Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.     

Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.     

Factors affecting cation-anion uptake balance and iron acquisition in peanut plants grown on calcareous soils

Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO₃) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO₃ content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO₃ is too high.     

Kinetic and molecular orbital studies on the rate of oxidation of monosubstituted phenols and anilines by lactoperoxidase compound II in comparison with the case of horseradish peroxidase

The second-order rate constant (k4) for the oxidation of monosubstituted phenols and anilines by lactoperoxidase compound II was examined by Chance's method [B. Chance, Arch. Biochem. Biophys. 71 (1957), 130–136]. When the electronic states of these substrates were calculated by an ab initio molecular orbital method, it was found that the log k4 value correlates well with the highest occupied molecular orbital (HOMO) energy level but not with the net charge or frontier electron density. These results are essentially similar to those reported previously in the case of horseradish peroxidase [J. Sakurada, R. Sekiguchi, K. Sato, and T. Hosoya, Biochemistry 29 (1990), 4093–4098], showing some dissimilar features which are considered to reflect the structural difference between the two enzymes.Abbreviations HOMO highest occupied molecular orbital - HRP horseradish peroxidase - LPO lactoperoxidase (EC 1.11.1.7) - LUMO lowest unoccupied molecular orbital     

An experimental test of the eddy correlation technique over a Mediterranean macchia canopy

Abstract. Flux densities of water vapour and carbon dioxide were measured for a Mediterranean macchia canopy. Results show good agreement between the measured available energy and the sum of latent sensible and heat flux densities determined with the eddy correlation technique. Joint evaluation of the Bowen ratio, aerodynamic resistance, canopy resistance and the 'omega factor' suggests that the macchia canopy is intermediate in aerodynamic roughness between coniferous and deciduous canopies. Maximum daytime carbon flux densities ranged from -14 to -22(μnol m⁻² s⁻¹ on a ground area basis. The ratio of transpiration to assimilation (E/A) was a function of incident photo-synthetic photon flux density below about 400 μmol m⁻²s⁻¹ and above it was fairly constant at 272 mol mol⁻¹ (H₂O/CO₂). The relationship between carbon influx and canopy conductance was linear. Results show promising applications of the eddy correlation technique for evaluating physiological features of canopies, treated as unitary functional systems.     

Chemiosmotic systems in bioenergetics: H+-cycles and Na+-cycles

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H⁺ cycle concept. The circulation of H⁺ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H⁺ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes.Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H⁺. It was found that, in ceertain bacteria, as well as in the outer membrane of the animal cell, Na⁺ effectively substitutes for H⁺ as the coupling ion (the chemiosmotic Na⁺ cycle). A precedent is set when the Na⁺ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H⁺ and Na⁺ cycles coexist in one and the same membrane (bacteria) or in two diffeerent membranes of one and the same cell (animals). The sets of and generators as well as and consumers found in different types of biomembranes, are listed and discussed.     

A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events

The initial (F₀), maximal (F_M) and steady-state (F_S) levels of chlorophyll fluorescence emitted by intact pea leaves exposed to various light intensities and environmental conditions, were measured with a modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the fluorescence signals contain only three terms, X=J₂p_2F/(1–G), Y=T/(1–G) and V, where V is the relative variable fluorescence, J₂ is the light absorption flux in PS II, p_2F is the probability of fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II units and for energy cycling between the reaction center and the chlorophyll pool: F₀=X, F_M=X/(1–Y) and F_S=X(1+(YV/(1–Y))). It is demonstrated that the amplitudes of the previously defined coefficients of chlorophyll fluorescence quenching, q_P and q_N, reflect, not just photochemical (q_P) or nonphotochemical (q_N) events as implied in the definitions, but both photochemical and nonphotochemical processes of PS II deactivation. The coefficient q_P is a measure of the ratio between the actual macroscopic quantum yield of photochemistry in PS II (41-1) in a given light state and its maximal value measured when all PS II traps are open (41-2) in that state, with 41-3 and 41-4. When the partial connection between PS II units is taken into consideration, 1-q_P is nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other hand, 1-q_N equals the (normalized) ratio of the rate constant of photochemistry (k_2b) to the combined rate constant (k_N) of all the nonphotochemical deactivation processes excluding the rate constant k₂₂ of energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the individual rate constants, k_N-k₂₂ and k_2b, is provided by the fluorescence ratios 1/F_M and (1/F₀)–(1/F_M), respectively. Although, in theory, 41-5 is determined by the value of both k_2b and k_N-k₂₂, experimental results presented in this paper show that, under various environmental conditions, 41-6 is modulated largely through changes in k _N, confirming the idea that PS II quantum efficiency is dynamically regulated in vivo by nonphotochemical energy dissipation.Abbreviations Chl chlorophyll - F₀, F_M and F_S initial, maximal and steady-state levels of modulated Chl fluorescence emitted by light-adapted leaves - PS I and II photosystem I and II - q_P and q_N (previously defined) photochemical and nonphotochemical components of Chl fluorescence quenching     

Evidence of excited state absorption in PS II membrane fragments

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.Abbreviations PS I photosystem I of green plants - PS II photosystem II of green plants - P700 primary donor of PS I - P680 primary donor of PS II     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways

The effects of lithium (Li⁺) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li⁺ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC₅₀ of approx. 20 mM. By contrast, half-maximal effects of Li⁺ on inositol monophosphate (InsP) accumulation in [³H]inositol labelled tissue slices occurred at about 1 mM. A similar IC₅₀ for Li⁺ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [³H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated corticol slices using a novel mass assay fro InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li⁺ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li⁺ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are comptable with the inositol depletion hypothesis of Li⁺ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.