The kinetics of formation of horseradish peroxidase compound I by reaction with peroxobenzoic acids. pH and peroxo acid substituent effects.

1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged 'electrostatic gate' controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.     

The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes

The reactivity with nitric oxide was investigated for a number of type-1, type-2 and type-3 copper proteins azurin from Pseudomonas aeruginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo medullosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77K. Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, photodissociation caused the reappearance of the original 625 nm absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 Cu1+-NO+ complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.     

The effects of cations on the structure and photochemistry of the photosystem II core complex

We have studied the effects of cations and detergents on the structure (molecular weight) and photochemistry of Triton X-100 Photosystem II subchloroplast particles (TSF-IIa). The effect of Mg²⁺ ions on activity depended on the Triton X-100 content of the preparation. If the residual Triton X-100 was not removed prior to assay, MgCl₂ increased the rate of electron transport, acting at a site on the reducing side of Photosystem II. Lowering the pH also increased the rate of electron transport. If the Triton X-100 was removed from the particles, both MgCl₂ and NaCl caused a decrease in the rate of electron transport. Addition of Triton X-100 caused a reversible decrease in the number of active Photosystem II reaction centers. Both cations and Triton X-100 had a profound effect on the molecular weight of the Photosystem II particles as determined by gel filtration. At 20 °C, addition of 0.05% Triton X-100 decreased the molecular weight from a high value (≥800,000) to 250,000. At 4 °C, addition of 1 mm MgCl₂ or 100 mm NaCl increased the molecular weight of the complex. In the absence of these salts 67% of the protein eluted with a molecular weight of 460,000 (the rest was >800,000-in the void volume). In the presence of these salts all of the material had a molecular weight of ≥800,000. A similar effect was observed when the pH was lowered from 8 to 6. Further work is needed to determine whether there is a correlation between the changes in molecular weight and activity.     

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.     

Surface photochemistry of the herbicide napropamide. The role of the media and environmental factors in directing the fates of intermediates.

The photochemical behaviour of the herbicide napropamide is studied on cellulose and silica surfaces, using steady-state and laser-flash diffuse reflectance techniques. The results are used to probe how the reaction sites of the host matrices influence the photo-reactive pathways. Napropamide undergoes reaction when irradiated with UV (lamps) or visible (sunlight) radiation on both solid supports. The nature of the intermediates and final products depend strongly on the presence or absence of molecular oxygen. The triplet state of napropamide adsorbed on cellulose is detected by both time-resolved luminescence and transient absorption spectroscopies. The triplet sate was not observed on silica, but transients which include the participation of molecular oxygen are detected during flash photolysis studies. The keto intermediates of the photo-Claisen rearrangement products are observed on both solids. Substituted 1-naphthols from photo-Claisen reactions and 1-naphthol are among the main reaction products. 1,4-Naphthoquinone is a major photoproduct in the presence of molecular oxygen and is expected to be prevalent when napropamide undergoes photodegradation in the environment (i.e., after being applied to plants and fields).     

Coumaric amide rotaxanes: effects of hydrogen bonding and mechanical interlocking on the photochemistry and photophysics.

Secondary amide derivatives of coumaric and ferulic acid are shown to undergo photoisomerization, forming a photostationary mixture of E- and Z-isomers. When the same chromophores are incorporated in rotaxanes, the extent of conversion to the Z-isomers is much smaller. Low temperature fluorescence experiments show that the energy barrier for non-radiative decay of the excited state is higher in the rotaxanes than in the corresponding threads, but the barriers are low in all cases.     

The role of reductants in the tyrosine hydroxylase reaction.

The role of several reducing systems in the tyrosine hydroxylase reaction has been studied. A significant dependence upon the reducing systems beyond that required to regenerate the oxidized cofactor has been observed. 2-Mercaptoethanol, NADPH, and ascorbate are each effective at reducing the cofactor, but their abilities to stimulate tyrosine hydroxylase vary over a threefold range. NADPH is a suitable reductant for the tyrosine hydroxylase reaction, even in the absence of pteridine reductase. A reducing system containing ascorbate, ferrous ion, and catalase gives unusually high enzyme activity and low blanks. This ascorbate system, in addition to being useful for in vitro enzyme assays, may serve as a model for the in vivo reaction. Ascorbate may play an important role in the hydroxylation of tyrosine in catecholaminergic tissues. This study demonstrates that an efficient reductant for the tyrosine hydroxylase reaction must, in addition to reducing the pterin cofactor, also interact effectively with the enzyme itself.     

The role of ascorbate in the prolyl hydroxylase reaction.

Binding isotherms of some anthracyclic derivatives with DNA have been determined by modern electrochemical techniques(Voltammetry - ac polarography). These techniques making use of the difference between the diffusion coefficients of DNA-drug complex and of free molecules of drug in solution allow the direct determination of the later ones. No striking differences can be noticed between the tested anthracyclines. No correlation can be established between the affinity of daunorubicin and some of its analogs for DNA and their more or less antitumoral activity.     

