Volume 30, Issue 9-10

The complexation of cadmium by isocitrate has been studied at 25 °C and pH 7.5 in a range of ionic strength from 0.01 to 0.16. The formation constant of the complex between cadmium and tribasic isocitrate varies from 860 M -1 at μ= 0.16 to approximately 24,500 M -1 at infinite dilution. These data allow the distribution of the chemical forms of cadmium added to the incubation mixtures for the assay of NAD-dependent isocitrate dehydrogenase to be calculated.

Sulfhydryl Groups, Methylmercury Containing Inactivator, Coenzyme Analogue Nicotinamide-(S-methylmercury-thioinosine) dinucleotide was formed by reaction of nicotin­ amide-(6-thiopurine) dinucleotide with methylmercury chloride. The compound exhibits coenzyme properties in the test with LDH (K m =1.5 × 10 -4 м , V max =12500) and LADH (K m =1.7 × 10 -4 м, V max =27) and inactivates YADH and GAPDH. From incubations with LDH and LADH the mercury containing coenzyme could be regained by column chromatography. The compound seems to be qualified for the X-ray structure analysis of the coenzyme-enzyme complex for some dehyrogenases based on the proportion of the heavy metal.

Arabinonucleosides, Spectrophotometric Titration, Sugar Hydroxyl Dissociation Spectrophotometric titration in the ultraviolet has been employed to determine the pK values for dissociation of the sugar hydroxyls in pyrimidine arabinonucleosides and some of their O′-methyl and O′-ethyl derivatives. The order of dissociation of the sugar hydroxyls in the arabinofuranose ring was 2′-OH > 3′-OH > 5′-OH. The higher acidity (lower pK) of the 2′-OH was interpreted in terms of formation of an intramolecular hydrogen bond of the form 5′-OH···2′-O(-) and the accompanying changes in conformation of the arabinose ring. The various factors affecting the dissociation of specific hydroxyls in some of the O′-alkyl derivatives are discussed in relation to steric, conformational and other effects.

The structure of β-chitin has been studied in detail. Atomic coordinates in the unit cell are determined. Various positions of O 6 atom have been studied in detail in order to reduce X-ray discrepancies. It is f duonthat O 6 atom lies near 140°.

Several optical and hydrodynamic properties of purified nucleohistone have indicated that the secondary structure of DNA in nucleohistone is slightly different both from the structures of native and denatured DNA in solution. It has been suggested that while histones stabilize the overall DNA-structure they incorporate into the latter some denaturation defects. Rotational diffusion studies of acridine-orange complexes of whole and dehistonized nucleohistones have indicated that nucleohistone should contain a single continuous DNA molecule. The contour length of the DNA molecule would be about 2 to 3 times the end-to-end distance of nucleohistone. Polarized fluorescence microscopy and linear dichroism studies of whole nucleohistone have given an average estimate (about 30% of the total DNA) of oriented DNA in nucleohistone. The degree and the direction of maximum polarization of fluorescence from the dye complexes of whole and partial (i. e. partially dissociated) nucleohistones have been studied. From these studies, it is proposed that no single supercoiled arrangement of the DNA in nucleohistone is tenable. The combined results of all these studies suggest that the basic nucleohistone molecule contains coiled and extended DNA regions. The proportion of the lengths of the DNA constrained in the coiled and the extended regions is about 7:3. Only two probable models or classes have this quantitative feature of the DNA-arrangement and have been discussed.

Actomyosin ATPase, Calcium Sensitivity, Sulfhydryl Groups SH group substitution by DTNB enabled natural actomyosin to split ATP (in the presence of Mg 2+ ) also in the absence of Ca 2+ , when assayed at low ionic strength. At higher KCl concentrations the ATPase activity of SH group substituted actomyosin was still Ca-dependent. Addition of unsubstituted myosin to natural actomyosin whose SH groups had been substituted increased the ATPase activity. This increase was Ca-insensitive indicating that SH group substitution of myosin in actomyosin can make the interaction of additional myosin molecules Ca-independent. In natural actomyosin Ca-insensitivity of ATPase activity was attained at a lower degree of SH group substitution when substitution was performed in the presence of EDTA. The part of ATPase activity which still remained Ca-sensitive after DTNB treatment could be activated by lower concentrations of free Ca 2+ than the Ca-sensitive ATPase of untreated actomyosin. In reconstituted actomyosin the Ca-sensitivity of ATPase activity could more easily be reduced when the myosin-actin ratio was high. For demonstrating remaining Ca-sensitivity in SH group substituted reconstituted actomyosin more tropomyosin-troponin was needed than for sensitizing unsubstituted actomyosin to Ca 2+ . - The similarities between the ATPase activity of SH group substituted actomyosin on the one hand and that of actomyosin at low concentrations of ATP on the other hand suggest that SH group substitution modifies actin-myosin interaction in a similar way as does nucleotide-free myosin (rigor myosin).

