Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Mediator structure and conformation change

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Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Genic homozygosity in an ancient reptile (Alligator mississippiensis)

Proteins assayed electrophoretically showed variation at only three of 49 presumed genetic loci in alligators from southwestern Louisiana. Average heterozygosity per individual was 0.021±0.012; proportion of polymorphic loci was 0.06. Data on the history, structure, and ecology of this alligator population are consistent with natural selection as the primary factor accounting for this low genetic variability. However, neither a historic population bottleneck nor some genetic mechanism limiting variability can be dismissed as a possible factor.The study was supported by NSF Grant BMS 73-0125 to H.C.D.     

VARIATION IN ALCOHOL DEHYDROGENASE ACTIVITY AND FLOOD TOLERANCE IN WHITE CLOVER,TRIFOLIUM REPENS

Flooding results in induction of anaerobic metabolism in many higher plants. As an important component of anaerobic energy production, alcohol dehydrogenase (ADH) activity increases markedly in response to flooding in white clover, Trifolium repens. Significant inter-individual variation in flood-induced ADH activity exists in natural populations of T. repens. The genetic basis of this variation was analyzed by offspring-midparent regression of data from 75 greenhouse reared families; the estimated heritability of flood-induced ADH activity was 0.55 (±0.13). Genetic variation in flood-induced ADH activity has pronounced effects on physiological response and flood tolerance in this species. ADH activity is positively correlated with the rate of ethanol production, indicating that observed in vitro activity differences are manifested in in vivo physiological function. T. repens plants with higher ADH activities during flooding have greater flood tolerance (measured as growth rate when flooded/unflooded growth rate). Variation in ADH activity during flooding accounts for more than 79% of the variance in flood tolerance. On the basis of a limited field survey of populations occupying three sites differing in exposure to flooding conditions, individuals from site C, the most frequently flooded site, expressed significantly higher average ADH activity when flooded than individuals from site A, a site with no history of flooding. Since ADH activity levels are not correlated with electrophoretic mobility variation in T. repens, this work supports previous suggestions that regulatory variation in enzyme activity may play a central role in biochemical adaptations to environmental stress.     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.Abbreviations Chl Chlorophyll - CL Continuous light - CPa the reaction center complex of PSII - CPI the reaction center complex of PSI - CPIa Chlorophyll protein complex containing the CPI and the light harvesting complex of PSI - fr w fresh weight - LDC Light dark cycles - LHC-I Light-harvesting complex of PSI - LHC-II Light harvesting complex of PSII - PS photosystem - PSI photosystem I - PSII photosystem II     

Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

Determination of “active” cytochrome P-450 from relaxation kinetics of product formation

We estimate the active part of cytochrome P-450, which is involved in a special substrate transformation, by measuring the initial change of the production rate as a function of the relaxation transitions between two different steady states of the reaction cycle of cytochrome P-450 using the light-reversibility of the carbon monoxide inhibition. The kinetic data of such relaxations are interpreted within a model cycle, which reduces the reaction cycle to three steps. The estimation of the rate constant of the first reduction step, derived from model simulation of the production rate, is confirmed by independent experimental study of the reduction kinetics.An application of our model to the O-deethylation of 7-ethoxycoumarin reveals that — in a time average — 10%–15% of the spectroscopically detectable cytochrome P-450 is involved in that transformation.Abbreviations Cyt. P-450 microsomal cytochrome P-450 - 7-EC 7-ethoxycoumarin