Molecular interactions of hormonal steroids: the participation of the 17 side chain of corticosteroids in the formation of complexes with Co (II.). II

The c₂₀-c₂₁ α-ketol system of the 176 side chain of deoxycorti-costerone is in equilibrium with its c₂₀-c₂₁ enediol. The apparent dissociation constant of this enol was determined by a photometric method using crystal violet indicator; p K_a¹ = 10.65 ± 044. Formation constants of the (1:1) deoxycorticosterone-cobalt (II) complex were determined by solvent extraction and in mixed solvent systems. The complex formation constant K_f in an aqueous medium was found by graphical extrapolation to be 2.5–3.0 × 10⁻¹ 1. mole⁻¹     

Characterization of the Bacillus subtilis W23 genome by sedimentation

Sedimentation measurements of DNA gently extracted from stationary-phase cells of Bacillus subtilis demonstrated molecules large enough to account for the total genome. The observed sedimentation coefficient of the putative genome showed a strong dependence on centrifuge speed and was, therefore, measured at very low speeds. The value for the sedimentation coefficient extrapolated to zero speed was 159 ± 19 s, from which was calculated molecular weights of (2.5 ± ^1.2_0.8) × 10⁹ a.m.u. for linear molecules and (1.8±^0.8_0.7) × 10⁹ a.m.u. for circular molecules (relative to the following values for T2 DNA: 57 s and 132±12 × 10 daltons; Leighton &; Rubenstein, 1969). The genome also showed extreme shear sensitivity.     

Dissociation constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a simple and sensitive stain method for glucan phosphorylase (EC 2.4.1.1) was developed. With this method 0.3–1.5 μg or 1–5 units of phosphorylase could be demonstrated as a sharp band within a few hours. Mobility of phosphorylase fraction was retarded in gels containing glycogen. From the change of mobility as a function of glycogen concentrations, the dissociation constants of phosphorylases of rabbit skeletal muscle, liver, and brain with rabbit liver glycogen was calculated. They were 6.1 × 10⁻⁴, 22 × 10⁻⁴, and 13 × 10⁻⁴m, respectively. From the electrophoretic mobility, rabbit tissue phosphorylases could be classified into two: those of brain and kidney, and those of skeletal muscle and liver. When the electrophoresis gel, however, contained glycogen in a considerable concentration, their mobilities were retarded, and the retardation was more marked with those of skeletal muscle and brain than with those of liver and kidney. Hence, all four tissue phosphorylases could be distinguished only by the disc gel containing glycogen.     

Insulin-receptor interactions in liver cell membranes

The specific binding of ¹²⁵I-insulin to liver cell membranes is a saturable process with respect to insulin. Binding is displaced by low concentrations of native insulin but not by biologically inactive insulin derivatives or by other peptide hormones. The rate constants of association (3.5 × 10⁶ mole⁻¹ sec⁻¹) and of dissociation (2.7 × 10⁻⁴ sec⁻¹) of the insulin-membrane complex can be determined independently. The dissociation constant of the complex, determined from the rate constants and from equilibrium data, is about 7 × 10⁻¹¹M. Complex formation does not result in degradation of the insulin molecule. The binding interaction is a dissociable process involving a homogeneous membrane structure which is almost certainly the biologically significant receptor. The kinetic properties, and the effects of enzymic perturbations of the membrane, suggest that the insulin receptors of liver and of adipose tissue cells may be very similar structures.     

南亚热带常绿阔叶林林冠不同部位藤本植物的光合生理特征及其对环境因子的适应

对南亚热带常绿阔叶林中着生在林冠层不同部位的4种藤本植物(白背瓜馥木(Fissistigma glaucescens)、瓜子金(Dischidia chinensis)、蔓九节(Psychotria serpens)和山蒌(Piper hancei))的光合生理生态特性进行比较, 探讨着生在林冠不同部位的藤本植物的光合生理特性随光照、温度、湿度等变化的规律。结果表明, 鼎湖山南亚热带常绿阔叶林微生境由上至下发生了较大变化, 相对于林内, 冠层顶部具有高温、高光强、低湿度的特征。受这些变化的环境因子的影响, 着生在林冠不同部位的藤本植物之间的光合生理特征存在着较大差异: 着生于林冠层中上部的瓜子金和蔓九节的最大净光合速率分别为(2.9 ± 0.6)和(6.3 ± 1.3) μmol CO₂·m^-2·s^-1, 光饱和点为(168.5 ± 83.4)和(231.4 ± 147.8) μmol·m^-2·s^-1, 显著小于位于冠层下部的白背瓜馥木和山蒌的最大净光合速率值(8.9 ± 2.9)和(8.6 ± 2.3) μmol CO₂·m^-2·s^-1以及光饱和点值(491.6 ± 230.8)和(402.3 ± 112.8) μmol·m^-2·s^-1。瓜子金和蔓九节的光补偿点值为(16.1 ± 5.9)和(10.1 ± 5.7) μmol·m^-2·s^-1, 水分利用效率值为(11.5 ± 3.9)和(8.7 ± 1.6) μmol CO₂·mmol^-1 H₂O, 显著大于林内的白背瓜馥木和山蒌的光补偿点值(5.6 ± 1.9)和(5.4 ± 1.7) μmol·m^-2·s^-1以及水分利用效率值(6.7 ± 1.8)和(6.8 ± 1.3) μmol CO₂·mmol^-1 H₂O。这些光合生理指标的变化显示出植物对不同的温度、光照、湿度的适应, 是植物适应环境条件的重要表现。     

