Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

Effect of actin concentration on the intermediate oxygen exchange of myosin; relation to the refractory state and the mechanism of exchange.

The effect of actin concentration on the myosin catalyzed exchange of phosphate oxygens with water accompanying ATP hydrolysis has been investigated. The extent of exchange was found to extrapolate to zero at infinite actin concentration at 23 and 0 degrees C for myosin subfragments S1(A1) and S1(A2). This result is consistent with actin associating directly with the product of the hydrolysis step and is not readily consistent with refractory state schemes in which the entire flow goes via a dissociating pathway. The possibility of a refractory state in the form of a phosphorylated intermediate or a bound metaphosphate state with hydrolysis occurring in the transition to the refractory state merits consideration. A full analysis of the dependence of intermediate exchange on the rate constants of the acto-S1 scheme is given and the errors arising from other methods of analysis are discussed. The rate of oxygen exchange was measured as 10 s-1 (23 degrees C) a value comparable with but slightly lower than the rate of reversal of the ATP cleavage step.     

Structure and activity of chloroplasts of sunflower leaves having various water potentials

Summary Changes in membrane integrity, conformation and configuration, and in photosystem II (PS II) activity (measured as dichloroindophenol photoreduction) of sunflower (Helianthus annuus L.) chloroplasts were studied after leaf tissue had been desiccated to various water potentials ( _w ). Fixatives for electron microscopy were adjusted osmotically to within 1 bar of the _w of the tissue to prevent rehydration during fixation. PS II activity decreased to 50% of the control activity at a _w of-26 bar. At this _w , leaf viability was being lost but there was virtually no loss of integrity of the thylakoid lamellar system. Even at extreme _w (below-100 bar), thylakoids retained much structural detail but were less stained. At-26 bar, intrathylakoid spacing (configuration) and lamellar thickness (conformation) were decreased in vivo. Upon isolation of the plastids, the differences in configuration disappeared but the differences in conformation remained. The decreases in membrane conformation and PS II activity both, in vivo and in vitro suggest that alterations in conformation may cause decreases in chloroplast activity at _w as low as-26 bar. Since structural detail was maintained, however, previous observations of altered membrane integrity, which involved tissue fixed without osmotic support, may have been affected by tissue rehydration during fixation.Abbreviations DCIP sodium 2,6-dichloroindophenol - PS II photosystem II - _w leaf water potential     

The rapid labeling of adenosine triphosphate by 32P-labeled inorganic phosphate and the exchange of phosphate oxygens as related to conformational coupling in oxidative phosphorylation.

Evidence is presented that extends and amplifies the concept that in oxidative phosphorylation energy input serves to bring about release of ATP formed at a catalytic site by reversal of hydrolysis. The evidence with beef heart submitochondrial particles includes additional demonstration of uncoupler insensitive Pi leads to HOH exhchange, demonstration that this exchange is sensitive to the specific phosphorylation inhibitor, oligomycin, and demonstration that the small burst of uncoupler-insensitive ATP, rapidly labeled after addition of a tracer of 32Pi, behaves in a manner consistent with its participation as a membrane-bound intermediate in the Pi leads to HOH exchange. In addition, data are presented showing that addition of hexokinase plus glucose to submitochondrial particles in presence of ADP and Pi considerably lowers the Pi leads to HOH exchange but that further addition of cyanide or 2,4-dinitrophenol or both has little additional effect. Such data are compatible with no energy requirement for formation of bound ATP. However, with a large excess of hexokinase, the rate of the Pi leads to HOH exchange is further depressed. This could reflect some use of energy to promote formation of ATP at the catalytic site or to maintain the integrity of the phosphorylation system. Relationships of these findings to related information in the field are discussed.     

Serum hormone levels following partial hepatectomy in the rat.

Serum levels of insulin, glucagon, growth hormone (somatotrophin) and thyroxine (TT₄) were measured by radioimmunoassay following both sham operation and 70% partial hepatectomy in the rat to evaluate changes in hormone levels during liver regeneration. An eleven fold increase in glucagon was observed (from 112 ± 10 pg/ml to 1500 ± 200 pg/ml) 6 hours following partial hepatectomy but not sham operation. In contrast, insulin levels remained unchanged compared to sham controls for up to 72 hr while growth hormone fell to low levels, 6 to 48 hr after partial hepatectomy. Both total thyroxine and free thyroxine levels also fell 24–72 hours after hepatectomy. These studies suggest that growth hormone, thyroxine and insulin are not primary stimulants of hepatic regeneration although the data suggests that glucagon may modify this growth process.     

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.     

Molecular cloning of an Escherichia coli plasmid determinant than encodes for the production of heat-stable enterotoxin.

A conjugative plasmid, ESF0041 was isolated from an enterotoxigenic strain of Escherichia coli from calves. ESF0041 was found to be 65 x 10(6) daltons in mass of a member of the F incompatibility complex. Acquisition of ESF0041 by E. coli K-12 was invariably associated with the capacity to produce heat-stable (ST) enterotoxin. ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase. Transformation of E. coli K-12 with the ligation mixture led to the isolation of an ST+ clone. Further analysis of the plasmid deoxyribonucleic acid from this clone showed that a structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 10(6)-dalton ESF0041 fragment in pSC101. In turn, 5.7 x 10(6)-dalton fragment was ligated to a multicopy COLE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Acetylene reduction (nitrogen fixation) and metabolic activities of soybean having various leaf and nodule water potentials

An apparatus was designed that permitted acetylene reduction (N₂ fixation) by root nodules to be measured in situ simultaneously with net photosynthesis, dark respiration, and transpiration of the shoot in soybean plants (Glycine max [L.] Merr. var. Beeson). Tests showed that acetylene reduction was linear with time for at least 5 hours, except for the first 30 to 60 minutes. Endogenous ethylene production did not affect the measurements. Successive determinations of acetylene reduction could be made without apparent aftereffects on the plant.