The non-equilibrium dialysis has been used for kinetic studies of ATP dependent calcium up­ take by the sarcoplasmic reticulum. The uptake displays two phases, which are defined as fast and slow uptake. The former is an exponential function of time, with a half-life time of approximately 15-20 sec, the latter presents the characteristics of an autocatalytic reaction. The fast uptake is characterized by its amplitude, the slow uptake by its rate. Compared with the fast up­ take, the slow uptake requires higher amounts of Mg 2+ or ATP, and is more sensitive to pH variations and aging. The reasons which formerly prevented the resolution of the second phase from the first are discussed. It is concluded that the fast uptake is not a simple binding reaction, and that the slow uptake is more sensitive to changes by the vesicular membrane.

Sarcoplasmic Membranes, Calcium Precipitating Protein, Fluram Fluram, a fast reacting reagent for primary amino groups does not label proteins enclosed in reconstituted sarcoplasmic vesicles (SR vesicles) when applied at low reagent/protein ratios. Hence, Fluram does not penetrate through SR membranes under these conditions. Likewise, the mem­ branes of erythrocytes prevent the reagent from reacting with hemoglobin. However, at high reagent/protein ratios the membranes of the SR vesicles disintegrate. In contrast, liposomal membranes prepared from SR lipids are not affected even at a high degree of labeling. Disintegration of SR membranes starts to occur when the phosphatidylethanolamine fraction is completely substituted by Fluram and the transport protein is labeled with 3-4 mol of Fluram per 100 000 d. When closed SR vesicles are labeled at low Fluram/protein ratios, the calcium precipitating protein is four times more intensely labeled than the transport protein. The labeling of the calcium transport protein in closed vesicles excludes its location in the intravesicular space. In SR vesicles solubilized with deoxycholate the relative degree of labeling of the calcium precipitating protein remains unchanged while the transport ATPase is more intensely labeled at the expense of the labeling of the amino lipids. The relative degree of labeling of the protein components depends not only on the number of labable groups but also on the rates with which these groups react with Fluram. Therefore, the experimental data do not give quantitative in­ formation concerning the distribution of the protein components in the SR membranes.

The elastic theory of a uniaxial membrane in an asymmetric environment predicts a spontaneous splay deformation. This spontaneous curvature of the membrane is discussed by the intrinsic splay of the membrane molecules (e. g. wedge shaped molecules) and their polar orientation. The chemoelastic effect is the polar orientation induced by the asymmetric environment in connection with the intrinsic splay. This effect is also discussed for polyelectrolytes where a small change of pH (~0.1) can lead to a spontaneous curvature of 10 4 cm -1 . The actual shape of red blood cells can be explained by the spontaneous splay and a change in environment induces the change in shape of these cells. A model is proposed for two conical bodies swimming in a uniaxial membrane which interact with each other through elastic coupling. The force between the bodies can be either attractive or repulsive. As an example of this model clustering of proteins is discussed.

The molecular weight and hydrodynamic properties of a polypeptide isolated from the lamellar system of Antirrhinum chloroplasts were determined in sodium dodecyl sulphate solution by measurement of sedimentation velocity, diffusion and effective partial specific volume. The polypeptide fraction exhibits a molecular weight of 25 000 which agrees with the apparent molecular weight found by polyacrylamide gel electrophoresis. The molecular weight of the polypeptidesodium dodecyl sulphate micelle was 54 000, with a friction ratio of 1.6 which indicates an effective asymmetric hydrodynamic shape. For binding measurements self-diffusion equilibrium dialysis with dodecyl [ 35 S] sulphate was used. In this case, dialysis equilibrium was reached within about 10 hours, in contrast to the dialysis with initial concentration differences which requires much longer times. A binding value of δ D = 1.15g sodium dodecyl sulphate per g polypeptide was obtained which corresponds to a molar binding ratio of 100 mol dodecyl sulphate bound per mol of polypeptide. After the removal of dodecyl sulphate the polypeptide is present in an aggregated state. In phosphate buffers of pH 6.8 and 7.5 the aggregates preponderantly have sedimentation coefficients of 11.7 and 6.8 Svedberg units respectively. Assuming equivalent spheres the molecular weights were calculated to be 340 000 and 150 000.

