Band 37, Heft 5-6

The structure of echinacoside, isolated from Echinacea angustifolia D.C. (Asteraceae) roots has been determided by 1 H-NMR-and 13 C-NMR-spectra to be β-(3′,4′-dihydroxyphenyl)-ethyl-O-α-ʟ-rhamnopyranosyl (1 → 3)-O-β-ᴅ-[β-ᴅ-glucopyranosyl (1 → 6)]-(4-O-caffeoyl)-glucopyranoside (cf. Fig. 1).

Several prenylated naphthalene derivatives were synthesized and characterized by spectroscopic methods. One of these compounds, 2-methoxycarbonyl-3-prenyl-1,4-naphthoquinone, was detected as a new natural product in the root system of Galium mollugo L. This new natural product is likely to be related to the biosynthesis of mollugin and anthraquinones.

13 C-NMR analysis of the isoflavonoid phytoalexins phaseollin and kievitone produced by feeding sodium [1,2- 13 C 2 ]acetate to wounded bean (Phaseolus vulgaris) cotyledons has demonstrated the incorporation of intact acetate units into the aromatic A rings. Phaseollin shows a specific folding of the polyketide chain, whereas kievitone exhibits a randomisation of label in accordance with the intermediacy of a 2′,4′,6′-trihydroxylated chalcone during its formation. In neither case was sufficient label incorporated into analysis.

Fluctuations of quinolizidine alkaloid content in leaflets of Lupinus polyphyllus, L. hartwegii, Baptisia australis, and Sarothamnus scoparius were studied over a 36 h period. The alkaloid contents reached their maximum at noon or early afternoon, and their minimum during the night. The amplitudes of diurnal variations in alkaloid levels lay within a range of 60 to 470% of the early morning contents of alkaloids (= 100%) in leaves. In photomixotrophic cell suspension cultures of L. polyphyllus and S. scoparius cultured under day-night regime, a similar increase of the alkaloid levels was observed within 5 to 8 h after onset of illumination. A subsequent excretion of the alkaloids produced, which also followed a diurnal rhythm, was found. S. scoparius cells excreted lupanine, L. polyphyllus cells 13-cinnamoyloxylupanine. From the cell culture and plant experiments it can be assumed that the alkaloids which are formed during illumination are translocated to some degree afterwards. In cell suspension cultures the alkaloids are subjected to rapid and rhythmic turnover. Exogenous alkaloids, fed to the cultures, are taken up by the cells and disappear usually within the first 72 h. A possible mechanism for a light-mediated regulation of quinolizidine alkaloid biosynthesis which was found to be localized in leaf chloroplast, is discussed.

Volatile cephalic secretions from females of four cleptoparasitic bee species, Epeolus cruciger (Pz.), E. variegatus, (L.) (Anthophoridae) and Coelioxys (Coelioxys) quadridentata (L.), C. (Schizocoelioxys) mandibularis Nyl. (Megachilidae) were analysed by combined gas chromatography/mass spectroscopy. There are considerable similarities between the two species within each genus, and also some qualitative correspondences between the two genera. The Epeolus species contain spiroacetals, 2-alkanols and pyrazines while the Coelioxys species contain spiroacetals, 3-alkanols and 3-ketones along with unsaturated alcohols. Two of the spiroacetals are first found in nature.

A glucosamine-P isomerase has been identified in Proteus mirabilis. The 113-fold purified enzyme exhibits a pH optimum of 7.5 with a secondary maximum at 8.5 and a temperature optimum at 37 °C. The apparent K m was 13.3 mᴍ for fructose-6-P and 18.8 mᴍ for ʟ-glutamine. Molecular weight of the enzyme has been estimated as 120000 and the protein can be dissociated in four subunits by SDS-polyacrylamide electrophoresis.

Most dyes related to trypan blue inhibited hemolytic activity of oligonucleotide-streptolysin S (SLS) complex, an exotoxin produced by Streptococcus pyogenes. Order of the inhibition was: trypan blue > benzo blue 2B > Congo red > Evans blue > benzo purpurine 4B > thiazine red > trypan red. When resting streptococcal cells were incubated with these dyes, significant amount of the hemolysin was produced. The carrier (or inducing) activity for SLS was further manifested in growing cell system and the potency of the compounds was as follows: Congo red > benzo blue 2B > trypan blue > Evans blue > Benzo purpurine 4B > thiazine red > trypan red. In this system, Congo red was more effective than oligonucleotide fraction rich in guanyl residue. Chromotrope 2B, H acid and o-tolidine were ineffective, as the carrier as well as the inhibitor. Based on these results, structure-function relationship among SLS, the carrier and the inhibitor was discussed.

