activity, only the phosvitin kinase P and appeared to be devoid of histone kinases I, II, and H. The occurrence and ratios of the protein kinases classified suggest an ordered distribution over the diverse subcellular fractions of HeLa cells. The overall pattern of soluble and membrane-bound histone and phosvitin kinases in extracts of cervix carcinoma tissue, the in vivo correlate of HeLa cells, closely resembled that of similar extracts of HeLa cells. HeLa cells hence appear, despite their long in vitro history, to express protein kinase activities similar to those of their in vivo ancestors, recommending them as a subject for the study of (certain) human protein kinase systems.

Fatty Acids, Spodoptera littoralis, Eggs Gas-liquid chromatographic analysis of egg lipids during development of Spodoptera littoralis showed the presence of 13 fatty acids ranging in carbon chain length from caproic (C6:0) to linolenic acid (C 18:3). Palmitic, palmitoleic, stearic, oleic and linoleic acids composed over 90% of the total fatty acids. Palmitic acid was always the predominant fatty acid and oleic acid was the second most abundant.

Under culture conditions the egg nucleus of Trichoplax adhaerens reaches an unusually high and variable DNA content. Before the "fertilization membrane" is formed, the nucleus undergoes fragmentation. It is assumed that culture conditions differ from natural conditions by preventing the switch-over from the S-phase to the G 2-phase of the cell cycle: DNA replication continues and nuclear division is impossible. Cleavage ceases at an earlier or later stage.

Large scale extraction and assay of the lymphocyte chalone (LC), the natural lymphocyte proliferation inhibitor, require optimized, reproducible and standardizable methods. To this end we compared the effects on LC yield of storing thymus at different temperatures after collection from calves: Both quick freezing on solid CO2 and slow freezing at -5 0 °C led to a 30% loss of LC yield relative to that from unfrozen tissue kept at 0°C . Moreover we found that the apparent decrease or total loss of LC activity upon storage of a purified LC fraction may result from an occasional lack of LC-responsiveness of human blood lymphocytes depending on the donor's physiological state, in spite of normal PHA reactivity. This suggested the use of LC-responsive, cryopreserved lymphocytes, the advantages of which are documented and discussed in detail.

When incubated for 30 min in vitro, 4-hydroxyalkenals in a 5 x 1 0 ~ 3 m solution react with SH-groups of soluble cytoplasmic and nuclear proteins (ProtL-SH) of Morris hepatomas 9618 A and 5123 tc, and of Ehrlich ascites tumor (EAT) cells. The extent of the reaction strongly increases with decreasing doubling time of the respective tumors. Former experiments with EAT cells have shown that the reaction with cytoplasmic ProtL-SH causes inhibition of respiration, glycolysis, and probably of other SH-controlled processes associated with cell division. The intranuclear reaction leads to an inhibition of DNA-and RNA-synthesis. In a 2 x 10-4 M solution of hydroxyalkenals, however, the reaction with cytoplasmic ProtL-SH diminishes almost completely so that no mea­ surable inhibition of respiration and only 3% inhibition of glycolysis are observed, while 20 to 30 percent of the nuclear ProtL-SH are still blocked. This corresponds well with a previous obser­ vation that a 2 x 10-4 m solution of hydroxypentenal inhibits DNA-synthesis by 90 percent.

The accessory sex glands (ASG) of adult male Cecropia contain an enzyme that methylates juvenile hormone acids (JH-acids) in the presence of S-adenosyl-L-methionine (SAM). The methyltransferase is highly specific. The reaction rates decrease in the order JH-I-acid, JH-II-acid and JH-III-acid; in each case the natural enantiomer is esterified predominantly. Methyltrans­ ferase activity with the same substrate specificity was also demonstrated in adult female corpora allata (CA). Male CA have only marginal methyltransferase activity. The CA of male H. cecropia contain substantial amounts of JH-I-acid and JH-II-acid (minimum: 5 pmol/pair). When kept in organ culture, they release JH-acids into the medium. Radiolabeled propionate and mevalonate are incorporated efficiently into the carbon skeletons of the JH-acids. The enzyme system performing these transformations cannot be forced to produce JH-III-acid even in the presence of high mevalonate concentrations, though homomevalonate may enhance biosynthesis of JH-I-acid and JH-II-acid more than tenfold. It becomes evident that the regulation of JH titer balances with regard to the homologous structures during insect development is not merely a question of the availability of low molecular weight precursors, but in addition that of highly specific enzymes acting as regulatory entities in the later steps of the biosynthetic sequence.

