Phosphorylation of the light-harvesting chlorophyll-protein regulates excitation energy distribution between photosystem II and photosystem I

The kinetics of thylakoid membrane protein phosphorylation in the presence of light and adenosine triphosphate is correlated to an incease in the 77 °K fluorescence emission at 735 nm (F735) relative to that at 685 nm (F685). Analysis of detergent-derived submembrane fractions indicate phosphorylation only of the polypeptides of Photosystem II, and the light-harvesting chlorophyll-protein complex serving Photosystem II (LHC-II). Although several polypeptides are phosphorylated, only the dephosphorylation kinetics of LHC-II follow the kinetics of the decrease of the $\text{F735}\text{F685}$ fluorescence emission ratios. The relative quantum yield of Photosystem II was significantly lower in phosphorylated membranes compared to dephosphorylated membranes. Reversible LHC-II phosphorylation thus provides the physiological mechanism for the control of the distribution of absorbed excitation energy between the two photosystems.     

The structure of a platinum(II) complex of cytidine-3'-monophosphate.

The structure of the cis-[Pt(NH₃)₂(3′-CMP)₂]^2? ion, isolated in a partially protonated form as its cesium salt, has been analyzed by single-crystal x-ray diffraction methods. The 3′-CMP ligands bind in a monodentate fashion through their N(3) atoms: in contrast to the structure of [Pt(en)(5′-CMP)]₂, no covalent platinum-phosphate bonding is found. This compound represents the first example of a 1:2 cis-metal/cytosine complex structurally characterized.     

The identification of myogenic cells in regenerating skeletal muscle. II. Early mammalian regenerates.

Most of the recent studies on skeletal muscle regeneration have used the criteria of cell shape and position as the primary means of identifying early presumptive myogenic elements or satellite cells. Studies of anuran muscle regeneration indicate, however, that macrophages can mimic early myogenic cells by adopting a fusiform shape and a sublaminar position during the initial stages of phagocytic invasion. The present study confirms these observations in injured mammalian muscle. Gastrocnemius muscle tissues from Sprague-Dawley rats were killed by lyophilization or repeated freezing and implanted subcutaneously to examine the cytology of the invading macrophages free from contamination by any endogenous myogenic cells. Within 2 days the implants are infiltrated by large numbers of fusiform macrophages. These cells form continuous cuffs around the degenerating myofibers but initially show little evidence of phagocytosis. They contain dense concentrations of free ribosomes but display few lysosomes, phagosomes, or pseudopodia. These distinctive phagocytic features do not appear until the macrophages penetrate the cores of the injured fibers and actually begin removal of the myofibrillar debris. These observations indicate that the criteria of cell shape and location cannot reliably distinguish between early mammalian macrophages and myogenic cells.     

The action of vasopressin and calcium on palmitate metabolism in hepatocytes and isolated mitochondria from rat liver

Vasopressin inhibits fatty acid oxidation and stimulates fatty acid esterification, glycogenolysis, and lactate production in hepatocytes from fed rats. In cells from fasted rats, the effect of the hormone on palmitate oxidation was absent, while gluconeogenesis was stimulated. The inhibitory action of vasopressin on palmitate oxidation was not due to the increased lactate production. Neither was it correlated to glycogen content or stimulation of glycogenolysis, which were restored earlier than the vasopressin effect on palmitate oxidation when previously fasted rats were refed a carbohydrate diet. The level of malonyl-CoA was moderately increased by vasopressin. Isolated mitochondria from rat liver were incubated in the presence of [U-14C]palmitate, ATP, CoA carnitine, glycerophosphate, ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, and varying amounts of calcium. The oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1 to 10 microM. Simultaneously, palmitate esterification was stimulated. This effect of calcium was observed also with mitochondria from fasted rats and with octanoate as well as palmitate as the substrate. Carnitine acylation was not affected by calcium. The possibility that the observed effects of calcium on mitochondrial fatty acid utilization is part of the mechanism of action of vasopressin on hepatocyte fatty acid metabolism is discussed.     

The reactivity of imidazole nitrogens in histidine to alkylation

Copper(II)-histidine complex was allowed to react at pH 6.0–6.1 at 22°C with bromoacetic acid. The reaction was followed by means of amino acid analysis of the histidine and N^im-carboxymethylhistidine derivatives. The results of the alkylation study indicate that the nucleophilic, active histidine molecule is coordinated to the copper(II) ion through the amino nitrogen and a carboxylate oxygen with the imidazole group turned away from the copper. This model of copper-bound histidine permitted the determination of the intrinsic nucleophilic activity of the imidazole nitrogens through their respective rate constants for alkylation. The tele-nitrogen is three times more reactive than the pros-nitrogen in the histidine and in the pros-carboxymethylhistidine-tele-carboxymethylhistidine systems. The carboxymethylation of copper(II)-histidine and bovine pancreatic ribonuclease have some analogies, which suggest that in pros-carboxymethylhistidine-119 ribonuclease the carboxylate unit of the alkylated histidine residue points into the active site.     