2-(α-hydroxybenzyl)-, 2-benzyl-, 2-phenyl-, 2-methyl-and 2-hydroxymethyl-benzimidazoles were screened for antiviral activity against TMV in tomato leaf discs, for cytokinin activity in the amaranthus betacyanin bioassay and the soya bean callus bioassay and for antagonism in the latter bioassay. None of the test compounds showed either cytokinin activity or antagonism. 2-(α-hydroxybenzyl)-benzimidazole at 23.3 μᴍ inhibited infectious virus production by 100% and no virus antigen was detectable. None of the other compounds showed similar antiviral activity. The realationship between the substituent at the 2-position and antiviral activity was as previously reported for 2-substituted benzimidazoles in animal virus chemotherapy.

In order to show that for a possible application in medical diagnosis NMR-relaxation experiments at low Larmor frequencies (v 0 ≤ 20 kHz) are more sensitive than the up to now done high field measurements in the MHz-range, we present dispersion curves (v 0 = 50 Hz to 50 MHz) of the proton longitudinal relaxation time T 1 and values of the transversal relaxation time T 2 for the example of amniotic fluids. Only for Larmor frequencies below ≃ 100 kHz the relaxation times for healthy amniotic fluid and pathological meconium solutions are significantly different, whereas at high Larmor frequencies, i. e. in the conventional MHz-range, the observed changes are rather small.

Anti 5-methyl-cytidine antibodies might be useful agents for the detection and localization of 5-methyl-cytidine of nucleic acids, but only if the antibodies recognize this nucleoside with sufficient specificity. A conjugate containing 18 moles of 5-methyl-cytidine per mole of BSA was prepared and antibodies directed against this nucleoside hapten were produced by immunization of rabbits (as determined by gel diffusion in agar containing excessive amounts of the carrier). A slight crossreaction of cytidine-BSA was eliminated by adsorption on the cross-reacting antigen. Further purification of the antibodies was effected by chromatography on DEAE-Sephadex A-50 and a method for the rapid quantitation of the antibodies showed that 12.7% of the IgG protein are monospecific against 5-methyl-cytidine-BSA. Hydrolysis of antibodies with insolubilized pa­pain produced monovalent Fab fragments which were identified by SDS-Disk-electrophoresis. A two stage method for cross linking the immunoproteins to ferritin by glutaraldehyde was used. The isolation of immunoferritin conjugates by Bio-Gel A 1.5 m column chromatography is described. The identification of the effluents was made by glycerin density gradient ultracentrifugation. The results were visualized by electron microscopy after the treatment of immunoferritin conjugates with (methylated and unmethylated) denaturated DNA, fractionation on the glycerine density gradient, and the spreading by a modification of drop technique.

Polyriboadenylic acid, polyribocytidylic acid and polyribouridylic acid were irradiated with 16 MeV electrons in aqueous solution. Part of the OH radicals generated during the radiolysis of water reacted with the sugar moieties of the polynucleotides. Subsequently formed peroxyl radicals reacted with each other thus forming oxyl radicals whose's decay led to main-chain scissions with the consequence of a decrease of the light scattering intensity (LSI). In the case of single-stranded polynucleotides, the analysis of the LSI decay curves revealed the occurrence of two processes. The rapid mode was assigned to the intramolecular and the slow mode to the intermolecular reaction of peroxyl radicals. For the rate constant of the rapid process (activation energy: 5 kcal/mol) a pronounced kinetic salt effect was observed. In the case of double-stranded polynucleotides (polyA and polyC at pH 4, poly(A+U) at pH 8) the same effects were observed as with native calf thymus DNA in a former study [9]: The LSI decreased only after a critical dose was surpassed. A rapid process was followed by a slow one. The slow process is assigned to the melting of H-bridges located at bases between main-chain scissons in the double helix. The rapid process corresponds to the separation of fragments formed by two single strand breaks laying very close to each other on opposite sites in the double helix.

