Bifunctional intercalation and sequence specificity in the binding of quinomycin and triostin antibiotics to deoxyribonucleic acid.

Quinomycin C, triostin A and triostin C are peptide antibiotics of the quinoxaline family, of which echinomycin (quinomycin A) is also a member. They all remove and reverse the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs, and the unwinding angle at I 0.01 is, in all cases, almost twice that of ethidium. Thus, as with echinomycin, they can be characterized as bifunctional intercalating agents. For the triostins this conclusion has been confirmed by measurements of changes in the viscosity of sonicated rod-like DNA fragments; the helix extension was found to be almost double that expected for a simple monofunctional intercalation process. For triostin A, further evidence for bifunctionality was derived from the cross-over point of binding isotherms to nicked circular and closed circular bacteriophage-PM2DNA. Binding curves for the interaction of quinomycin C and triostin A with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis, but triostin C was too insoluble in aqueous solution to make this method applicable. For quinomycin C the highest binding constant was found with Micrococcus lysodeikticus DNA, and its pattern of specificity among natural DNA species was broadly similar to that of echinomycin, although the binding constants were 2--6 times as large. For triostin A the highest binding constant was again found for M. lysodeikticus DNA, but the specificity pattern was quite different from that of the quinomycins. In particular, triostin A bound better to poly(dA-dT) than to the poly(dG-dC) whereas this order was reversed for quinomycin C. There was also evidence that the binding to poly(dA-dT) might be co-operative in nature. No significant interaction could be detected with poly(dA).poly(dT) or with RNA from Escherichia coli. Poly(dG).poly(dC) gave variable results, depending on the source of the polymer. The different patterns of specificity displayed by the quinomycins and triostins are tentatively ascribed to differences in their conformations in solution.     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.     

CALCIUM ION DEPENDENCE OF THE 2-SITE IMMUNORADIOMETRIC ASSAY OF MOORE''S S-100 PROTEIN

Abstract— The two-site immunoradiometric assay (two-site IRMA) for a specific protein of the nervous system, S-100, is carried out by reaction of the S-100 protein solution with a solid-phase anti(S-100) followed by a second reaction in which the insoluble product is incubated with purified, radioactive anti(S-100). Unreacted labeled antibodies remain in solution and are washed away. As the amount of S-100 increases, the radioactivity in the solid-phase increases. The most significant assay variable is the effect of calcium on the assay dose-response. 0.1 mM-EDTA causes a total inhibition of the dose-response curve which is reversed by increasing the concentration of calcium ions. The dose-response reaches a maximum at 1.0mM-Ca²⁺. then becomes progressively inhibited as the Ca²⁺ concentration is increased further. Previous immunochemical studies of S-100 which did not allow for this effect must now be interpreted with caution.     

A method for demonstrating the heterogeneity of pigeon red cell membrane vesicles based on their glycine transport activity

A procedure is described which is capable of detecting the changes in size and/or density of small membrane vesicles resulting from solute uptake. Vesicles which have taken up solute sediment more slowly in a density gradient, the ratio of glycine uptake/vesicle-trapped space is not uniform in the vesicle population, and vesicles with higher uptake/space ratios are preferentially retarded upon centrifugation.Alanine transport activity is associated with glycine transport activity in that retardation of vesicles due to glycine uptake equally retards vesicles possessing alanine uptake activity.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Indole alkaloid biosynthesis: Partial purification of “ajmalicine synthetase” from Catharanthus roseus

The “ajmalicine synthetase” system of $\text{Catharanthus roseus}$ has been partially purified from callus, seedlings and mature plants. On gel filtration of the cell-free extract, four β-D-glucosidase isozymes were observed in seedlings and plants. Only two were present in the callus. A protein peak at 55,000 daltons in all three materials was capable of synthesizing ajmalicine from tryptamine and secologanin in the presence of NADPH. This “ajmalicine synthetase” rapidly lost its ability to synthsize ajmalicine, but retained the β-glucosidase activity.