Antibodies to neoxanthin agglutinate stroma-free swellable chloroplasts from tobacco (Nico-tiana tabacum var. John William’s Broadleaf) and Antirrhinum (Antirrhinum majus) whereas stroma-freed chloroplasts, which have lost the swellability are not agglutinated despite the fact that antibodies to neoxanthin are specifically adsorbed. In this latter case the agglutination is hindered for sterical reasons. From this it is concluded that neoxanthin is located in the outer surface of the thylakoid membrane. The antiserum to neoxanthin inhibits the ferricyanide photo­ reduction in chloroplasts when water is the electron donor by 15%. With diphenylcarbazide in tris-treated chloroplasts no inhibition is observed. Hence, just as in the case of the antiserum to lutein the site of inhibition is on the donor side of potosystem II namely between water and the site of electron donation of diphenylcarbazide. Benzidine/ascorbate is another artificial electron donor system of photosystem II reported in the literature. The photoreduction of anthraquinone-2-sulphonate with this donor system is inhibited. In contrast to the antiserum to lutein the antiserum to neoxanthin inhibits DCMU-sensitive photophosphorylation reactions in the system H 2 O → ferricyanide and benzidine/ascorbate → anthraquinone-2-sulphonate. Therefore, the electron transport coupled to photophosphorylation is inhibited by the antiserum.

1. Ethanol-extracted spinach chloroplasts were carefully sonified in a mixture of buffered urea, TRITON X-100 and dithiothreitol. This procedure solubilized the membrane-bound b-type cytochromes 559 and 563 to approx 90%. The crude extract contained both the high-potential (HP) and the low-potential form (LP) of cytochrome b-559 in a molar ratio of 1:1. 2. Strong sonification doubled the amount of low potential cytochrome b-559 in the extract. There­fore, it is assumed that Cyt b-559 HP was completely converted into the low-potential form. 3. Cyt b-563 was solubilized in the same concentration by both methods. 4. In the same medium (see No. 1), cytochrome f (= Cyt c-554) was dissolved by strong sonification only and is then essentially present in the extract in a dithionite-reducible form.

The enhancement of CO 2 fixation in isolated, intact spinach chloroplasts by ascorbate or by low sulfite concentrations (< 1 mм) is strongly reduced or even abolished by the addition of superoxide dismutase (SOD). By the use of 35 SO 3 2- it is demonstrated that the rate of sulfate formation is much lower than the sulfite induced increase in CO 2 fixation. This indicates that the superoxide radical is the chain initiating event; and, in parallel to ascorbate (Elstner and Kramer, 1973), the HSO 3 radical, acting as a Hill reagent for photosystem I, is reduced to sulfite in turn. The inhibitory action of sulfite at concentrations > 1 mм is not relieved by SOD, since this effect is mainly based on a competitive inhibition of ribulosediphosphate carboxylase. SOD itself stimulates the CO 2 fixation, if the reaction is started after 3 min of pre-illumination. This effect is discussed with respect to factors linked with the isolation procedure.

For 3T3, PY-3T3 and SV40-3T3 cells, the contents of total lipid, cholesterol and phospholipid were determined. Weight of total lipid per cell correlates closely with cellular volume as deter­ mined microscopically. This correlation is not observed for cholesterol or phospholipid content per cell. Cholesterol content per cm 2 cellular surface area is found about fifty percent higher in the transformed cell lines compared to 3T3 cells. This result is discussed in relation to thermal transitions observed in physical properties of the plasma membrane of these cells as well as in lamellar phospholipid/water model systems containing cholesterol.