Membrane-Phloretin Interaction, Infrared Raman, ESR Spectroscopy The transport inhibitor phloretin was bound to human red cell membrane and the concomitant structural changes were observed by spectroscopic methods. By the spin labeling method a decrease in fluidity of the membrane was found at 1 and 10 |iM concentrations of the reagent. This result was obtained with the 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and the 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl lipid spin labels. Infrared spectroscopy of modified membranes revealed an intensity increase of the POO~ band at about 1250 cm-1. Moreover, a shift of the peak at 1050 cm -1 to 1100 cm-1 was observed in the presence of phloretin. Raman spectroscopy of the membranes did not contradict the results found with infrared and ESR spectroscopy: In the phloretin modified membrane we observed a lack of the band at 1085 cm-1, which leads to suggest that the POO" and/or C-C regions are less fluid. Changes of the extracted red cell membrane lipids were less characteristic, and the results differed from those found in red cell membrane.

Investigations on the dose response of cell fusion, induced by ionfree aqueous polyethylene glycol (PEG) solutions, reveal distinct lowest fusogenic PEG concentrations for different permanently growing mammalian cell lines. Part of the requisite PEG can be replaced by carbo­ hydrates, preserving the fusogenity of the solutions. This discriminates two effects of PEG solutions causing cell fusion: a) cell shrinkage, the required hyperosmolality of the solutions may be provided by PEG or by carbohydrates, is supposed to cause intracellular processes necessary for consolidating polycaryons; b) membrane alterations, which can not be induced by carbo­ hydrates, enable intimate cell-cell contact via particle-free membrane areas. Depending on cell line salts can not only raise the osmolality of PEG solutions but are able to co-operate with PEG in generating membrane alterations.

Various drugs known or expected to increase the levels of cyclic nucleotides in cells were applied to isolated superfused frog retinae, and their influence on the aspartate-isolated a-wave was studied. Isobutylmethylxanthine (IBMX), triacetylguanosine (TAG), and dimethylaminopurine (DAMP) strongly influenced the responses elicited from dark-adapted retinae by flashes of light With all three drugs the response amplitude was increased, and latency and time to peak were prolonged. If, on the other hand, the retinae were light-adapted by background light of various intensities, the drugs showed different effects on the response amplitude: IBMX either did not influence the amplitude at all or even caused a decrease (4 of 6 experiments), DAMP decreased the amplitude and TAG caused an increase of the amplitude in 2 of 3 experiments. But latency and time to peak were still prolonged by all three drugs. When dark-adapted retinae were superfused with IBMX or TAG Ringer solution and simul­ taneously calcium concentration was raised, different effects of calcium on the three measured parameters of the a-wave were observed: By increasing the extracellular calcium concentration the increase of the amplitude caused by the drugs was reversed, down or even below the control level, whereas latency and time to peak remained prolonged. Thus, both an increased calcium level and light adaptation had the same effect, namely to reverse only that part of the drug effect concerning the amplitude but not latency or time to peak of the response. The data suggest that calcium and cyclic nucleotides act through different ways in the rod cells.

Notholaena fendleri, Polypodiaceae, Farinose Exudate, Novel Flavanone Notholaena fendleri is a rare representative of the genus, growing mainly in Colorado/USA. The white farinose exudate on the lower surface of its pinnules is composed partly of waxy material and partly of flavonoid aglycones. The flavonoid moiety consists mainly of methyl derivatives of the flavanones naringenin and eriodictyol. The eriodictyol methyl ethers are rare compounds; eriodictyol-7 methyl ether is a novel natural flavanone. Three flavones and two flavonols are found as trace constituents only. — The farina flavonoids of N. fendleri form a very typical pattern that is characteristic for this species.

Dryopteris filix-mas (L.) Schott, Polypodiaceae, Flavan, Dihydrocoumarin, Lactone From acetone extracts of fresh aerial parts of Dryopteris filix-mas (L.) Schott a flavan-3-ol was isolated. Its structure was elucidated as 5-(3,4-dihydroxyphenyl)-3,3a,4,5-tetrahydro-4,8-dihydro-xy-2H-pyrano[4,3,2-ife]-l-benzopyran-2-one by means of spectroscopic methods including l3C-NMR.

a-Formylphenylacetic Acid, Phenylmalonic Semialdehyde, Tropic Acid Biosynthesis a-Formylphenylacetic acid, the postulated immediate precursor of tropic acid, has been synthesized by deacetalization of a-diethoxymethylphenylacetic acid in the presence of silica gel. The compound was reasonably stable in organic solution. In aqueous media, however, a pronounced lability of this semialdehyde was observed at various pH-values (t/2 = 4.5 min at pH 7.4). It is thus very unlikely that this compound can be employed successfully in biosynthetic studies.