The binding of the dye cations acriflavine AF, tetramethylacriflavine TMAF and acridine orange AO (scheme of structures) to calf thymus DNA has been investigated by means of absorption spectroscopy, Table I. In order to avoid dye association we used very low dye concentrations and sufficiently high DNA concentrations. In this case we got linear Scatchard isotherms. The formal Scatchard binding constant K strongly depends on the salt concentration Cs (S = NaCl) of the solution and the temperature T (278 - 303 K), K (C S , T). The average value of binding sites per mononucleotide is n = 0.17. It is independent of the dye species and of C S and T. The value of r (bound dye cations per mononucleotide) diminishes with growing salt concentration C S (C S ≲ 1 ᴍ). At sufficiently high salt concentrations r is approximately constant (C S ≳ 1 ᴍ). Obviously there are two types of binding of the dye cations to DNA even in the domain of linear Scatchard isotherms. They can be distinguished experimentally with the competitive salt effect. To describe r(C S ,T ) or K (C S , T) we used a simple model with three equilibria: 1. Noncompetitive binding 1 (intercalation) of dye cations to n 1 C N binding sites (C N = concentration of mononucleotides), equilibrium constant K 1 . 2. Competitive binding 2 (external binding) of dye cations to n 2 C N binding sites, equilibrium constant K 2 . In contrast to type 1 binding, the dye cations in type 2 binding can be replaced by metal cations M of S (M = Na ⊕ ) at sufficiently high salt concentrations C S . 3. Competitive binding 3 of M to the same sites of 2 and the dye cations as competitor, equilibrium constant K 3 . The model agrees very well with the experiments on the condition n 1 = n 2 = n. Therefore the dye can be bound to one of the n C N binding sites either non-competitively or competitively. Type 1 and type 2 binding exclude one another at the same binding site in the domain of linear Scatchard plots. The binding constants K i (i = 1, 2, 3) have been determined by means of the competitive salt effect, Table II. They only are T dependent. From K(T) i we got the binding enthalpies ΔH i 0 and binding entropies ΔS i 0 , Table III. AF and AO cations are bound non-competitively and competitively, TMAF only competitively. In comparison with AF or AO the competitive binding of TMAF is much weaker. In the case of AF and AO K 1 is approximately one power of ten smaller than K 2 , K 1 ⪡ K 2 ! The binding enthalpies of the non-competitive and the competitive binding are nearly equal, ΔH 1 0 ≅ ΔH 2 0 . Therefore the difference in the binding constants K 1 , K 2 can be attributed to the difference in the binding entropies, ΔS 1 0 ⪡ ΔS 2 0 . Thermodynamically type 2 binding (external binding) is preferred to type 1 binding (intercalation). The binding enthalpy of Na ⊕ to DNA is in all cases nearly zero, ΔH 3 0 ≅ 0. Only the increase of entropy S 3 0 > 0 enables binding 3. From the thermodynamic data follows that type 1 and type 2 binding of AF and AO are produced by electrostatic and hydrophobic interaction which are intensified by hydrogen bonding. In contrast to this the weaker competitive binding of TMAF is caused by electrostatic and hydrophobic interaction only. Our investigations agree with former works on ethidium bromide E and tetramethylethidium bromide TME (scheme of structures, Tables II and III). They are consistent with the assignment non-competitive binding = intercalation, competitive binding = external binding.

The fatty acid chains of the lipid A component of lipopolysaccharides are hexagonally packed with a lattice periodicity of 4.1 Å. The smallest subunit of this lattice consists of a disaccharide to which seven fatty acid chains are linked representing the hydrophobic part. Carbohydrates and substituted phosphate residues linked to them form the hydrophilic part of the molecule. Because of sterical reasons and because of the necessity of a separation of hydrophobic and hydrophilic part, we could derive that the angles of the hydrocarbon chains with the planes of the two sugar residues should be as vertical as possible and the planes of both sugar residues should be approximately coplanar forming an angle of about 180° with one another. Conformation angles for all the theoretically possible linkages of the disaccaride and of the linkages of the fatty acid residues have been calculated. Between the two-N-acylglucosamine residues theoretically β-1,4, α -1,6 or β-1,6 linkages are possible. The experimentally found β-1,6 linkage has the largest degrees of freedom for its conformation angles.