Positional effects of the hydroxy substituent on the photochemical and photophysical behavior of 3- and 4-hydroxystilbene.

The photophysical and photochemical behavior of meta- and para-hydroxy- and methoxystilbene are reported and compared to the aminostilbenes. Absorption spectra of all four studied compounds in organic solution display a featureless, intense long-wavelength absorption band around 300 nm. In basic aqueous solution, meta-hydroxystilbene displays a less intense band with a long-wavelength shoulder, a consequence of configuration interaction from the O(-) substituent. Emission from the meta-substituted stilbenes is much stronger than from the para isomers, with an especially large effect for the hydroxystilbenes in solvents that are good hydrogen bond acceptors. Emission from the hydroxystilbenes is weak in aqueous solution, and even weaker in basic solution, a consequence of facile nonradiative decay. The excited state lifetimes of the meta isomers are longer than those with para substituents. Ground and excited state acidity constants are reported for the hydroxystilbenes. In the ground state, the para derivative is an order of magnitude more acidic than the meta isomer, however, its short excited state lifetime precludes excited state deprotonation. In contrast, 3-hydroxystilbene does exhibit deprotonation in its excited state.     

Reciprocal influence of the graft vs. host reaction and pregnancy]

The graft versus host reaction (GVHR) was induced in mouse females-hybrids F1 (CBA X C57BL/6) by intravenous injection of suspension of the lymphoid cells of the spleen and of lymphoid nodes from C57BL/6 mouse females. Pregnancy resulted from interbreeding of the test females with syngenic males 1--5 days before, and 1--10, 10--20, 30--40 and more than 40 days after the moment of the lymphoid cells injection, aggravated the GVHR induced transplantation disease. At the same time the GVHR under these conditions decreased the percentage of pregnant animals and brought to child-bearing disfunction of the test animals (stillbirth, death of pregnant females, miscarriage). In some of the test mice aggravation of the GVHR was observed after delivery. Survival of the progeny decreased.     

The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction.

The hairpin ribozyme is an example of a small catalytic RNA that catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. We have utilised chemical synthesis of RNA to create mutants of the hairpin ribozyme in which a nucleoside analogue replaces one of the essential pyrimidines in the ribozyme. Individual pyrimidine nucleosides were substituted by 4-thiouridine, O4-methyluridine, O2-methyluridine or 2-pyrimidinone-1-beta-d-riboside. To facilitate the synthesis of oligoribonucleotides containing 4-thiouridine, we have devised a new synthetic route to the key intermediate 5'-O-(4, 4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-S-cyanoethyl-4-thiou ridine. The ability of the modified ribozymes to support catalysis was studied and the steady-state kinetic parameters were determined for each mutant. The range of analogues used in this study allows the important functional groups of the essential pyrimidines to be identified. The results demonstrate that each pyrimidine (U41, U42 and C25) plays an important role in hairpin ribozyme catalysis. The findings are discussed in terms of the various models that have been proposed for loop B of the hairpin ribozyme.     

The role of major histocompatibility complex genes in the reaction of macrophages and thymocytes during induction of T-effectors of the graft vs host reaction. III. Effect of antisera against H-2 complex antigens on the realization of the process

The data are presented on the influence of antisera to different products of the histocompatibility gene complex on the interaction between macrophages and thymocytes during the formation of T-cell GVH effectors. The intensity of the local GVH reaction was evaluated by the index of the lymph nodes enlargement. The close physical contact between cells was necessary for the induction of T-effectors GVH. Treatment of macrophages or thymocytes with anti-H-2 and anti-H-2K antibodies, prior to the beginning of combined incubation, or addition of these antisera to the cultural media suppressed the formation of T-cell inducing GVH. Anti-H-2 I and anti-H-2 D sera did not affect the formation of GVH T-effectors.     

Effects of the measuring light on the photochemistry of the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

The bacterial reaction center (RC) has become a reference model in the study of the diverse interactions of quinones with electron transfer complexes. In these studies, the RC functionality was probed through flash-induced absorption changes where the state of the primary donor is probed by means of a continuous measuring beam and the electron transfer is triggered by a short intense light pulse. The single-beam set-up implies the use as reference of the transmittance measured before the light pulse. Implicit in the analysis of these data is the assumption that the measuring beam does not elicit the protein photochemistry. At variance, measuring beam is actinic in nature at almost all the suitable wavelengths. In this contribution, the analytical modelling of the time evolution of neutral and charge-separated RCs has been performed. The ability of measuring light to elicit RC photochemistry induces a first order growth of the charge-separated state up to a steady state that depends on the light intensity and on the occupation of the secondary quinone (Q(B)) site. Then the laser pulse pumps all the RCs in the charge-separated state. The following charge recombination is still affected by the measuring beam. Actually, the kinetics of charge recombination measured in RC preparation with the Q(B) site partially occupied are two-exponential. The rate constant of both fast and slow phases depends linearly on the intensity of the measuring beam while their relative weights depend not only on the fractions of RC with the Q(B) site occupied but also on the measuring light intensity itself.     