The different metabolic behaviour of endogenously produced [1-14C] pyruvate derived from [3,4-14C]glucose and of exogenously added [1-14C]pyruvate were studied with synchronous yeast cell populations, under conditions which differentially influenced the activities of pyruvate decarboxylase in the cytoplasm and pyruvate dehydrogenase in the mitochondria. Endogenously produced [1-14C]pyruvate is decarboxylated almost exclusively by PDC under anaerobic condi­ tions, in contrast to added [1-14C]pyruvate which is decarboxylated under aerobic conditions by the action of PDH mainly. Whereas 14CO 2 evolution from exogenous [l-14C]pyruvate can be diluted proportionally by addition of pyruvate, this is not the case for 14CO 2 evolution from endogenous [1-14C]pyruvate. It is suggested that glycolysis or at least some constituents of it might be arranged in such a manner that the resulting complex behaves as a functional compartment, meaning that it is almost inaccessible to exogenously added pyruvate.

Chalcone synthase activity was demonstrated in enzyme preparations from flowers of defined genotypes of Matthiola incana (stock). The product formed from 4-coumaroyl-CoA and malonyl-CoA was naringenin and not the isomeric chalcone, because chalcone isomerase was also present in the reaction mixture. Chalcone synthase activity could be detected only in flower extracts of genotypes with wild-type alleles at the locus f Thus, the interruption of the anthocyanin pathway in white flowering lines with recessive alleles (ff) of this gene is clearly due to a lack of this enzyme activity. Independent on the genetic state of the locus b which controls the formation of pelargonidin or cyanidin, respectively, in the flowers, 4-coumaroyl-CoA was the only suitable substrate for the condensation reaction.

L-Alanine: aldehyde aminotransferase occurs ubiquitously in higher plants. The enzyme catalyzes the reaction: L-alanine + monoaldehyde -> monoamine + pyruvate; it is responsible for the formation of aliphatic plant amines and involved in the biosynthesis of hemlock alkaloids as shown by Roberts. A continuous coupled photometric test was developed to determine the low activities of the transaminase. The enzyme from the "amine-free" plant Spinacia oleracea was purified 77-fold and separated from other aminotransferases. A comparison of the Spinacia enzyme with that isolated from spadix-appendices of the amine-producing Arum maculatum during anthesis revealed very similar characteristics in pH-dependence, ATm-values for alanine and aliphatic aldehydes, and inhibition by 2-oxoacids. In contrast to the Spinacia enzyme the Arum aminotransferase is rapidly inactivated in the absence of pyridoxal-5'-phosphate. The enzymes of S. oleracea, A. maculatum and Mercurialis perennis are localized in mitochondria, but not in chloroplasts or peroxisomes. The results are discussed in relation to the function of alanine: aldehyde aminotransferase in secondary metabolism. It is suggested that some enzymes may be expressed in plants at low levels, even in the absence of any metabolic function.

Nitrodiphenylethers substituted with Cl, CF3, and other comparatively simple substituents are compared with respect to their primary effects on the photosynthetic apparatus. These are direct electron-transport inhibition, energy-transfer inhibition (of ATP synthesis), and pigment bleach­ ing, the latter accompanied by decrease of electron transport activity. Certain substituents and their positions at the diphenylether skeleton can be correlated with a particular influence on these phytotoxic effects. The unicellular alga, Scenedesmus, is an appropriate species to fumisch quantitative data on these primary herbicidal modes of action within a 24-h assay period.