A plasmid containing promoter-deleted inactive β-galactosidase gene [1] was used to select promoters of the pEP121 plasmid [2]. Colonies of cells harboring reactivated β-galactosidase gene were identified by their red color on McConkey plates. The quantitative amounts of β-galactosidase produced in each clone were estimated by assaying enzyme activity and by measuring the specific β-galactosidase protein following fractionation of total cells′ proteins on polyacrylamide gel. A wide range of enzyme activities was observed. The most active promoter isolated was shown to promote β-galactosidase production more efficiently, compared with the original β-galactosidase promoter, amounting to 20% of all cell proteins. Such highly active promoters may be utilized in the future, to promote expression of cloned genes in bacteria.

In highly purified bc 1 -complex from baker’s yeast, the reduction of cyt c 1 and partial reduction of cyt b is obtained by catalytic amount of succinate dehydrogenase and succinate in the presence of 7 μᴍ antimycin. After the addition of ferricyanide the c 1 is re-oxidized and a increase in the reduction of b is observed. Using stopped-flow we established that the oxidation of c 1 by ferricyanide proceeds as a pseudo-first order reaction and the reduction of b is faster and with two phases. Our observation suggests that these two processes are not directly interconected and that other component than c 1 must be the “control factor” in the anomalous reduction of cyt b. This component must be, by exclusion, the iron-sulfur protein.

Algae, when slowly cooled down to around -5 °C, undergo a change in the fluorescence emission spectrum subsequently taken at liqu. nitrogen temperature. This change resembles the magnesium effect described by Murata [BBA 189,171 - 181, (1969)] for isolated chloroplasts. Evidence is shown, that both effects are indeed analogous. Cooling the organisms seems to in­ crease the permeability of the thylakoids for cations and, thus, a depletion with concomitant changes in membrane structure. The system serves as a model for the probable in vivo control of pigment interaction through alteration of membrane properties.

Inhibition of photosynthesis by simazine or diuron, as well as disturbance brought about by darkness, fungal infections, heat, high concentrations or lack of CO 2 , lead to a luminescence of chloroplasts after irradiation with photosynthetically active light. This effect is the basis of phytoluminography, a new method which may replace in many cases the tedious procedures used at present to determine the photosynthetic capacity of living plant tissue. Both qualitative analyses of luminescence images produced by an image intensifier as well as quantitative measurements using a photomultiplier are possible. Autoradiographic studies have shown that luminescence images of herbicide-damaged leaves are identical with autoradiographs of the same leaf obtained after labelling with radioactive CO 2 . In contrast to autoradiography, phytoluminography does not damage the tested parts of plants nor is there danger of contamination. There is no need for expensive chemicals and skilled technicans, and immediate results are obtained. Quantitative measurements of phytoluminescence after the application of simazine and diuron (DCMU) to Elodea canadensis in submersed culture showed that approximately 1 ᴍ of inhibitor per 20 ᴍ of chlorophyll (a + b) is needed to obtain maximal luminescence, which indicated total inhibition of photosynthesis. Both luminescence and inhibition are reversible if the inhibitor is removed by dialysis.

Pre-illumination of Chlorella cells at room temperature with light primarily absorbed by PSII (650 nm) produces a state called state II. This is characterized by high fluorescence at 715 nm (high F715/F685) at liquid nitrogen temperature. Alternately if the cells are pre-illuminated by light primarily absorbed by PSI (710 nm) then state I with high fluorescence at 685 nm (low F715/ F685) at 77K is produced. We have investigated the role of photophosphorylation in the development of state I/II in chlorella cells using uncoupler and energy transfer inhibitors. The results suggested that the development of state I depends upon the energization of the membrane. Both DCCD and TPTC which permit build up of proton gradient block the adaptation to state I in­ dicating that proton gradient formation alone is not sufficient to develop state I. The data obtain­ ed by us as also published in the literature indicate that redox states o f the electron transport carriers are responsible for the development of state II.