Impact of ZnSO and ZnO Nanoparticles on Seed Germination and Seedling Growth of Lettuce

Micronutrient nanoparticles (NPs) are currently an option for chemical fertilization and biostimulation in crops. However, there is little information on the phytotoxic or biostimulatory effects of NPs at low concentrations of some elements, such as Zn. In this study, the effect of low concentrations of Zn oxide (ZnO) NPs on germination, growth variables, and nutritional attributes of lettuce (Lactuca sativa L.) was evaluated in comparison to Zn sulfate. Romaine lettuce seeds were treated with ZnSO₄^-- × 7H₂O and ZnO NPs at Zn molar concentrations of 1 × 10⁻³, 5 × 10⁻³, 1 × 10⁻⁴, 5 × 10⁻⁴, 1 × 10⁻⁵, 5 × 10⁻⁵, 1 × 10⁻⁶, and 5 × 10⁻⁶. The seeds treated with ZnSO₄⁻ at 5 × 10⁻⁶ registered the highest radicle length, 73% more than the control treatment. The seeds treated with ZnSO₄⁻ at 5 × 10⁻³ registered the lowest values, with 50% less than the control treatment. ZnO NPs at 5 × 10⁻⁶ significantly increased content of chlorophyll A and B and total phenolics. These results indicate the possible existence of a mechanism related to the intrinsic nanoparticle properties, especially at low concentrations.

     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.     

Naphthoquinone inhibitors of Periplaneta americana and Scolytus multistriatus feeding: ultraviolet difference spectra of reactions of juglone, menadione, and 1,4-naphthoquinone with amino acids and the indicated mechanism of feeding inhibition

The relative reactivity of 3 naphthoquinones, which are feeding inhibitors for Periplaneta americana and Scolytus multistriatus, with each of 7 amino acids was measured by ultraviolet difference spectroscopy. Juglone (5-hydroxy-1,4-naphthoquinone), menadione(2-methyl-1,4-naphthoquinone) or 1,4-naphthoquinone produced difference spectra immediately upon mixing with cysteine, but not with valine, serine, glutamic acid, arginine, tryptophan or proline in phosphate buffer (pH 7.0). The K_s values for the reactions indicated that the affinities of 1,4-naphthoquinone (K_s = 4.4 · 10⁻⁴M) and juglone (K_s = 8.3 · 10⁻⁴M) for cysteine were comparable, but were both approx. 10 times greater than that for menadione (K_s = 3.2 · 10⁻³M). The extinction coefficient of the complex formed by cysteine with juglone (A = 3.448 · 10⁻¹M) was approx. 2 times greater than that of 1,4-naphthoquinone (A = 1.290 · 10⁻¹M) or menadione (A = 1.176 · 10⁻¹ M). The importance of these results to explaining the mechanism of chemoreception in P. americana and S. multistriatus is discussed.     

Joining of bacteriophage T4D heads and tails: A kinetic study by inelastic light scattering

We have used inelastic laser light scattering to study the kinetics of the spontaneous assembly of heads and tails of bacteriophage T4D to form noninfectious tail fiberless particles. For interpretation of the kinetics, it was first necessary to determine the physical properties of the strongly scattering phage parts. For heads, these are D_{20,w = 3.60 × 10⁻⁸cm²/s, 820,w = 1025 S, M = 1.76 × 10⁸}. For tail fiberless particles, D_20,w = 3.14 × 10⁻⁸cm²/s, 8_20,w = 968 S, and M = 1.95 × 10⁸. The kinetics of the head-tail joining process was followed by measuring the time variation of the homodyne scattering autocorrelation function. This was interpreted as a sum of exponentials whose decay constants were known from the scattering angle and the diffusion coefficients, and whose amplitudes were related to the concentrations of reactants and products. Scattering experiments at 22 °C gave a bimolecular rate constant of 1.02 × 10⁷m⁻¹ s⁻¹, while infectivity assays at 30 °C gave a rate constant of 1.28 × 10⁷. Adjustment of both rate constants to 20 °C, assuming diffusion controlled reaction, gave 0.97 × 10⁷ and 0.98 × 10⁷m⁻¹ s⁻¹, respectively. This rate is about $\text{1}\text{500}$ that predicted by Smoluchowski theory for a diffusion controlled reaction between two spherical particles; the discrepancy is largely explicable from orientational factors.     