Evidence for suitability of glutathione peroxidase as a protective enzyme: studies of oxidative damage, renaturation, and proteolysis

The stability of glutathione peroxidase was assessed in vitro via oxidative inactivation by peroxides and a peroxidizing fatty acid and by renaturation and proteolysis. The stability of glutathione peroxidase to methyl ethyl ketone peroxide, H2O2, linoleic acid hydroperoxide, and peroxidizing methyl linolenate was compared with the stability of several other enzymes. Sulfhydryl enzymes were the most labile to all four treatments. Some of the enzymes tested were very stable to methyl ethyl ketone peroxide but very labile to linoleic acid hydroperoxide treatment. Glutathione peroxidase in the absence of glutathione was relatively slowly inactivated by each treatment. Linoleic acid hydroperoxide damage to glutathione peroxidase was characterized by release of a nonstoichiometric amount of selenite from the protein. Glutathione peroxidase samples lost all of their activity when (i) acidified to pH 2, (ii) heated 5 min at 100 degrees C, and (iii) treated with 6 M guanidinium hydrochloride or 8.5 M urea and heated 5 min at 100 degrees C. When the pH 2 sample was neutralized or the guanidinium hydrochloride-treated sample was diluted 101-fold, about 80% of the original activity was recovered in 30 min. The samples treated with urea and heat recovered no activity when diluted 101-fold. No loss of glutathione peroxidase occurred during treatment for 24 h within trypsin or thermolysin. Based on these results, glutathione peroxidase appears to be a relatively stable enzyme, and thus is is well-suited to perform its role in peroxide detoxification and prevention of oxidative deterioration of cells.     

Zn(II)-induced dimerization of human carbonmonoxy hemoglobin

Binding of Zn(II) to the carbon monoxide complex of human hemoglobin was shown by equilibrium sedimentation and sedimentation velocity experiments at pH 7.0 to induce the dissociation of liganded tetramers to dimers but not to monomers. These results provide direct confirmation of previous kinetic and gel filtration experiments (R. D. Gray, (1980) J. Biol. Chem.255, 1812–1818) that Zn(II) binding to liganded hemoglobin produces a change in aggregation state of liganded hemoglobin.     

An RNA species involved in Escherichia coli ribonuclease P activity. Gene cloning and effect on transfer RnA synthesis in vivo

The isolation and characterization of a plasmid capable of complementing the temperature-sensitive transfer RNA biosynthetic mutation rnpA49 (ribonuclease P) is described. The DNA segment responsible for complementation codes for an RNA species, approximately 340 bases long. Hybridization-selection experiments indicated that all rnp mutants were deficient in the production of the complementing RNA at high temperature; these defects were not due to the accumulation of a precursor form of this RNA. Examination of tRNA species synthesized in vivo indicated that the plasmid clone did not completely relieve the deficiency in RNase P activity of rnpA49 since some tRNA precursors still accumulated in strain A49 containing the plasmid. At least one other tRNA precursor was no longer detectable in plasmid-containing rnpA49 cells.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

Effect of temperature on functional properties of carp hemoglobin

Precise oxygen equilibrium curves of carp hemoglobin have been obtained in 0·1 m-phosphate from 10 to 25 °C. The equilibrium data were analyzed according to the stepwise oxygenation model of Adair (1925) to obtain the enthalpy change (ΔH_i), entropy change (ΔS_i) and free energy change (ΔG_i) for the i (= 1, 2, 3, 4) individual oxygenation steps. The values of ΔH_i are definitely non-uniform with dependencies on i and the pH of the medium. The co-operative effects in carp hemoglobin are due mainly to enthalpic contributions under the conditions studied here. The thermodynamic properties suggest a structural transition with pK ~8·5 as was also seen in other functional and spectroscopic measurements.     

Chloride ion transport and its inhibition in thylakoid membranes

Cl- translocation across energized and nonenergized thylakoid membranes was found to be inhibited by piretanide, an inhibitor of active Cl- transport in fish intestinal epithelia. Piretanide has no effect on photophosphorylation catalyzed by phenazine methosulfate or on Ca2+-dependent ATPase activity of isolated chloroplast coupling factor (CF1). Light-dependent Cl- uptake, contrary to H+ uptake, is severalfold greater at pH 8.0 than at pH 6.7.     