Experiments were designed to investigate the low-dose side of the so-called dose-effect relation of mercuric chloride on the proliferation of a murine lymphoblastic cell strain (MB VIa). Three concentration ranges, from 0.9 × 10 -5 м to 0.9 × 10 -18 м, from 0.9 × 10 -12 м to 0.9 × 10 -25 м , and from 0.9 × 10 -15 м to 0.9 x 10 -21 м, in which the concentration decreased stepwise by a factor 10, were studied in 61, 74 and 58 experimental repetitions, respectively. In the first range, the concentrations 0.9 × 10-5 M and 0.9 x 10 -6 м HgCl 2 proved to be definitely toxic as was expected. However, also 0.9 × 10 -16 м and 0.9 × 10 -17 м appeared to be growth-inhibiting, the latter results being confirmed in the second and the third concentration ranges. These differences in cell growth were statistically significant.

Oxidative decarboxylation of p-hydroxybenzoic acids in plant cell suspension cultures is catalyzed by peroxidases. This reaction has been characterized in vivo and in vitro. Decarboxylation of substituted benzoic acids yields monomeric, dimeric and oligomeric benzoquinones. All peroxidases obtained from soybean (Glycine max) cell suspension cultures by gel electrophoresis are equally capable to decarboxylate p-hydroxybenzoic acids as indicated by their rather low differences in specific activity for various benzoic acids.

Incorporation of ʟ-[U- 14 C] phenylalanine, ʟ-[U- 14 C] tyrosine and ᴅʟ-[β- 14 C] 3,4-dihydroxyphenyl-alanine (DOPA) into stizolobinic acid, ʟ-β-(6-carboxy-α′-pyron-3-yl) -alanine, and stizolobic acid. ʟ-β-(6-carboxy-α′-pyron-4-yl)-alanine, was investigated as test precursors for these new types of amino acids in the epicotyls of etiolated seedlings of Stizolobium hassjoo. Radioactive stizolobinic acid and stizolobic acid resulted from administering labelled precursors were identified by colour reactions, radioautography and/or recrystallization to constant specific radioactivity. Radiolabelled stizolobic acid was subjected to alkaline hydrolysis and distribution pattern of the radioactivity was further examined in a degradation fragment of the amino acid. Proposed biosynthetic routes to stizolobinic or stizolobic acid from DOPA were empirically con­ firmed and the results are further discussed in relation to the possibility that phenylalanine may also be introduced into reaction sequences leading to the synthesis of both heterocyclic amino acids.

The amount of sinapine in immature seeds of Sinapis alba is very low; it increases during ripening. Most of the sinapine is located in the cotyledons. Hypocotyls and roots contain only traces of it. During the early development of seedlings the amount of sinapine in the cotyledons decreases in 26 °C very rapidly. Simultaneously the amount of a substance X increases. Darkness retarded both these processes. The same effect has a temperature of 10 °C. In both cases the increase of X is completly proportional to the decrease of sinapine. Therefore X is probably a derivative of sinapine. After pretreatment with 26 °C the decrease of sinapine in the cold is stronger. The amount of sinapic acid increases only in the at a very low level for a long period.

The toxicity of two mushroom species found in Finland, Cortinarius speciosissimus and Cortinarius gentilis, on the rat was studied. Dried, homogenized mushroom was given orally by stomach tubing. A dose of 500 mg dried mushroom/kg body weight, was used. It was demonstrated that both species caused renal damage. No damage could be shown in other organs. The renal histopathological changes corresponded to those of tubulo-interstitial nephritis. The sensitivity of different individuals to the fungal toxins varied greatly.

Disparlure, (cis-7,8-epoxy-2-methyloctadecane) the sex attractant of the gypsy moth, Porthetria (Lymantria) dispar, attracts the male nun moth, Lymantria monacha, in the field and is a highly effective olfactory stimulus in electroantennogram (EAG) and single-cell recordings. We have now analyzed the extract of 2000 abdominal tips of the female nun moth. Physical and chemical tests, which included gas-chromatographic retention times, elution volumes from silica gel and silica gel-silver nitrate columns, mass spectra, epoxide functionality, EAG-activity of chromatographic fractions with gypsy moth antennae, presence of disparlure precursor, all indicated that disparlure is present in the extract of nun moth sex glands. The optical activity of the natural disparlure of the two species has not yet been determined. Several authors have reported 1-0 that disparlure,

