Sull'attivazione Funzionale dei Semi Germinanti. Nota v. l'attività Nicotinamide Adenina Dinucleotide Ridotto: - e Nicotinamide Adenina Dinucleotide Fosfato Ridotto: 2,6 - Diclorofenolo Indofenolo Ossidoreduttasica

Abstract

On the functional activation of germinating seeds. Note V. NADH₂:— and NADPH₂: DIP oxidoreductase activity. - NADH ₂:— and NADPH ₂ : DIP oxidoreductase activities are ascertained, in Pisum sativum and Zea mays, in the acetone precipitates obtained from extracts of dry and germinating seeds.

Such activities are always present and increase during the first days of germination.

In the mays, where in the embryo the reduction of the DIP is particularly high, at the point that it could be measurable on less than 1 mg of material, the NADPH ₂ : DIP oxidoreductase activity prevails. In the pea, the NADH ₂ : DIP oxidoreductase activity prevails.

Endosperm of mays and cotyledons and embryonic axis of pea have NADH ₂ : DIP and NADPH ₂ : DIP oxidoreductase activities which, referred to the dry weight, are of the same order of extent and about thirty-fifty times inferior to those of the embryo of mays.     

Reduced nicotinamide-adenine-dinucleotide-pyrophosphorylase: A novel enzyme in seeds

An enzyme which splits reduced NAD has been partially purified from pea (Pisum sativum, Kelvedon Wonder) seeds. The activity requires orthophosphate and the products are ADP and probably NMN (dihydro NMN?). The enzyme splits the NADH₂ at the pyrophosphate bond and incorporates the phosphate into the AMP residue. NAD, NADP or NADPH₂ could not replace NADH₂. The enzyme is unstable during storage, is activated by Mg²⁺ and by Mn²⁺, and inhibited by Ca²⁺. K⁺, Li⁺ and NH₄⁺ have no effect. The possible role of this enzyme in the synthesis of ATP in seeds at the early stage of germination is discussed.     

The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH₂ of high purity, the maximal specific rates were similar to those obtained with NAD and NADH₂, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH₂, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH₂ do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH₂. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH₂ than NAD and NADH₂. 5. Phosphate behaves as a competitive inhibitor towards NADP.     

Nonenzymatic tight binding of radioactivity to macromolecular fractions as a source of error in labeling experiments

A highly sensitive recording method is developed for determining the activity of the CoB-12-dependent enzyme, bacterial glycerol dehydratase. The method is based on the ability of alcohol dehydrogenase, in the presence of NADH₂, to reduce aldehydes formed from glycols by glycerol dehydratase. The rate of the coupled reaction is measured spectrophotometrically by the decrease in optical density at 340 nm or by measuring the decrease of H⁺ concentration with a sensitive pH-metric method. The conditions for coupling these two reactions, method of application, and its advantages and limitations are discussed. The method is highly sensitive and makes it possible to detect 0.5–2.5 × 10^?3 glycerol dehydratase units.     

Les nitrate-réductases bactériennes

Résumé Les formes réduites du benzyl-viologène, du méthyl-viologène et du FMN sont utilisées comme donneuses d'électrons par la nitrate-réductase A. Le benzyl-viologène est le plus efficace de ces 3 transporteurs: c'est lui qui fournit la plus faible constante de Michaelis. Nos résultats appuient l'opinion selon laquelle les indicateurs viologènes réduits et le FMNH₂ céderaient directement leurs électrons à l'enzyme.Les extraits bruts d'A. aerogenes et M. denitrificans contenant la nitrate-réductase A catalysent, en anaérobiose, l'oxydation du NADH₂ par le nitrate. Dans les mêmes conditions, le NADPH₂ ne sert pas de donneur d'électrons. Les traitements suivants font perdre aux extraits de M. denitrificans leurs activités NADH₂-nitrate-réductase et NADH₂-oxydase sans inactiver l'enzyme A (dosé en présence de benzyl-viologène): (a) action d'un détergent: le désoxycholate de sodium à la concentration 0,1 M; (b) chauffage à 55° C pendant 4 min. Ces résultats, ainsi que les observations faites par d'autres chercheurs, démontrent que la nitrate-réductase A n'est pas un enzyme à pyridine-nucléotide, et que les électrons issus du NADH₂ lui sont transférés indirectement par l'intermédiaire d'une chaîne de transporteurs et non pas directement. L'oxygène inhibe de 60% la réduction de NO₃ ^- en NO₂ ^- aux dépens du NADH₂ catalysée par un extrait de M. denitrificans.