Role of Allosteric Switch Residue Histidine 195 in Maintaining Active-Site Asymmetry in Presynaptic Filaments of Bacteriophage T4 UvsX Recombinase

Cyanophycin, or poly-l-Asp-multi-l-Arg, is a non-ribosomally synthesized peptidic polymer that is used for nitrogen storage by cyanobacteria and other select eubacteria. Upon synthesis, it self-associates to form insoluble granules, the degradation of which is uniquely catalyzed by a carboxy-terminal-specific protease, cyanophycinase. We have determined the structure of cyanophycinase from the freshwater cyanobacterium Synechocystis sp. PCC6803 at 1.5-Å resolution, showing that the structure is dimeric, with individual protomers resembling aspartyl dipeptidase. Kinetic characterization of the enzyme demonstrates that the enzyme displays Michaelis-Menten kinetics with a k_cat of 16.5 s^− 1 and a k_cat/K_M of 7.5 × 10^− 6 M^− 1 s^− 1. Site-directed mutagenesis experiments confirm that cyanophycinase is a serine protease and that Gln101, Asp172, Gln173, Arg178, Arg180 and Arg183, which form a conserved pocket adjacent to the catalytic Ser132, are functionally critical residues. Modeling indicates that cyanophycinase binds the β-Asp-Arg dipeptide residue immediately N-terminal to the scissile bond in an extended conformation in this pocket, primarily recognizing this penultimate β-Asp-Arg residue of the polymeric chain. Because binding and catalysis depend on substrate features unique to β-linked aspartyl peptides, cyanophycinase is able to act within the cytosol without non-specific cleavage events disrupting essential cellular processes.     

The binding of antifibrinolytic amino acids to kringle-4-containing fragments of plasminogen

A monoclonal antibody to human plasminogen, 10-F-1, was found to interact with the lysine-binding site (LBS) on the kringle 4 (K 4) region of the molecule. This observation has been employed to measure the binding of various antifibrinolytic amino acid analogs of ?-aminocaproic acid (?ACA) to its site on K 4 in appropriate elastolytic-derived fragments of human plasminogen and to other species of plasminogen to which antibody 10-F-1 cross-reacts. By analysis of the concentration dependence of ?ACA displacement of [¹²⁵I]10-F-1 from human Glu₁Pg, a K_D for ?ACA of 7.1 ± 1.0 mm was calculated. Similar experiments with K 4-containing fragments of Glu₁Pg, viz., Lys₇₇Pg, K 4, Lys₇₇H and Val₃₅₄Pg, yielded K_D values of 6.6 ± 1.0, 7.5 ± 1.0, 6.6 ± 1.0, and 12.0 ± 2.0 mm, respectively. When baboon, goat, monkey, rabbit, and sheep plasminogens were substituted for human plasminogen, the K_D values calculated ranged from 2.1 to 7.1 mm. The K_D values for several analogs of ?ACA, i.e., 4-aminobutyric acid, 5-aminopentanoic acid, 8-aminooctanoic acid, l-lysine, and trans-aminomethyl cyclohexane-1-carboxylic acid, were measured to the K 4 region of Lys₇₇Pg. The values obtained were 11.3 ± 1.5, 9.0 ± 1.0, 71.0 ± 10, 38.0 ± 5.0, and 1.1 ± 0.4 mm, respectively. Additionally, the K_D of trans-aminomethylcyclohexane-1-carboxylic acid towards the K 4 region of Glu₁Pg, Lys₇₇Pg, and isolated K 4 was found to be 2.4 ± 0.5, 1.1 ± 0.3, and 2.0 ± 0.6 mm, respectively. These studies show directly that the LBS on the K 4 domain of plasminogen represents one of its 4–5 weak binding sites and that this site can be specifically probed with the use of monoclonal antibody 10-F-1. Furthermore, it appears as though this site is conserved in several important proteolytic fragments of plasminogen, providing additional evidence that these fragments exist as independent domains in the native molecule. Finally, this weak LBS on the K 4 domain of human plasminogen is also present in other species of plasminogen.     

Columns for rapid chromatographic separation of small amounts of tracer-labeled transfer ribonucleic acids

A small reversed-phase chromatographic system for the separation of [³H] or [¹⁴C] aminoacyl-tRNA's is described. This system has the advantages of speed (chromatographic runs can be completed in 30 to 60 min), sensitivity (samples containing as little as 3.5 × 10⁻⁹ gm or 60 × 16⁻⁶A₂₆₀ unit of phenylalanyl-tRNA have been resolved as a chromatographic peak), and excellent resolution of isoaccepting tRNA's. The system has been applied to samples of E. coli tRNA's and calf liver tRNA's.     