Subjecting pea chloroplasts to hydrogen ion gradients results in small but significant changes in the absorption and circular dichroic spectra. The relative intensities of the fast and slow fluorescence decays are modified by the hydrogen ion concentration. The life time of the slow components is only slightly altered. At pH 11 the circular dichroic peak at 690 nm, assigned to Photosystem I chlorophyll-protein complex, is clearly resolved. Also, there is a splitting of the peak at 650-640 nm, attributed to Photosystem II chlorophyll-protein. At pH 3.9 splitting of the 650-640 nm peak is observed. There is also a positive component at 705 nm which may be associated with an aggregated form of chlorophyll or pheophytin. The changes in absorption spectra are determined by measuring the difference in spectra between identical chloroplasts suspensions one at pH 7.6 and the other at alkaline or acidic pH. At pH 2 and pH 3.7 there are time dependent increases in absorbance at 430, 445, 520, 538, 670 nm, and a decrease in absorbance at 606 nm. After a delay of 11 min at pH 2, there is also an increase in absorbance at 700 nm. The latter is interpreted as possible formation of aggregated pheophytin. At pH 11.0 there are time dependent increases in absorbance at 638, 454 and 440 nm, and decreases in absorbance at 675, 660, 508 and 472 nm. The changes in absorbance of the pigment around 520 nm (associated with an electrochromic effect) is interpreted as resulting from a leakage of hydrogen ion across the thylakoid membrane. The spectral changes that occur during the first 9 min are reversible. After the chloroplasts have incubated for more than 15 min most of the spectral changes are not reversible.

Halogenated naphthoquinones act as inhibitors of photosynthetic electron flow. I50 concentra­ tion for inhibition of methylviologen reduction were found to range between 2 × 10-5 m to 2 × 10-6 M. Comparing their effects on several partial reactions of electron flow, the inhibition site of the naphthoquinones was found to be at the reducing site of PS II. Studies of fluorescence transients in presence of halogenated naphthoquinones give further evidence for a site action similar to that of diuron and different to that of DBMIB. All naphthoquinones act as quenchers of chlorophyll fluorescence with pure chlorophyll a, and with much higher efficiency in green algae and chloroplasts. It is concluded, that the halogenated naphthoquinones act similar to PS II-inhibitors like diuron, but do not share a common binding site at the PS II-complex. Implications of a possible involvement of phylloquinone K 1 in photosynthetic electron transport are discussed. The synthesis of 2-chloro-as well as 2-bromo-3-isopropyl-1,4-naphthoquinone is described.

Electron Acceptors, Photosystem II, Quinones and Quinonediamines Dichlorodimethoxy-/?-benzoquinone (DCDMQ) was tested for its site of action in the photo­ synthetic electron transport chain. Hill reaction mediated by DCDMQ was insensitive to DBMIB (1 nm) but sensitive to DCMU, suggesting its site of action before plastoquinone but after Q -the primary electron acceptor of photosystem II. Extraction of freeze-dried chloroplasts with heptane and analyzing their capacity to photo-oxidize water using various Hill oxidants revealed that silicomolybdate (SiMO) and DCDMQ could effectively restore the activity. Diaminodurene (DAD) in the presence of ferricyanide could restore 40% of the activity. But ferricyanide alone failed to restore the ability to photo-oxidize water in heptane extracted chloroplasts. Similarly, N a2S 0 3 which is known to cause a bottleneck in the electron flow at plastoquinone affected the ferricyanide Hill reaction. Hill reactions mediated by SiMO and DCDMQ were insensitive to the addition of Na2SO3, suggesting that both these oxidants intercept electrons before plastoquinone. But 50% of the activity was lost when sulfite was added to the Hill reaction mediated by DADox. DNP-INT, melittin and picrylhydrazyl were recently introduced as photosystem II inhibitors inhibiting the electron flow between Q and the PQ pool. While DCBQ and DCDMQ Hill reactions were insensitive to DNP-INT, ferricyanide was highly sensitive. The quinonediamines TMPD and DADox showed 50% decrease in the electron transport rate, similar to heptane extracted or sulfite inhibited chloroplasts. Melittin increased the electron transport rate when ferricyanide or TMPD was the Hill oxidant, while DCBQ and DCDMQ reduction remained unaffected. However, DADox Hill reaction showed 50% inhibition in the presence of melittin. Picrylhydrazyl - which inhibits the electron flow between Q and the PQ pool - inhibited the Hill reaction of all the PS II electron acceptors except that of DCDMQ. It is possible that there is another site of intercepting electrons between Q and plastoquinone before the site where most of the quinonediamines accept electrons.