Adaptation In saturating light radish seedlings grown in high-light growth conditions (90 W · m -2 ) possess a much higher photosynthetic capacity on a chlorophyll and leaf area basis than the low-light grown plants (10 W · m -2 ). The higher CO 2 -fixation rate of HL-plants is due to the presence of HL-chloroplasts which possess a different ultrastructure and also different levels of the individual chlorophyll-carotenoid-proteins than the LL-chloroplasts of LL-seedlings. 1. Ultrastructure: The high-light adapted chloroplasts are characterized by fewer photo­ synthetic membranes per chloroplast section, by low grana stacks (only few thylakoids per granum), a lower stacking degree of thylakoids, a higher proportion of non-appressed membranes (stroma thylakoids + end grana membranes) and a high starch content. The LL-chloroplasts possess no starch, their grana stacks are higher (up to 17 thylakoids per granum) and also significantly broader than that of HL-chloroplasts. 2. Chlorophyll-proteins: The photosynthetic apparatus of HL-chloroplasts contains a larger proportion of chlorophyll a-proteins of photosystem I (CPIa + CPI) and of photosystem II (CPa, the presumable reaction center of PS II) than the LL-chloroplasts which possess a higher proportion of light-harvesting chlorophyll a/b-proteins (LHCP 1 , LHCP 2 , LHCP 3 , LHCP y ). The higher levels of LHCPs in LL-plants are associated with a higher ground fluorescence fo and maximum fluorescence fp of the in vivo chlorophyll. 3. Chlorophyll and carotenoid ratios: The chloroplasts of HL-plants possess a higher proportion of chlorophyll a and β-carotene (higher values for the ratios chlorophyll a /b and lower values for a/c and x/c) which reflect the increased level of the chlorophyll a/β-carotene-proteins CPIa, CPI and CPa. The higher level of light-harvesting chlorophyll a/b-xanthophyll-proteins (LHCPs) in LL-plants is also indicated by an increased content of xanthophylls and chlorophyll b as seen from lower a/b and higher x/c and a/c ratios. 4. The results indicate that plants possess the capacity for an ontogenetic adaptation of their photosynthetic apparatus to the incident light intensity. The HL-chloroplasts of HL-plants which contain less antenna chlorophyll, are adapted for a more efficient photosynthetic quantum conversion at light saturation than the LL-chloroplasts with high grana stacks. The correlation between higher levels of light-harvesting chlorophyll a/b-proteins (LHCPs) and a higher stacking degree of thylakoids, and the involvement of LHCPs in stacking is discussed.

Completely dissolved components of native prolamellar body/primary thylakoids PLB/PT were induced to reaggregate into tubular structures in vitro. Incubation of these pchlide containing PLB-like tubules under red light with NADPH : pchlide oxidoreductase led to a specific association of the enzyme protein to the tubules. Photoconversion of the pigment initiates the dispersal of reaggregated PLB-tubules as indicated by changes in the content of their main constituent saponin.

The ability of the sarcoplasmic (endoplasmic) reticulum (SR, ER) of smooth muscle cells to accumulate Ca was demonstrated by measuring the uptake of 45 Ca in fibers which were chemically skinned with saponin, and by electron cytochemistry of the accumulated Ca. The Ca uptake was dependent on ATP and it was stimulated by oxalate, as it is the case in SR of striated muscle. Electron microscopy of the skinned smooth muscle preparations revealed the presence of calcium oxalate deposits in the reticulum. The SR vesicles were isolated from several smooth muscles. The purification was carried out by taking advantage of the density increase of the SR vesicles after loading with calcium in the presence of oxalate. Among the muscles investigated the smooth muscle of the pig stomach was found to be the most suitable and it was selected for further biochemical and morphological characterization of the SR vesicles. These vesicles, which contain calcium oxalate crystals, were able to accumulate an additional amount of Ca. The Ca uptake was supported by several energy yielding substrates. Their order of potency was ATP > dATP ≃ UTP > ITP > GTP ≃ CTP. The rate of Ca uptake was two orders of magnitude slower than that in SR of skeletal muscle. The measurement of the level of phosphorylated Ca transport intermediate showed that this difference is due to smaller number of calcium transport sites per vesicle. The distribution of intramembrane particles in freeze-fractured specimens is in agreement with this conclusion.

