Kinetics of Leptin Binding to the Q223R Leptin Receptor

Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR), a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: k_a 1.76×10⁶±0.193×10⁶ M⁻¹ s⁻¹, k_d 1.21×10⁻⁴±0.707×10⁻⁴ s⁻¹, K_D 6.47×10⁻¹¹±3.30×10⁻¹¹ M; Q223R: k_a 1.75×10⁶±0.0245×10⁶ M⁻¹ s⁻¹, k_d 1.47×10⁻⁴±0.0505×10⁻⁴ s⁻¹, K_D 8.43×10⁻¹¹±0.407×10⁻¹¹ M). Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.     

Cytokinin activity induced by thidiazuron

The diphenylurea derivative thidiazuron induces a variety of cytokinin responses. Levels above 5 × 10⁻⁹ molar and 4 × 10⁻⁷ molar stimulate maximum soybean callus growth and radish cotyledon expansion, respectively. A wider range of dose response related effects follows thidiazuron induced tobacco plant regeneration. Analysis of soybean callus extracts strongly suggests that thidiazuron treatment creates an accumulation and/or synthesis of purine cytokinins, able to induce the growth, expansion and regeneration, mentioned above.     

Cytokinins of the Developing Mango Fruit : Isolation, Identification, and Changes in Levels during Maturation

The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N⁶-(Δ²-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.

The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.

A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 × 10³ micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 × 10² micromolar) and naphthalene acetamide (3.1 × 10 micromolar) at the young fruit stage.

     

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×10⁶ M⁻¹ and 1.66×10⁶ M⁻¹, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC₅₀ 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.     

Localization of cytokinin biosynthetic sites in pea plants and carrot roots

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-¹⁴C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-¹⁴C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N⁶-(Δ²-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N⁶-(Δ²-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N⁶-(Δ²-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.     

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca²⁺-binding properties of the light chains. The Ca²⁺-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH₂ thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca²⁺-binding properties of light chains and generation of tension. When the SH₂ moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca²⁺-binding properties of light chain LC₂ is lost; under these conditions the Ca²⁺-binding affinity value of SH₂-N-ethylmaleimide-blocked myosin (3.3×10⁴m⁻¹) decreases to near that expressed with the dissociated light chain LC₂ (0.7×10⁴m⁻¹). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH₂ thiol does not affect Ca²⁺ binding. The native secondary and tertiary structure of myosin seem to be required for Ca²⁺ binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH₂-N-ethylmaleimide-blocked myosin normal Ca²⁺- and (Mg²⁺+actin)-stimulated ATPase activities are expressed; however, there is a loss in K⁺-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca²⁺ with alterations in pH values. In the absence of Ca²⁺/EGTA buffer the biphasic Ca²⁺-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2×10⁶m⁻¹ and site two: 0.4×10⁶m⁻¹) as compared with values obtained at pH6.5 (site one: 0.64×10⁶m⁻¹ and site two: 0.2×10⁶m⁻¹). The Ca²⁺-binding affinity of light chain LC₂ and S₁, where the (S-1)–(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca²⁺-binding affinity, approx. 0.7×10⁴m⁻¹, whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca²⁺-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S₁ myosin subfragment light chain LC₂ was lost and thus was added back to the purified S₁ fraction. Light chain LC₂ was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)–(S-2) junction is needed for the positioning of light chain LC₂ and thus influences its essential conformation for Ca²⁺ binding.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [³H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K_0.5 = 2.8 × 10⁻⁷ molar. The K_0.5 for NPA under these conditions is 2 × 10⁻⁸ molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [³H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [³H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

Control of logarithmic growth rates of tobacco callus tissue by cytokinins

The growth rates of tobacco callus tissues on media containing 10⁻⁶ to 10 μm 6-(γ,γ-dimethylallylamino)purine (2iP) were measured. At concentrations of 10⁻⁴ μm and above growth rates were exponential and dependent on cytokinin concentration. At 2iP concentrations of 10⁻⁴ to 0.33 μm, the exponential rate was maintained for 4 to 5 doublings of fresh and dry weight. After this period a linear phase, resulting in approximately 1 doubling of weight, occurred. The growth of tissues on media containing higher than 0.33 μm 2iP was exponential for only about 15 days. At the end of this time, and well before they achieved half their final weight, they exhibited growth which was less rapid than logarithmic but more rapid than linear. Comparisons with zeatin, 6-benzylaminopurine and kinetin indicated that, although the maximum growth rates obtained with relatively high concentrations (0.1-1 μm) were similar, the naturally occurring cytokinins, 2iP and zeatin, promoted faster rates at lower concentrations (10⁻³-10⁻² μm) than did 6-benzylaminopurine and kinetin.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Investigation of the role of phosphorus in symbiotic dinitrogen fixation