A new set of tobacco mutants was obtained by selfing a single variegated plant which emerged in a seed lot of Nicotiana tabacum var. Consolation. The seeds obtained from this mutant give rise to four phenotypes: variegated, yellow, yellow-green, and green seedlings. The green, yellow-green and yellow characters are due to two interdependent nuclear gene factors. The yellow-green phenotype is the homozygous (aabb) true breeding condition, whereas the green and the yellow phenotype are heterozygous (AaBb) with respect to both nuclear factors, the difference in the yellow and green phenotype being the addition of a labile gene factor pair, Cc, in the yellow condition. If photorespiration is measured as the Warburg effect or as 180 2-consumption by mass spectrometry it appears that the heterozygous green phenotype is the defective condition with high photorespiration. The three phenotypes differ with respect to chlorophyll content and photosynthetic unit sizes, the photosynthetic unit size in the yellow phenotype being approxi­ mately 1/10 of that of the green type. The gene expression for photorespiration (measured as 180 2-uptake for example) in the heterozygous green type is suppressed by the addition of the labile gene factor pair Cc in heterozygous condition which leads to the yellow phenotype. In the yellow and green phenotype the photosynthetic unit size is different but not the ratio of photosys­ tem I/photosystem II activity. Moreover, from the present studies it appears that the Warburg effect i. e. an increase of photo­ synthetic rate upon anoxia, can only partly be due to an inhibition of ribulose 1,5-biphosphate oxygenase or glycolate oxidase.

The uptake of auxin (LAA), abscisic acid (ABA) the synthetic cytokinin benzylaminopurine (BA), gibberellic acid (GA3) and mevalonic acid (lactone) (MVA) into intact chloroplasts and protoplasts from spinach mesophyll cells was measured and the permeability coefficients Ps of the chloroplast envelope and the plasmalemma were calculated. With all solutes tested uptake and P s values were considerably higher in the chloroplast system than in the protoplast system. At an external pH of 7.0, rates of uptake exhibited the order BA > LAA > MVA > ABA > GA3 in both systems. However, the P s values (corrected for the undissociated species of the solutes) exhibited the order LAA > GA3 > (ABA or BA) > MVA. This corrected sequence indicates the theoretical capacity of penetration under the assumption that preferentially the protonated species of phytohormones are capable of readily penetrating membranes. P s values for phytohormones appeared largely to be determined by the distribution coefficient K d and to a lesser extent by the molecular weight (Mr). In the Collander-plot the relation between the logarithm of Ps values for phytohormones and some other solutes such as acetate, glycerol, glucose, sorbitol and sucrose and the logarithm of K J M T 1.5 approached linearity.

A compartmental analysis of the distribution of the plant hormone indole-3-acetic acid (IAA) was performed with mesophyll cells of spinach leaves. The results indicated that up to about 45% of the total amount of free IAA in the laminae of spinach is localized within the chloroplasts, although these organelles occupy only about 7% of the tissue volume. This distribution is due to the dissociation properties of IAA which has a pK a of 4,7. The chloroplast envelope is easily permeable for the undissociated acid (IAAH) and less permeable for the anion (IAA"). The rate of uptake of LAA by chloroplasts was linearly dependent on the IAAH concentration gradient between medium and stroma. Uptake was increased by lowering the extraplastidic pH or by alkalization of the stroma during illumination. In illuminated chloroplasts, IAA was accumulated up to 4 fold compared to the surrounding medium owing to the pH gradient between the medium (pH 7.6) and the stroma (pH 8.0). The unexpectedly high plastidic IAA concentration observed in the dark (concentration ratio stroma/medium was about 2) suggested binding of IAA to chloroplast components. Due to this binding, the IAA distribution between medium and chloroplasts must not be used for calculating the stroma pH, as is possible with ABA. However, from the different distribution of IAA in light and darkness the light-induced alkalization of the chloroplast stroma can be calculated.