In the present investigation different finestructural and histochemical procedures were employed to demonstrate normal morphology and Ca 2+ -transport mechanism in bullfrog atrial myocardium. For normal morphology specimens were fixed in 1% OsO 4 , 2.5% glutaraldehyde or liquid propane at - 185 °C before they were prepared for conventional embedding and freeze-etching, respectively. Special interest was focussed on the caveolae system, which is composed of single, spherical membrane invaginations (diameter, 85 nm), randomly distributed at the entire cell periphery. The caveolae enlarge the cell surface by 24.5% and occupy 8.5% of the cellular volume. The caveolae membrane contains a few intramembraneous particles with a diameter of 8.4 nm comparable to the size of ATPases as found in other cells. For histochemistry the specimens were first stabilized by treatment with 50% glycerol, 0.025% glutaraldehyde or 0.15% formaldehyde and then incubated in a medium containing 4 mᴍ CaCl 2 , 4 mᴍ EGTA, 5 mᴍ MgCl 2 , 5 mᴍ K 2 C 2 O 4 , 5 mᴍ ATP and 20 mᴍ histidine at pH 7.0. This incubation always succeeded in the formation of electron-dense deposits with elliptical shape, measuring 100 -200 nm in length and 10 -30 nm in diameter. According to X-ray spectra they deliver a characteristic calcium-peak and can be found within two different cellular compartments: in small invaginations of the sarcolemma, i. e. the caveolae-system, and in the intrafibrillar sarcoplasmic reticulum. The elliptical deposits can be clearly distinguished from round electron-dense granules measuring 16 nm in diameter, which are located within randomly distributed small vesicles and composed of potassium and phosphate. Contrary to the elliptical deposits the round granules are also present in controls and seem to be identical with the so-called atrial granules. In comparison to observations obtained with the same method in other muscular systems and derived from various control experiments the results of this study favour the existence of an ATP-dependent Ca 2+ -pumping mechanism in frog atrial muscle bound to both, the sarcoplasmic reticulum and the caveolae system.

Single bovine ventricular myocytes were superfused with Tyrode solution containing 1.8 mᴍ CaCl 2 . The cells did not bear external load and contracted isotonically. Contraction and relaxation were characterized by the shortening and relengthening of the sarcomeres which resembled in their time course the isometric twitches of bovine papillary muscles. Resemblance was also found in regard to positive inotropic interventions as increase in the stimulation frequency, exposure to elevated [Ca] 0 or to adrenaline. A two-microelectrode voltage-clamp technique was applied to the single myocyte. The trans­membrane Ca inward current I Ca was defined as difference current sensitive to 5 mᴍ Ni or to 2 μᴍ D600. During a voltage step from -45 to + 5 mV, I Ca peaked within 3 ms to -6 nA, afterwards it decayed to 15% of peak amplitude (incomplete inactivation with a 2 exponential time course). Experiments in Na-free media suggested that Na entry does not significantly contaminate I Ca . Therefore, Ca entry could be calculated from I Ca . The increment in total intra­ cellular Ca concentration (Δ[Ca] i T ) was estimated by referring Ca entry to the cell volume (50 pl). Within 100 ms Δ[Ca] i T came to 25 μᴍ at control conditions, to 55 μᴍ at [Ca] 0 = 3.6 mᴍ and to 88 μᴍ when 0.1 μᴍ adrenaline were present. The Δ[Ca] i T values were sufficient to activate contraction without the necessity of Ca-release from SR. Despite the new data, the relationship between Ca entry and activation of contraction was complex: during the “positive Herztreppe” I Ca slightly attenuated but contractility doubled. Therefore, the old EC-model (M. Morad and Y. Goldman, Progr. Biophys. Mol. Biol. 27, 257 (1973)) was adapted. The Ca-entry’s capability to load and to overload the intracellular Ca store (SR) is discussed.

We measured fluorescence energy transfer between ATPase monomers labeled with either a donor or an acceptor fluorophore, in order to test the hypothesis that the presumably oligomeric sarcoplasmic reticulum ATPase would dissociate into monomers within the membrane on the addition of low nonsolubilizing amounts of a nonionic detergent, C 12 E 18 . We found that at low nonsolubilizing detergent concentrations below the critical micellar concentration (c.m.c.), there was indeed direct interaction between the detergent and the protein; however, only solubilizing detergent concentrations reduced fluorescence transfer to a minimum. Considering the current literature, fluorescence energy transfer proved insufficient, at the present time, to confirm or reject the hypothesis.

There is both structural and functional evidence for protein-protein interaction between Ca 2+ -ATPase polypeptide chains in the sarcoplasmic reticulum membrane. Studies on detergent solubilized ATPase indicate that the monomeric form is capable of performing a normal cycle of ATP hydrolysis, but some of the modulatory effects of the substrates disappear after detergent solubilization. However, the necessity of protein-protein interactions for the Ca 2+ transport function remains unclarified. A new approach is described, employing ATPase reconstituted with a large excess of phospholipid, which may help to resolve this question.

