Preparation of algal extracts for bacterial luciferase assay of pyridine nucleotides

Pyridine nucleotides in the cyanobacterium Synechococcus leopoliensis (Racib) Komárek and in the green algae Scenedesmus obtusiusulus Chod. and Ankistrodesmus braunii (Naegeli) Brunnth. were extracted with either hot NaOH, hot ethanol, hot acidic or alkaline methanol, perchloric acid (PCA) or trichloroacetic acid (TCA). Reduced pyridine nucleotides were determined with NADH- and NADPH-dependent bacterial luciferase. Oxidized nucleotides were determined after enzymatic conversion to the reduced forms. The yields of pyridine nucleotides in the extracts were compared to the relative extraction efficiency for ATP. Extraction with hot NaOH appeared satisfactory for reduced nucleotides in the green algae but less so in Synechococcus. Extraction with PCA seemed preferable for the oxidized nucleotides. The sensitivity of the bacterial luciferase assay was lowered by all extractants. Criteria for assessment of optimal extraction procedures for pyridine nucleotides are discussed.     

Studies of regulatory metabolism in Moniezia expansa: Effects of cambendazole and mebendazole

Effects of cambendazole and mebendazole on the respiratory metabolism of the anterior portion of Moniezia expansa were investigated in vitro. Anaerobically and in the presence of glucose, both drugs inhibited glucose uptake and increased glycogen utilisation. They reduced succinate production, by inhibiting fumarate reductase and (in the case of cambendazole) phosphoenolypyruvate carboxykinase activities, and increased lactate production. The additional lactate formed was accumulated in the worms. The drugs diminished ATP synthesis and/or turnover of adenine nucleotides. Aerobically, the drugs exerted similar effects on glucose uptake, glycogen utilisation and adenine nucleotides but the formation of end products was unaffected. Hexokinase and phosphofructokinase activities were inhibited by the drugs in vitro, but were not inhibited in extracts of parasites preincubated with the drugs.     

Enzymatic oxidation of p-nitrophenol.

A Moraxella sp. capable of growth with p-nitrophenol was isolated from activated sludge. Differential centrifugation of crude cell extracts gave a membrane preparation that oxidized p-nitrophenol to hydroquinone and nitrite. Enzymatic activity was dependent on the presence of oxygen and reduced pyridine nucleotides and was stimulated by the addition of flavin adenine dinucleotide. Experiments with ¹⁸O₂ showed that the incoming hydroxyl group was derived from molecular oxygen. The soluble fraction prepared from crude cell extracts oxidized hydroquinone to a product whose spectral properties were identical to those reported for γ-hydroxymuconic semialdehyde.     

Purification of leaf nucleotides and nucleosides on insoluble polyvinylpyrrolidone

Of the 19 nucleotides and nucleosides tested, all were eluted by 1 mM HCl in less than 60 ml from 2 × 6-cm columns of Polyclar AT (an insoluble polyvinylpyrrolidone). Recoveries were good and, with the possible exceptions of ADPG and UDPG, the presence of cotton leaf extract did not decrease recovery of known nucleotides and nucleosides.Passing leaf extracts through Polyclar AT removed most, but not all, of the uv-absorbing impurities that interfere with quantitation of nucleotides and nucleosides. The optimum pH for purification of HClO₄ extracts from leaves of alfalfa, cotton, grape, and orange appeared to be between 2.0 and 3.0. In this pH range Polyclar AT removed from 59 to 91% of the substances in leaf extracts that absorbed at 230 nm and from 93 to 97% of the substances that absorbed at 320 nm.Extraction of leaf extract with isoamyl alcohol was relatively ineffective and extraction with ether was almost completely ineffective in removing uv-absorbing impurities.Because nucleotides and nucleosides quickly pass through a short column of Polyclar AT at pH 3.0 while plant phenols are retained, this procedure provides a simple and rapid method for bulk purification of leaf extracts prior to chromatography and assay of nucleotides and nucleosides.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.     