Qualitative X-ray microanalysis of frozen-dried cyclo-stome, teleost and amphibian yolk platelets has demon­ strated the general and undoubtable presence of P, S, Cl (!) and K, the probable presence of Na and the irregular presence of Ca and Mg. Iron occurred only in trace amounts if at all. Details of the process of yolk utilization by the embryo are still enigmatic. Indirect evidence ob­ tained from particular features of the glycolipophos-phoprotein molecules building up the yolk platelets as main constituents has, therefore, become a matter of recent interest. Thus, the highly phosphorylated phosvitins ("cation exchange-resin") may play a role as carrier of cations essential for embryonic growth (Taborsky [1]); studies on cation-binding as well as on the entailing conformational changes of the phos­ vitin molecule are numerous (Taborsky [1, 2]). Re­ cent observations on yolk platelets in this laboratory, however, do not fit into the frame of a very specific role of yolk-phosphoproteins in the transport of selected cations. Although the investigation has been performed only on the usual qualitative basis, the broad collection of material and the regularity of findings render the observations significant. Cryosections of whole eggs (Lampetra planeri [Bloch], Cyclostomata; Pelvicachromis pulcher, Cich-lidae, Teleostei; Triturus sp., Salamandridae, Am­ phibia) or isolated yolk platelets (M yxine glutinosa L., Cyclostomata) were dried at subzero temperatu­ res onto pure carbon specimen supports for scanning electron microscopy. Lamprey and teleost material fixed in Na-phosphate buffered glutaraldehyde was used for comparison. Elemental analysis was carried out at 25 kV in a Philips PSEM 500 scanning elec­ Reprint requests to Prof. Dr. R. H. Lange. 0341-0382/81/0700-0686 $01.00/0 tron microscope equipped with energy-dispersive Edax system (3 -4 spectra -100000 to 400000 total counts each — per species, point probe diameter 1 |im). Ooplasmic inclusions corresponding both in size and frequency to yolk platelets of the various species were identified in the scanning mode and a point probe was then positioned on their interior. Ele­ ments markedly and undoubtedly present in all un­ fixed samples and absent in the carbon support be­ tween single platelets were: P, S, Cl, and K. Ca was also present in Lampetra, Pelvicachromis and Triturus (as judged from its clear K β peak at 4.01 keV, while Ca-K or at 3.69 keV fuses with K-K β at 3.59 keV). Due to the peculiar background spectrum (Lange and Blödom [3]) we shall not enter into detailed dis­ cussion of the probable presence of some Na and Mg. Fe was never unequivocally demonstrated, but traces might have occurred in the cyclostomes. Whereas P and S probably represent covalently bound protein constituents, the presence of chloride (only irregularly present following fixation, abso­ lutely absent from neutral underground) in this highly anionic protein ambiance is remarkable. Re­ sults with respect to iron were disappointing, espe­ cially considering recent biochemical work (Tabors­ ky [2]). It is concluded that K+ is regularly and undoubt­ edly present in considerable amounts in yolk pla­ telets; this holds true for Ca2+, but to a lesser degree (not found in M yxine). The occurrence of Na+ (and in Lampetra some Mg2+) is probable but evidence is at present unsatisfactory for technical reasons. The presence of traces of Fe cannot be excluded. Due to the presence of chloride, the above cations can only in part be bound by the phosphate groups of the highly anionic yolk-platelet proteins. By combining the recent results in yolk-platelet crystal research (Lange [4]; Ohlendorf et al. [5]) with morphometry of the ooplasm and attempts at micro-analytic quantitation, important quantitative data on both a relative and absolute scale will eventually become available.

The authors have suggested (Can. J. Bot. 49, 631 (1971) that the — COOH group of the C4-P carbonyl compound of the thermostable P-enolpyruvate acid carboxylase reaction may transcarboxylase with a pentose phosphate as ac­ ceptor. We now have considerable evidence supporting this consideration. In an assay system (0.8 ml) containing soni­ cated chloroplast extract in 0.1 m Tris-HCl, pH 6.3; and PEP, 0.1 |imol; Mg2+, 0.5 nmol; NADH, 0.25 nmol; sugar phosphate, 0.5 nmol. The amount of 14C 0 2 fixation is con­ siderably enhanced by either ribose-5-phosphate, fructose-1,6-bisphosphate or ribulose-l,5-bisphosphate in the pres­ ence of PEP. The products of the reaction include malate as a product of /?-carboxylation, and glycerate or 3-phosphate glycerate, their proportion being determined by the ac­ ceptor sugar phosphate. The results provide evidence for a "transcarboxylase" presented in the crude extract of Maize chloroplasts.

Agmatine ureo: hydrolase, which produces both putres-cine and urea from agmatine, and urease, which hydrolyzes this urea, are highly restricted to the phycobiont cells in E. prunastri thallus. Arginase and L-arginine decarboxylase, which catabolize L-arginine, have a more extensive dis­ tribution between both symbiotic partners.

Normal Drosophila males produce a volatile pheromone which inhibits courtship. This chemical cue is not identical to the pheromone made by mated female flies, which also inhibits male sexual behavior. Mutant olfC males, which fail to respond to several organic compounds, are not inhibited by the pheromone made by males; hence, unlike normal sexually mature males, olfC males court each other vigorously.