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Bacterial nitrate reductasesII. Behaviour of enzyme A towards electron donors

Summary Reduced forms of benzyl viologen, methyl viologen and FMN are utilized as electron donors by nitrate reductase A. Benzyl viologen is the most efficient donor of these 3 electron carriers; it has the lowest Michaelis constant. Our results suggest that the reduced viologens and FMNH₂ donate their electrons directly to the enzyme.Crude extracts of A. aerogenes and M. denitrificans containing nitrate reductase A catalyze the oxidation of NADH₂ by nitrate under anaerobic conditions. Under the same conditions NADPH₂ does not serve as an electron donor. The following treatments cause the loss of NADH₂-nitrate reductase and NADH₂ oxidase activities in extracts of M. denitrificans without inactivating enzyme A (estimated in presence of benzyl viologen): (a) action of sodium deoxycholate 0,1 M; (b) heating at 55° C for 4 min. These results, as well as observations of other authors, show that the nitrate reductase A is not a pyridine nucleotide enzyme and that the electrons from NADH₂ are transferred to nitrate reductase through intermediate electron carriers. The reduction of NO₃ ^- to NO₂ ^- at the expense of NADH₂ catalyzed by an extract of M. denitrificans is inhibited 60% by the presence of O₂.

     

Role of Copper Ions in the Regulation of L-Glutamate Biosynthesis

Cell-free extracts of Brevibacterium thiogenitalis culture grown in the presence of copper catalyzed the oxidation of NADH₂ and succinate through an electron transport chain which contained menaquinones and cytochromes a, b and c. On the other hand, extracts of cells grown in the absence of copper lacked cytochromes a and c, and contained cytochrome d.

These findings, as well as the results obtained in inhibition experiments, suggest that in copper-deficient cells the major part of NADH₂ was oxidized via a bypass in which the electrons were transferred directly from flavoprotein or cytochrome b to molecular oxygen.

Electron transport from these substrates to molecular oxygen resulted in ATP synthesis. The average P/O ratios in extracts of the copper-sufficient cells were 0.33 for generated NADH₂, 0.20 for added NADH₂, and 0.34 for succinate, and those in extracts of the copper-deficient cells were 0.15, 0.13 and 0.21, respectively. In addition, a linear relationship was found between the yield of L-glutamate from acetate and the P/Ο ratios with both NADH₂ and succinate as substrates.

From these results, it is reasonable to consider that the poor yield of L-glutamate from acetate in copper-deficient cells was due to a reduction in energy supply, which was caused by the low efficiency of oxidative phosphorylation.     

Enzyme des Fettsäureabbaus in den Organen des Flußkrebses Orconectes limosus Rafinesque

Zusammenfassung In den Organen des Flußkrebses wurden drei Enzyme der Fettsäureoxydation festgestellt: Crotonase (E.C.: 4.2.1.17), Hydroxyacyl-CoA-dehydrogenase (HAD. E.C.: 1.1.1.35) und Ketoacyl-thiolase (KAT, E.C.: 2.1.3.9).Eigenschaften der HAD und KAT bei unseren Testbedingungen werden geschildert. Im großen und ganzen ähneln die Enzyme in ihren Eigenschaften den Wirbeltierenzymen. Im Gegensatz zu den Wirbeltierenzymen zeigt die HAD des Flußkrebses auch mit NADPH₂ Aktivität (50–75% der Aktivität mit NADH₂). Mit 10^–3 und 2·10^–3 M o-Phenanthrolin wird nur die Aktivität gegen NADPH₂, nicht die gegen NADH₂, gehemmt.Bei unseren Präparationsbedingungen befinden sich 80–90% der Gesamtaktivität der HAD und KAT im 20000 g-Überstand.Hohe spezifische Aktivitäten der HAD und KAT findet man in Herz, Antennendrüse und Darm, niedrige vor allem im Schwanzmuskel.HAD und KAT sind auch in den Organen des Flußkrebses proportionskonstante Enzyme. Durch Quotienten aus Aktivitäten von Schlüsselenzymen des Fettsäureabbaus, der Glykolyse und des Citratcyclus wurden die Organe hinsichtlich ihres energieliefernden Stoffwechsels charakterisiert.

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Enzymes of fatty acid oxidation in the organs of the crayfish orconectes limosus Rafinesque

Summary Three enzymes of fatty acid oxidation have been detected in the organs of the crayfish: Crotonase (E.C.: 4.2.1.17),Hydroxyacyl-CoA-dehydrogenase (HAD, E.C.: 1.1.1.35) and Ketoacyl-thiolase (KAT, E.C.: 2. 1. 3. 9.).The properties of HAD and KAT have been investigated at the conditions of our tests. In general the enzymes of the crayfish resemble to those of vertebrates. In contrary to the HAD of vertebrates the HAD of the crayfish shows activity not only with NADH₂ but also with NADPH₂ (50–75% of the activity with NADH₂). The activity with NADPH₂ is inhibited by 10^–3 and 2·10^–3 M o-phenanthroline, the activity with NADH₂ is not.In our preparations 80–90% of the total activity was localized in the 20 000 g — supernatant.High specific activities of HAD and KAT are found in the heart, antennal gland and the intestine, low predominantly in the abdominal muscle.As in vertebrates and insects HAD and KAT in the different organs have constant proportions in their activities. The organs of the crayfish are characterized with regard to their energy supply by help of quotients of the activities of keyenzymes of fatty acid oxidation, glykolysis and citrate cycle.