Compartmentation of Nucleotides in Corn Root Tips Studied by P-NMR and HPLC

Corn (Zea mays L.) root tips were subjected to different conditions so that nucleotide levels varied over a wide range. Levels of nucleotides in corn root tips were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography. Results indicate: (a) Similar amounts of NTP and sugar nucleotides were observed by in vivo NMR and in extracts. In contrast, a significant amount of NDP observed in root tip extracts was not detected by in vivo NMR. Thus, for a given sample, [NTP]/[NDP] ratios determined in vivo by ³¹P-NMR are always higher than ratios observed in extracts, deviating by ~4-fold at the highest ratios. The NMR-invisible pool of NDP appeared quite metabolically inert, barely changing in size as total cell NDP changed. We conclude that NDP in corn root tips is compartmented with respect to NMR visibility, and that it is the NMR-visible pool which responds dynamically to metabolic state. The NMR-invisible NDP could either be immobilized (and so have broad, undetectable NMR signals), or be complexed with species that cause the chemical shift of NDP to change (so it does not contribute to the NMR signal of free NDP), or both. (b) ³¹P-NMR cannot distinguish between bases (A, U, C, and G) of nucleotides. HPLC analysis of root tip extracts showed that the relative amount of each base in the NTP and NDP pools was quite constant in the different samples. (c) In extracts, for each of the nonadenylate nucleotides, [NTP]/[NDP] was linearly proportional to [ATP]/[ADP], indicating near equilibrium in the nucleoside diphosphokinase (NDPK) reaction. However, the apparent equilibrium constants for the phosphorylation of GDP and UDP by ATP were significantly lower than 1, the true equilibrium constant for the NDPK reaction. Thus, for a given sample, [ATP]/[ADP] ~ [CTP]/[CDP] > [UTP]/[UDP] > [GTP]/[GDP]. This result suggests that the different NDPs in corn root tips do not have equal access to NDPK.     

Enzymatic formation of intermediates in the biosynthests of ajmalicine: Strictosidine and cathenamine

Pre-purified enzymes isolated from Catharanthus roseus suspension cultures synthesize strictosidine and cathenamine from tryptamine and secologanin. Whereas strictosidine showed metabolic activity, cathenamine accumulates during the cell-free incubations in the absence of reduced pyridine nucleotides. In the presence of δ-d-gluconolactone (0.1 M), strictosidine accumulates in a yield of ca 50%. Optimum conditions for its accumulation in crude extracts were found to be at pH 4.1, 0.25 mM tryptamine and 1.25 mM secologinin. Strictosidine synthase is stable for more than 1.5 months at 4°. The optimum conditions for the enzymatic synthesis of cathenamine are 1.54 mM tryptamine and 7.7 mM secologanin at pH 7.5. In the presence of NH₄⁺ the formation of the latter alkaloid decreases due to the synthesis of unidentified compounds.     

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO₄^?, subjected to preliminary purification on a Cu²⁺-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.     

Cellulose synthesis by Acetobacter xylinum I. Low molecular weight compounds present in the region of synthesis

An analysis has been made of the low molecular weight fraction present in the region of cellulose synthesis in Acetobacter xylinum suspensions.A number of nucleic acid bases, nucleosides and nucleotides, together with α-glucose 1-phosphate and UDPG, were detected in various extracts of washed cells supplied with glucose. Since glucose-6-P could be detected in extracts of ultrasonically disrupted cells, but not in extracts of whole cells, it was concluded that separate pools of hexose phosphate exist in A. xylinum. Preferential release of α-glucose-1-P, UDPG and nucleotides was observed during ethanol and EDTA treatment of bacteria. Electron microscopic examination of treated and untreated cells revealed that extensive modification of the cell wall region occured during such treatments. The results support the proposal that α-glucose-1-P, UDPG and nucleotide pools are localised in the cell envelope region, possibly in the periplasm, and that A. xylinum possesses a second permeability barrier outside the cytoplasmic membrane. Nucleic acid bases and nucleosides were observed to diffuse freely through the cell wall and accumulate in the medium, probably as the result of nucleic acid breakdown. The results imply that the effects of cell damage caused by the isolation of the bacteria from the surface pellicle of the culture medium, together with nutrient deprivation, should be considered in work using the non-proliferating system. A study of the variation in concentration with time of α-glucose-1-P and UDPG, during cellulose synthesis, indicated that both components mau play an immediate role in cellulose synthesis.Glycosylated lipid compounds were detected in both cell wall extracts and supernatant fluid, but it is not certain whether these compounds are constituents of the supernatant fluid in vivo.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

Specific enzymatic determinations of adenosine triphosphate.

The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).     

Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [¹⁴C]NaHCO₃, [¹⁴C]carbamylaspartate, and [¹⁴C]orotic acid into uridine nucleotides; (b) the isolation of [¹⁴C]orotic acid when [¹⁴C]NaHCO₃ and [¹⁴C]carbamylaspartate were used as precursors; (c) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.     