The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C.

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.     

The effects of training in hyperoxia vs. normoxia on skeletal muscle enzyme activities and exercise performance.

Inspiring a hyperoxic (H) gas permits subjects to exercise at higher power outputs while training, but there is controversy as to whether this improves skeletal muscle oxidative capacity, maximal O(2) consumption (Vo(2 max)), and endurance performance to a greater extent than training in normoxia (N). To determine whether the higher power output during H training leads to a greater increase in these parameters, nine recreationally active subjects were randomly assigned in a single-blind fashion to train in H (60% O(2)) or N for 6 wk (3 sessions/wk of 10 x 4 min at 90% Vo(2 max)). Training heart rate (HR) was maintained during the study by increasing power output. After at least 6 wk of detraining, a second 6-wk training protocol was completed with the other breathing condition. Vo(2 max) and cycle time to exhaustion at 90% of pretraining Vo(2 max) were tested in room air pre- and posttraining. Muscle biopsies were sampled pre- and posttraining for citrate synthase (CS), beta-hydroxyacyl-coenzyme A dehydrogenase (beta-HAD), and mitochondrial aspartate aminotransferase (m-AsAT) activity measurements. Training power outputs were 8% higher (17 W) in H vs. N. However, both conditions produced similar improvements in Vo(2 max) (11-12%); time to exhaustion (approximately 100%); and CS (H, 30%; N, 32%), beta-HAD (H, 23%; N, 21%), and m-AsAT (H, 21%; N, 26%) activities. We conclude that the additional training stimulus provided by training in H was not sufficient to produce greater increases in the aerobic capacity of skeletal muscle and whole body Vo(2 max) and exercise performance compared with training in N.     

The role of the protein in the photochemistry of the retinal chromophore of visual pigments and the purple membrane protein

First various models that have been proposed for the primary photoevent in vision are critically discussed. It is concluded that the classical picture of a single cis-trans isomerization step is the only one which satisfactorily accounts for all the available experimental data. Experiments are performed showing that this process is temperature independent over a range of 200 C. In contrast to the efficient and wavelength independent photobleaching of rhodopsin, the yields of the 11-cis all-trans isomerization of the free protonated Schiff base chromophore are small, exhibiting a marked dependence on the excitation wavelength. Potential energy curves for both ground and excited states of rhodopsin are derived from the analysis of the accumulated experimental data (Rosenfeld, Honig, Ottolenghi, and Ebrey, Pure Appl. Chem., in press). In variance with the behavior of model compounds, photoisomerization in the pigment proceeds via the quantitative population of a common, barrierless, thermally relaxed excited state along the 11–12 torsional coordinate separating the 11-cis (rhodopsin) and all-trans (bathorhodopsin) configurations. In the ground state, interactions with the protein destabilize the all-trans isomerization product, leading to storage of a significant fraction of the photon's energy in the primary step.Next, the photochemistry of the purple membrane protein of Halobacterium halobium is discussed. It is suggested that, as in the case of visual pigments, the primary photoevent is a geometrical change in the chromophore. Also, as with visual pigments, the resulting primary photoproduct must have the highest free energy of any form of the pigment. The quantum yield for the formation of the 412 nm intermediate (M) is c. 0.26, while that for the back reaction is c. 0.68 (Becher and Ebrey, Biophys. J., in press). The sum of these two quantum yields is c. 1.0, suggesting, again like rhodopsin, that the primary photochemistry proceeds through a common excited state shared by the pigment and its bathoproduct.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Epigenetic and genotype-specific effects on the stability of de novo imposed methylation patterns in transgenic mice

The chloramphenicol acetyltransferase gene under the control of the late E2A promoter of adenovirus type 2 (Ad2) was introduced as transgene into the B6D2F1 mouse strain with mixed genetic background and became extensively de novo methylated. The methylation of this pAd2E2AL-CAT (7-1A) transgene was regulated in a strain-specific manner apparently depending on the site of integration. Transmission of the 7-1A transgene into an inbred DBA/2, 129/sv, or FVB/N genetic background led to a significant loss of methylation in the transgene, whereas C57BL/6, CB20, and Balb/c backgrounds favored the de novo methylation in very specific patterns. The newly established patterns of de novo methylation were transmitted to the offspring and remained stable for many generations, regardless of the heterozygosity of strain-specific DNA sequences present in these mouse strains. Segregation analyses showed a non-mendelian transmission of methylation phenotypes and suggested the involvement of dominant modifiers of methylation. The genotype-specific modifications of the transgene were followed for 11 backcross generations. These observations reflect an evolutionarily conserved mechanism directed against foreign, e.g. viral or bacterial, DNA at least in the chromosomal location of the 7-1A transgene. In seven additional mouse lines carrying the same transgene in different chromosomal locations, strain-specific alterations of methylation patterns were not observed.