This paper summarizes true equilibrium measurements for some partial reactions of the sarcoplasmic reticulum calcium pump transport cycle. The most important result is the estimation of the equilibrium constant for the interconversion of the two major conformational states of the protein, E (Ca 2+ binding sites facing the cytoplasm) and E′ (Ca 2+ binding sides facing the sar­coplasmic reticulum lumen). The value of K 0 = [E′]/[E] cannot be evaluated directly by any method available at present, but observed cooperativity in the binding of Mg 2+ and Ca 2+ to unliganded protein strongly indictes that K 0 ⪢ 1. The most probable value, valid within an order of magnitude, is K 0 ≃ 10 3 , i.e., the E′ state is more stable than the E state by about 4 kcal/mol.

The effect of organic solvents on the phosphorylation of the Ca 2+ -dependent ATPase of sarcoplasmic reticulum by inorganic phosphate in the absence of a calcium gradient was investigated. Kinetic analysis of the reaction in water and water-organic solvent media according to a bireactant scheme shows no correlation between changes in kinetic parameters and the dieletric constant of the mixed solvents. The pronounced increase in equilibrium levels of phosphoenzyme in water-solvent mixtures is attributed to changes in the water activity of the medium.

The average ATPase activity in homogenates of the cat carotid body is found to be 20 nmol P i liberated per minute per mg fresh tissue weight at 25 °C. ATPase activity in the nodose ganglion and the superior cervical ganglion is found in the same range. Most of the activity remains in the supernatant after removal of the mitochondria by centrifugation. The activity is inhibited by ouabain only marginally (ganglia) or not at all (carotid body). In all these organs up to 80% of the activity is seen in the absence of Mg 2+ and the presence of increasing concentrations of Ca 2+ .

Chlorochalcone, Chloroisoflavone, Oxidation with Thallium(III) Nitrate An unambiguous synthesis of 6-chlorogenistein (5,7,4′-trihydroxy-6-chloroisoflavone) has been worked out via the oxidation of 2′-hydroxy-4,4′,6′-trimethoxy-5′-chlorochal-cone with thallium(III) nitrate followed by the demethylation of the 5,7,4′-trimethoxy-6-chloroisoflavone.

The relative rate of DNA synthesis increases over control level and the synthesis of inorganic pyrophospha­tase is stimulated when cells of E. coli are cultured in the presence of penicillins which inhibit cell division and cause filamentation of the cells. In contrast to that in temperature sensitive cell division mutants filamentation at 42 °C does not change the DNA/cell mass ratio and the synthesis of inorganic pyrophosphatase is not stimulated. This suggests that the change in the synthesis of the enzyme is not correlated with the filamentation but is a consequence of the supernormal DNA/cell mass ratio that appears in the presence of penicillins.

Hyperglycemia, Oziotelphusa senex senex, Hyperglycemic Hormone, Hexachlorocyclohexane (BHC) Benzene hexachloride (1,2,3,4,5,6-hexachlorocyclohe-xane) (BHC) produced a significant increase in the hemolymph sugar level of intact crabs. Oziotelphusa senex senex, apparantly by triggering release of the hypergly­ cemic hormone (HGH).

pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pK a in the range 6.8 -8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotina-mide-ribosyl-P 2 -ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD + give evidence for the participation of a group in the mitochondrial enzyme with pK a 6.8, deprotonation of which is necessary for detectable association of NAD + . In contrast the fixation of NAD + to the cytoplasmic enzyme is independent of pH.

In membrane fractions from pumpkin hypocotyls AT­Pase activity was stimulated by a combination of CCCP (carbonyl cyanide m-chlorophenylhydrazone), a protonophore, and valinomycin, a K + -ionophore. Singly, these ionophores stimulated ATPase activity much less. Nigericin, an H + /K + -antiporter, and nystatin, a cation pore, had similar effects as the combination of CCCP and valinomycin. The results suggest the presence of a cation-translocating ATPase which is stimulated by ionophores by dissipating cation gradients formed in the vesicles. A major part of the ionophore-stimulated ATPase activity correlated with marker enzymes for plasma membranes but part of it could be located in other compartments.