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [³H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 10¹⁴Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10⁻⁹ molar and 1.85 × 10⁻⁹ molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10⁻⁹ molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [³H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe.     

Polycyclic Aromatic Hydrocarbons (PAHs) in Indoor Dusts of Guizhou,Southwest of China: Status,Sources and Potential Human Health Risk

Polycyclic aromatic hydrocarbons (PAHs) were analyzed for 136 indoor dust samples collected from Guizhou province, southwest of China. The ∑18PAHs concentrations ranged from 2.18 μg•g-1 to 14.20 μg•g-1 with the mean value of 6.78 μg•g-1. The highest Σ18PAHs concentration was found in dust samples from orefields, followed by city, town and village. Moreover, the mean concentration of Σ18PAHs in indoor dust was at least 10% higher than that of outdoors. The 4–6 rings PAHs, contributing more than 70% of ∑18PAHs, were the dominant species. PAHs ratios, principal component analysis with multiple linear regression (PCA-MLR) and hierarchical clustering analysis (HCA) were applied to evaluate the possible sources. Two major origins of PAHs in indoor dust were identified as vehicle emissions and coal combustion. The mean incremental lifetime cancer risk (ILCR) due to human exposure to indoor dust PAHs in city, town, village and orefield of Guizhou province, China was 6.14×10⁻⁶, 5.00×10⁻⁶, 3.08×10⁻⁶, 6.02×10⁻⁶ for children and 5.92×10⁻⁶, 4.83×10⁻⁶, 2.97×10⁻⁶, 5.81×10⁻⁶ for adults, respectively.     

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl⁻¹ 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl⁻¹ 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl⁻¹ N₆-benzylaminopurine (BA) and 1 mgl⁻¹ NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5–4 mgl⁻¹) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

Structure-Activity Relationships in Microbial Transformation of Phenols

The second-order rate constants for the microbial transformation of a series of phenols were correlated with the physicochemical properties of the phenols. The compounds studied were phenol, p-methylphenol, p-chlorophenol, p-bromophenol, p-cyanophenol, p-nitrophenol, p-acetylphenol, and p-methoxyphenol. Phenol-grown cells of Pseudomonas putida U transformed these compounds. Microbial transformation rate constants ranged from (1.5 ± 0.99) × 10⁻¹⁴ liter · organism⁻¹ · h⁻¹ for p-cyanophenol to (7.0 ± 1.3) × 10⁻¹² liter · organism⁻¹ · h⁻¹ for phenol. Linear regression analyses of rate constants and electronic, steric, and hydrophobic parameters showed that van der Waal's radii gave the best coefficient of determination (r² = 0.956). Products identified by thin-layer chromatography and liquid chromatography indicated that the phenols were microbially oxidized to the corresponding catechols.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

Kinetin inhibition of h-uracil and C-leucine incorporation by tobacco cells in suspension culture

The rate of ¹⁴C-leucine and ³H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 × 10⁻⁵m. Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N⁶-methyl-aminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N^6,6-dimethyl-aminopurine, and 2-aminopurine, did not relieve kinetin inhibition.     

Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen

O₂ irreversibly inactivates hydrogenase from Chlamydomonas reinhardi. The mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of O₂ (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres⁻¹ minute⁻¹ (1.4 × 10⁵ molar⁻¹ minute⁻¹). At levels above 0.01 atmosphere O₂, the increased numbers of O₂ molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one O₂ molecule and resulting in lowered rates of inactivation.     

Active cytokinins: photoaffinity labeling agents to detect binding

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N⁶-benzyladenines, -N⁶-(Δ²-isopentenyl)adenines, and -zeatins, and N⁶-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10⁻⁵ micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10⁻⁶ micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10⁻⁵ micromolar, representing a sensitivity rarely attained.