Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver.

An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.     

Age-dependent content of polymerized lipids in Sphagnum fuscum

The polymerized lipids of Sphagnum fuscum cell wall fragments were found to be composed of long chain hydroxy acids, long chain dicarboxylic acids, fatty alcohols and fatty acids. Their content, on a dry weight basis, was low in the topmost 3 cm of the shoot and increased with shoot age (and depth). A pronounced increase (16-fold) occurred in the contents of hydroxy acids which comprised 76% of the totals at the depth of 40–43 cm. The increase at the depth of 40-43 cm is considered to be at least partly associated with the frequently found destruction of the most suscptible part, the thin-walled stem center. The results suggest that aliphatic lipid polymers are present and acumulated in cell walls resistant to breakdown.     

Cycling of organic and inorganic sulphur in a chestnut oak forest

Summary Sulfur (S) cycling in a chestnut oak forest on Walker Branch Watershed, Tennessee, was dominated by geochemical processes involving sulfate. Even though available SO ₄ ^2- was present far in excess of forest nutritional requirements, the ecosystem as a whole accumulated 60% of incoming SO₄–S. Most (90%) of this accumulation occurred by SO ₄ ^2- adsorption in sesquioxide-rich subsurface soils, with a relatively minor amount accumulating and cycling as SO ₄ ^2- within vegetative components. Organic sulfates are thought to constitute a large proportion of total S in surface soils, also, and to provide a pool of readily mineralized available S within the ecosystem.Research sponsored by Division of Biomedical and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with Union Carbide Corporation. Soil ester sulfate work sponsored by contract RP-1813-1 with the Electric Power Research Institute. Publication No. 1990, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830     

Oscillations and efficiency in glycolysis

We suggest that temporal oscillations of concentrations of intermediates in biochemical reaction systems may enhance the efficiency of free energy conversion (reduce dissipation) in those reactions. Experiments on glycolysis are used to estimate the Gibbs free energy changes along the glycolysis mechanism, and to postulate a construct for the glycolysis "machine" which involves: the PFK reaction as the primary oscillophor; the GAPDH reaction as a phase-shifting device; and the PK reaction with the property of intrinsic oscillatory response at resonance with the driving frequency. Analysis of a simple reaction mechanism with these postulated properties shows that the conversion of free energy from reactants to products is more efficient in an oscillatory than a steady state operation. The efficiency of free energy conversion in glycolysis from glucose + ADP to products + ATP is estimated to be increased by 5--10% due to oscillations. This may have been relevant for the evolutionary development of oscillations such as in glycolysis, especially in anaerobic cells.     

Ribosomal DNA of the fly Sciara coprophila has a very small and homogeneous repeat unit

Summary In this report we show by hybridization of restriction fragments and by Miller spreads that the unit repeat of the fly Sciara coprophila is only 8.4 kb which is the smallest known for a multicellular eukaryote. The 8.4 kb EcoR1 fragment containing a complete unit of Sciara rDNA was cloned in pBR322, and mapped by the method of Parker (1977) and also by double digestion. The coding regions for 28S, 18S, and 5.8S RNA were localized by the method of Berk and Sharp (1977). From these data we conclude that the nontranscribed spacer, external transcribed spacer, and internal transcribed spacer are all shorter than in other organisms, thereby giving rise to the shorter overall rDNA repeat unit of Sciara.At least 90% of the Sciara rDNA repeats are homogeneous, with a length of 8.4 kb, but a 700 bp ladder of minor bands can also be found in digestions of total genome DNA. This profile of major and minor bands is identical between the X and X chromosomes, as seen by a comparison of several genotypes.There are only 45 rRNA genes per X chromosome of Sciara (Gerbi and Crouse, 1976). These can easily be counted by low magnification Miller speads which show that virtually all gene copies are actively being transcribed in the stage of spermatogenesis examined. This is the first demonstration for any reiterated gene family where all copies are shown to be simultaneously active.Present address same as last author     

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

A negative influence of manure on calcareous soils

Summary On a soil rich in CaCO₃ in a semiaride climate in Algeria a hard soil layer, impermeable for plant roots, was formed in a depth of 20–40 cm after farmyard manure application and irrigation. To find the reason soil samples of this field were taken and leaching experiments were carried out in the laboratory, with the result that much more Caions and HCO₃-ions were leached out of the soils with farmyard manure application than from the soils without manure. Probably the high amount of CO₂, being liberated by the organic matter, and the irrigation water dissolved the CaCO₃ in the soil, and the formed Ca- and HCO₃-ions followed the movement of water in the soil. Where the Ca(HCO₃)₂ reached soil layers with a less amount of CO₂, CaCO₃ precipitated and formed the hard soil layer. In order to avoid the formation of such calcareous crusts on irrigated, limy soils in a dry climate it is recommended to fertilize rather often small quantities instead of rarely big quantities of farmyard manure.     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.