A protein-binding assay for phallotoxins using muscle actin

The high affinity of phallotoxins to filamentous actin (K_D = 3.6 × 10⁻⁸m) has been used to assay small amounts of phallotoxins. The limit of detection was as low as 160 ng of phallotoxins. This is 50 times lower than the limit of all other procedures known so far. The assay was successfully applied to the analysis of phallotoxins in crude extracts of Amanita pholloides mushrooms.     

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography.

The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10⁻¹¹m and 5 × 10⁻¹⁰m, respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10⁻⁶m for catalase and 1.8 × 10⁻⁶m for superoxide dismutase, which agree with previously reported values.     

A sensitive method for measuring oxygen consumption

A technique is presented whereby oxygen consumption rates of the order of 10⁻⁶ μl/hr can be measured, thus providing a means for studying the respiration rates of single cells, even slowly respiring ones. The technique is based on the principle of incubating cells in extremely small chambers containing highly concentrated hemoglobin solutions. The absorbance shift occurring when hemoglobin is transformed from its oxygenated to its deoxygenated form is recorded microspectrophotometrically. The results obtained by this technique seem to be well in accordance with those of the Warburg manometric method. The technique is convenient and easy to handle and the sensitivity can be varied within wide limits, permitting different types of materials to be studied. In experiments using yeast cells, respiration rates from 1.0 × 10⁻⁶ to 1.8 × 10⁻⁶ μl O₂/cell/hr were revealed.     

Cyclic 3'.5'-nucleotide phosphodiesterase. Effect of binding protein on the hydrolysis of cyclic AMP

The rate of cyclic AMP hydrolysis by a cyclic 3′,5′-nucleotide phosphodiesterase was diminished by the presence of a cyclic AMP binding protein in the reaction mixture. The reduction was proportional to the concentration of the binding protein; and was more pronounced at 0° than at 30°, presumably because the affinity of cyclic AMP to the binding protein was greater at 0° (“apparent dissociation constant” = 3 × 10⁻⁸ M) than at 30° (“apparent dissociation constant” = 4 × 10⁻⁷ M). These experiments indicate that cyclic AMP bound to the binding protein is not susceptible to the action of phosphodiesterase. It is hydrolyzed only when dissociated from the protein, and the rate of dissociation appears to be the limiting factor. The possible physiological significance of these results is discussed.     

Characterization of DNA from the cellular slime mould Dictyostelium discoideum after growth of the amoebae in different media

Amoebae of the cellular slime mould Dictyostelium discoideum Ax2 grown on Aerobacter aerogenes as food source have a DNA content (36.0 ± 0.9 × 10⁻¹⁴ g/cell) approximately twice that of the same amoebae grown axenically (16.8 ± 0.4 × 10⁻¹⁴ g/cell). Isolation and characterization of DNA from amoebae grown either axenically or on bacteria, by several methods (melting curve, density gradient centrifugation, DNA/DNA hybridization) suggests that not more than 16% of the DNA content of bacterially grown amoebae is of bacterial origin. Studies of the rate of reannealing of DNA samples isolated from amoebae grown either axenically or on bacteria and of the degree to which they hybridize with ribosomal RNA, suggests that the ‘extra’ DNA that bacterially grown cells contain is biologically similar to that contained in axenically grown cells. It is therefore concluded that amoebae growing exponentially on bacteria have, on average, 2.4 to 2.7 genome equivalents per cell and amoebae growing exponentially in axenic medium have 1.3 to 1.4 genome equivalents per cell. Since it is believed that amoebae of this strain growing on bacteria are haploid and since these differences in DNA content persist during their subsequent differentiation, it is concluded that axenically grown amoebae differentiate whilst in the G1 phase of the cell cycle and bacterially grown amoebae differentiate whilst in the G2 phase of the cell cycle.     

Radiation-induced human chromosome aberrations. II. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes

Whole blood cultures from humans and from the New World primate, Saguinus fuscicollis, were irradiated with various doses of 250 kV X-rays. The resulting centric ring plus dicentric aberration yields were fitted to the three models, Y = a+bD, Y = a+bD+cD², and Y = a+cD², by least squares regression. In both instances the best fit was to the model Y = a+bD+cD², with coefficients of the one- and two-track components for human and marmoset being: b = (0.78 ± 0.09)·10⁻³, c = (5.92 ± 0.31)·10⁻⁶, and b = (1.11 ± 0.36)·⁻³, c = (7.7 ± 1.7)·10⁻⁶, respectively.