Pyruvate oxidation by the Reiter strain of Treponema phagedenis.

Spectrophotometric assays of pyruvate oxidation catalyzed by extracts of the Reiter strain of Treponema phagedenis indicated that viologen dyes, flavin nucleotides, and a ferric iron chelate, but not pyridine nucleotides, were utilized as electron acceptors. Benzyl viologen-linked activity partially sedimented during ultracentrifugation and appeared similar to clostridial pyruvate:ferredoxin oxidoreductase with respect to the spectral properties of the enzyme chromophore. Electron carrier activity in treponemal extracts was quantitated by a metronidazole-linked assay in which the oxidation of pyruvate by carrier-depleted extracts led to the reduction of electron carrier in the crude extracts which then reduced metronidazole. The rate of metronidazole reduction was proportional to the amount of electron carrier present in the assay. Electron carrier activity in Triton X-100-solubilized, crude extracts partially purified by DEAE-cellulose chromatography and gel filtration was attributed to a protein possessing the spectral and physical properties of a ferredoxin. A similar protein appeared to be present in extracts of Treponema denticola ST10.     

Inhibition of female mating receptivity by male-derived extracts in two Callosobruchus species: Consequences for interspecific mating

We investigated the effects of injecting male-derived extracts on congeneric female receptivity in two species of Callosobruchus beetle, C. chinensis and C. maculatus. We also examined the influence of interspecific mating on female remating behaviour in these two species. Male-derived extracts reduced congeneric female receptivity in both species. As quick-acting components, extracts of C. chinensis male seminal vesicles reduced the receptivity of C. maculatus females, whereas extracts of C. maculatus male testes reduced the receptivity of C. chinensis females. As slow-acting components, extracts of male accessory glands of other species reduced the receptivity of both C. maculatus and chinensis females. After interspecific mating, the sperm of C. maculatus males were transferred to the reproductive organs of C. chinensis females, thereby reducing their receptivity. In contrast, no C. chinensis sperm were transferred to the reproductive organs of C. maculatus females; accordingly, the latter's receptivity was not reduced. Furthermore, the survival rate of C. chinensis females decreased markedly after interspecific mating. These results raise the possibility that under circumstances where populations of these two species share the same habitat, reproductive interference would occur only in the interactions between C. maculatus males and C. chinensis females.     

Chromatographic separation and identification of nucleotides from animal tissues and yeast cells on a polyethyleneimine cellulose column.

A procedure has been developed for the separation and identification of nucleotides by anion-exchange chromatography. The method includes column chromatography on PEI-cellulose with an unbuffered exponential salt gradient. This easily handled procedure gives a high degree of resolution and reproducibility and demands no personal attendance during elution. Application of the method is demonstrated with a mouse liver extract and extracts of yeast cells (Saccharomyces cerevisiae).     

Quantitative extraction and estimation of intracellular nicotinamide nucleotides of Escherichia coli.

The conditions of extraction of nicotinamide nucleotides from Escherichia coli were systematically investigated. The optimum pH for NAD⁺ and NADP⁺ appeared to be around 1.5 while that for NADH and NADPH was around 12.5. Seven minutes at 60°C was chosen for the extraction of all four nucleotides. Addition of CuCl to the alkaline extract improved considerably the recovery of the reduced forms. Two sensitive (10–100 pmol) assay methods for nicotinamide nucleotides were compared. A spectrophotometric cycling method based on the measurement of reduced thiazolyl blue formation appeared to be more sensitive and more reproducible and gave better recovery than a fluorometric cycling method used earlier in bacterial studies.     

Ribose Utilization by Veillonella alcalescens

The utilization of ribose by Veillonella alcalescens has been further investigated. Nonfermentation of ribose is not a result of a phosphorylation lesion since ribose-phosphorylating activity was measured in cell extracts. Resting cells accumulated ribose-5-phosphate and nucleotides when ¹⁴C-ribose was provided; no other sugar phosphates were detectable. Resting cells that were shifted to growth conditions polymerized rather than degraded the accumulated ribose compounds. Cell extracts contained a fructose diphosphate phosphatase. Ribose-5-phosphate, glucose-6-phosphate, and fructose-6-phosphate were not hydrolyzed. It is postulated that the nonfermentation of ribose is not due to any metabolic lesions, but is a consequence of metabolic control at the fructose diphosphate level of glycolysis.     

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.