Sex pheromones of European species of Lymantria, Euproctis, Stilpnotia, Orgyia, and Dasychira (Lymantriidae) have been cross-checked by recording male electroantennogram (EAG) responses to excised female pheromone glands. Within the same genus, there was invariably full reciprocity of the gland effects. Between different genera, however, in all species combinations investigated the males strongly preferred their own species. From this pattern it is concluded that the major pheromone constituents are different for the five genera. In accordance with these results, several species of Lymantria are either known or supposed to produce the same sexual attractant, cis-7,8-epoxy-2-methyloctadecane (disparlure), whereas for one species of Orgyia the sex pheromone was recently identified (Smith et al., Science 188, 63 [1975]) as cis-6-heneicosen-11-one. None of the additional lymantriid pheromones have yet been chemically defined. In EAG screening tests, some species of this family were specifically responsive to hydrocarbons related to cis-7,2-methylocta-decene, the olefinic precursor of disparlure.

A device is described which allows withdrawing of samples of 1 to 2 ml ± 0.03 to 0.06 ml from a lollipop at 1 sec intervals.

The number of amino residues present in the proteins of the sarcoplasmic reticulum which can react with Fluram has been determined in native and sonicated SR vesicles. Sonication increases the number of amino groups accessible to Fluram from 0.57 to 0 .8 7 μmol·mg prot. -1 . This increase indicates that 66% of the amino residues are present in the external and 34% in the internal membrane leaflet. The distribution of the amino phospholipids is computed from the distribution of Fluram in the membrane proteins in con­junction with the relative distribution of Fluram between protein and lipid in native and sonicated vesicles. The distribution of the calcium transport protein has been approximated under different assumptions concerning the distribution of the residual protein and taking into account that 15% of the membranes of the SR vesicles might have changed their sideness during preparation.

Chlorella cell envelopes were isolated by a mechanical procedure. The obtained morphological structure shown in an EM-picture has a mean density of ρ = 1.42. A characteristic chemical property of the envelope is the percentage of mono-and di-unsaturated C 18 -fatty acids which is considerably higher than that of intact cells. With 14 C labelled sugars a selectivity of the hexose uptake could be shown, galactose uptake amounting only to about one fifth that of glucose and mannose. The uptake shows a temperature maximum. Part of the 14 C registered after a 30 min uptake peroid is released by high concentration of unlabelled glucose, 20% are irreversibly fixed.

Induced by soy bean tissue cultures in socalled “tissue chambers”, Rhizobium japonicum str. 61-A-96 developed nitrogenase activity separated from the plant cells. The activity proceded for 48 h with a rate of 1 × 10 -8 nmol C 2 H 4 h -1 cell -1 , which is about 6% of the activity measured for bacteroids from Rhizobium japonicum in nodules of Glycine max.

In contrast to slower heating rates, a temperature jump reveals complex rise phases in the heat induced chlorophyll fluorescence emission increase in intact plants. Three rise phases have been detected which indicate the stepwise loss of different quenching mechanism of system II fluorescence. Two of the phases appear to reflect heat deactivation of the system II reaction centers, while the other may be associated with the induction of hydrogenase activity. Variations in T max of the jump, for the increase in different plant varieties, suggest a correlation with membrane lipid phase transitions affecting thylakoid membrane structure and the fluorescence increase.

Physiological Optics, Crustacean Superposition Eye Histological and optical observations in the eye of crayfish lead to a new hypothesis of the superposition ray path in this eye. Rays from an object point entering through different facets are superimposed not by refracting systems as in other superposition eyes but by a radial arrangement of orthogonal reflecting planes which are formed by the sides of the crystalline cones and purine layers surrounding them.

Spectral and spatial sensitivity were determined in several retinula cells of the worker bee eye during one penetration with a microelectrode. Pairs of retinula cells were found, which differed in their spectral sensitivity, but had nearly identical fields of view. From this result it is concluded that ommatidia in the worker bee eye are not colour specific, but consist of different colour receptor typs. The consequences for the functional organisation of the fused rhabdom are shortly discussed.

The duration of the pulsed distress calls of Rana r. ridibunda ranges from 200 to 600 msec. Calls with regularily built pulses exhibit a mixed spectrum (up to 8 kHz) consisting of unharmonious and harmonious frequencies. The latter ones are frequency-modulated.

A method is described for the preparation and use of Lissamine Rhodamine B conjugated antibody for the detection of Tobacco Mosaic Virus antigen in mesophyll protoplasts of tomato.

A suspension tissue culture producing large amounts of Friend leukemia virus was developed. Properties of the cells and the virus are described.