Utilization of the Synthetic Phosphagen Cyclocreatine Phosphate by a Simple Brain Model During Stimulation by Neuroexcitatory Amino Acids

The ability of 1-carboxymethyl-2-imino-3-phosphonoimidazolidine (cyclocreatine-P), accumulated by a simple brain model, to function as a supplemental synthetic phosphagen and respond to the decreases in cytosolic ATP/free ADP ratios that occur during prolonged stimulation by various excitatory amino acids was investigated. Suspensions of chopped whole brain from 11- to 14-day-old chick embryos were incubated with 30 mM cyclocreatine for 90 min, resulting in accumulation of 100 mumol/g dry weight of cyclocreatine-P, and then incubated for up to 1 h with a series of excitatory amino acids of widely differing potencies. Under these conditions net utilization of cyclocreatine-P was detected in response to stimulation by the following neuroexcitatory compounds at the indicated threshold concentrations: kainate (20 microM), N-methyl-DL-aspartate (20 microM), L-homocysteate (20 microM), L-glutamate (200 microM), D-glutamate (200 microM), L-aspartate (2 mM), DL-2-amino-3-phosphonopropionate (2 mM), and DL-2-amino-4-phosphonobutyrate (2 mM). Significant increases in water content of chick embryo brain minces accompanied stimulation by excitatory amino acids. It is suggested that changes in water content or cyclocreatine-P levels in this sensitive brain model might be utilized in automatable screening procedures for detecting novel antagonists and/or new agonists of excitatory amino acids.     

Sequence analysis of a Dictyostelium discoideum gene coding for an active dihydroorotate dehydrogenase in yeast

A Dictyostelium discoideum DNA fragment isolated on the basis of its ability to complement the ural mutation of yeast, codes for a dihydroorotate dehydrogenase activity. The complete nucleotide sequence of this 1898 bp fragment has been determined and reveals an open reading frame capable of coding for a 369 amino acid polypeptide of molecular mass 47.000. The gene shows preferential use of codons with weak pairing forces. Eleven codons, mainly those with a G in the third position, are absent. The flanking sequences are unusually rich in A + T (80%). Several direct and inverted repeats exist in the 5' flanking sequence.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Metabolism of [6-14C]orotate by shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus

Incorporation of radioactivity from [6-¹⁴C]orotate into the pyrimidine constituents of shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus was examined with special reference to the unusual pyrimidine constituents. With each species, although 80% of the orotate supplied was catabolized to β-alanine, all the pyrimidine derivatives became radioactively labelled. With Pisum, the major part of the radioactivity incorporated into pyrimidines was located in UMP and the uracil derivatives, including the uracilyl amino acids willardiine and isowillardiine. With Phaseolus, UMP and the uracil derivatives were again the major radioactive products; incorporation of radioactivity into 5-ribosyluracil (pseudouridine), which accumulates in Phaseolus tissues, was comparable to the incorporation into orotidine and twice that found in cytidine. Lathyrus incorporated a substantially larger part of the presented [6-¹⁴C] orotate into pyrimidine derivatives than did the other two species. CMP was the most highly radioactive product, followed next by lathyrine and UMP. Surprisingly, 20% of the total radioactivity incorporated into pyrimidines by Lathyrus was located in the pyrimidine amino acid lathyrine. This confirms previous evidence that lathyrine is essentially a product of the orotate pathway. The overall recovery of radioactivity in all three species was 93–95%. The data emphasize the necessity of including the less common pyrimidine constituents, as well as the common ones, in quantitative studies of pyrimidine metabolism in plants.     

Supercomputer analysis of purine and pyrimidine metabolism leading to DNA synthesis

A model-system is established to analyze purine and pyrimidine metabolism leading to DNA synthesis. The principal aim is to explore the flow and regulation of terminal deoxynucleoside triophosphates (dNTPs) in various input and parametric conditions. A series of flow equations are established, which are subsequently converted to differential equations. These are programmed (Fortran) and analyzed on a Cray X-MP/48 supercomputer. The pool concentrations are presented as a function of time in conditions in which various pertinent parameters of the system are modified. The system is formulated by 100 differential equations.     

Site-directed mutagenesis of the T4 endonuclease V gene: mutations which enhance enzyme specific activity at low salt concentrations

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well in E. coli and not only significantly enhanced the UV survival of repair-deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129----Trp and Tyr-129----Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129----Phe and Tyr-129----Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129----Phe mutant and to 20% that of wild type for the Tyr-129----Trp mutant.     

Synthesis of methyl

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).