Determination of mixtures of benzo[a]pyrene, 2,6-dimethylnaphthalene and their metabolites by high-performance liquid chromatography with fluorescence detection

Nonradiometric techniques were used to identify and quantitate two aromatic hydrocarbons and several of their metabolites in biological samples. High-performance liquid chromatographic separation combined with ultraviolet fluorescence detection provided a sensitive and selective method of trace analysis. Metabolites of benzo[a]pyrene and 2,6-dimethylnaphthalene were prepared by in vitro incubation of these substrates, singly and together, with liver microsomes of coho salmon (Oncorhynchus kisutch). Individual metabolites were separated by high-performance liquid chromatography and quantitated at fluorescence excitation and emission wavelengths characteristic of the corresponding parent hydrocarbon. Future refinement of these techniques may allow the determination of more complex mixtures of xenobiotics and their metabolites in marine organisms.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.     

Circular dichroism and spin-label studies of carp hemoglobin

Circular dichroism (c.d.) spectra were obtained for deoxy, oxy, carboxy, nitrosyl, aquomet and azidomet derivatives of carp hemoglobin. The spectra of the hemolysate and its two major components are virtually identical. Binding of diatomic ligands induces large changes in the 287 nm ellipticity. In the case of oxygen binding this change appears to be proportional to the free energy of co-operation. The changes of L-band ellipticity and Soret rotational strength with ligation reflect tertiary structural alterations and bear no relationship to quaternary transitions. The c.d. results indicate that carp deoxyhemoglobin has very similar tertiary and quaternary structures between pH 6·4 and 8·0, whereas the oxyhemoglobin undergoes continuous conformational adjustment in response to pH changes. The effect of inositol hexaphosphate on c.d. spectra is much smaller than it is on the functional properties. Electron paramagnetic resonance spectra of iodoacetamide nitroxide label are sensitive to ligation, the label is probably attached to Cys142β.     

Biophysical studies on circle formation by Sindbis virus 49 S RNA

Bacteriophage P2-infected cell extracts lacking the M gene product can package linear (mature) P2 DNA, but not its closed circular precursor, into phage particles. Thus the P2 M gene product is required for site-specific DNA cleavage (ter cleavage) during DNA packaging. The P2 M and P gene products have been extensively purified using an in vitro complementation-packaging assay. When studied in vitro, the site-specific DNA cutting which generates mature P2 DNA requires P2 head structures in addition to purified M and P proteins, spermidine, and ATP.     

Detection of two essential sulfhydryl residues in Escherichia coli B glycogen synthase

Results presented indicate that two distinct essential sulfhydryl residues are present in the Escherichia coli B glycogen synthase. One residue is modified by iodoacetic acid and can be protected by ADP or ADPglucose. The other site can be modified by 5,5′-dithiobis (2-nitrobenzoic acid) and is protected by glycogen. Each reagent appears to be specific for a given site and thus allows the two sites to be distingushed.     

Sensitive enzyme assays based on the production of chemiluminescent leaving groups

A new type of synthetic peptide substrate for amidase assay has been devised. The substrates are luminogenic, with potential for extremely high sensitivity, and are here exemplified by Boc- and Z-Ala-Ala-Phe-isoluminol amide. The synthetic substrates were designed to release isoluminol when hydrolyzed by enzyme; isoluminol production was determined by measuring its chemiluminescence. Kinetic constants of the luminogenic substrates were measured with α-chymotrypsin; and levels of the enzyme as low as 50 ng were determined conveniently. A comparison of similar luminogenic, chromogenic, and fluorogenic substrates is presented.     

Scanning electron microscopy of Drosophila melanogaster embryogenesis: III. Formation of the head and caudal segments

The formation of both the anterior most and posterior most segments in higher dipteran embryos involves complex movements of primordia which can be best visualized with the scanning electron microscope. During head formation, the gnathocephalic segments partially involute through the stomodeum. The labial segment forms the floor of the mouth, and the fused maxillary and mandibular segments form the lateral sides of the mouth. The involuted clypeolabrum forms the roof of the mouth. Invaginations of cells for segmentally derived sense organs can be found prior to involution on all the gnathocephalic and thoracic segments as well as on the labrum. The antennal sense organ derives from the lateral surface of the procephalic lobe. Following involution of the mouth parts, the dorsal ridge, which arises just anterior to the first thoracic segment, is drawn over the dorsal procephalic lobe producing the deep dorsal sac. The optic lobes of the brain invaginate anterior to the dorsal ridge just prior to the covering over of the head. The formation of the anal segment is similarly complex. Two rudimentary segments are found posterior to the eighth abdominal segment. During shortening of the germ band, the posterior most segment is drawn around the posterior tip of the embryo to lie ventrally. Two large anal pads form lateral to the anus from this segment. The next segment, following dorsal closure, produces a pair of anal sense organs and a central tuft of setae. Finally, the eighth abdominal segment gives rise to the posterior spiracles. Following dorsal closure these three segments fuse to produce the terminal (anal) segment of the larva.     

Exogenous versus endogenous acceptors in photosystem ii in inhibited chloroplasts

The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.