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Fräulein C. Kretschmer, Herrn H. Förstel und meiner Frau danke ich für zuverlässige Mitarbeit.     

Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)     

Optical activity of single-stranded polydeoxyadenylic and polyriboadenylic acids; dependence of adenine chromophore cotton effects on polymer conformation

Circular dichroism (CD) curves are reported for poly dA, (pdA)₆, (pdA)₂, poly A, ApAp, ApA, AMP, dApA, pdApA, A-2′-O-methyl pA, and A-2′-O-methyl pAp. Analysis of these curves indicated the presence of single CD bands at 228–230 mμ and at 278–280 mμ in oligomers longer than dinucleotides. In the case of dinucleotides and mononucleotides (from the literature, in addition to those studied here), the 230 mμ CD of band appears but the 280 mμ CD band does not. We assign the 230 mμ band to a very weak π–π* transition at this wavelength. From theoretical considerations, we show that the 280 mμ band is not an exciton component of the strong π–π* transition at 260 mμ in adenine. We conclude that the 280 mμ CD band must be assigned to a distinct absorption, not previously reported, which we suggest arises from an n–π* transition. The fact that the n–π* CD band at 280 mμ is not seen in mononucleotides or dinucleotides is ascribed to solvation of the adenine ring by water, which shifts the band to shorter wavelengths. Therefore, only interior residues of oligomers have the 280 mμ band, and the optical activity of a polymer cannot be computed from that of a dinucleotide, by using a nearest-neighbor approximation. The existence of this end effect hag been tested, by taking it into account in computing the rotational strengths of the 278 mμ n–π* transition for several oligomers; it is pointed out that a more sensitive test of this end effect would require CD data for the oligo dA series of 3 to 5 residues. We speculate about the structural and optical differences between poly dA and poly A, and point out the need for a theoretical treatment of n–π* Cotton effects in polynucleotides.     

Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

The screening of the enzyme and isoenzyme patterns in seeds ofAllium cepa L.

The screening of enzyme patterns in seeds ofAllium cepa cv. Všetatská revealed the presence of the following enzymes: alcohol dehydrogenase, lactate dehyd ogenase, NAD+- and NADP+-glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase NAD+- and NADP+-malate dehydrogenase, NADH₂- and NADPH₂-tetrazolium reductase catalase, Superoxide dismutase, acid and alkaline phosphatase, L-leucine aminopeptidase, glutamate dehydrogenase, non-specific esterase, and cholinesterase. Altogether 17 enzymes were detected in onion seeds, nine of which had more than three isoenzymes, NAD+-malate dehydrogenase had 8, and non-specific esterase 9 isoenzymes. The demonstration of cholinesterase and Superoxide dismutase activities is remarkable.     

Demonstration of a NADPH-linked Δ1-pyrroline-5-carboxylate-proline shuttle in a cell-free rat liver system

These studies indicate that the interconversions of Δ¹-pyrroline-5-carboxylate and proline can function as a shuttle that generates extra-mitochondrial NADP⁺ and transfers hydride ions into mitochondria in a cell-free rat liver system. A phosphate-free buffer with high concentrations of triethanolamine and 2-mercaptoethanol prevented the cold inactivation of pyrroline-5-carboxylate reductase (ED 1.5.1.2) in liver extracts. This enzyme had an apparent K_m^NADPH that was 2% of the apparent K_m^NADH. V_max^NADPH was approx. 50% of V_max^NADH. Unlabeled proline was converted to [5-³H]proline in incubations containing liver soluble fraction, mitochondria and a [4S-³H]NADPH generating system. This demonstrated one turn of the proposed shuttle in a homologous liver system. [5-³H]Proline production increased linearly over 60 min and decreased by 87% or more when specific components were eliminated. Rotenone was required for maximal activity, suggesting that inhibition of Δ¹-pyrroline-5-carboxylate efflux would be required for significant shuttle activity in vivo. Both the relative concentrations of NADPH and NADH in liver cytosol and the kinetic characteristics of liver pyrroline-5-carboxylate reductase predict that the described shuttle should be overwhelmingly linked to NADPH rather than NADH. A NADPH-linked Δ¹-pyrroline-5-carboxylate-proline shuttle may occur in hepatocytes and function at specific times to regulate pathways limited by cytosolic [NADP⁺].     

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k_cat/K_m, NADPH) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k_cat/K_m, NADPH 1000-fold but had little effect on k_cat/K_m, NADH. The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.     

NADH2- and succinate-dependent O2 uptake in chromatophores from Chromatium vinosum

The O₂ uptake linked to NADH₂ and succinate oxidation was observedin chromatophores from photosynthetically grown Chromatium vinosum. The maximal rate was 60–120 nmoles of O₂ uptake per minper {diaeresis}mole of bacterio-chlorophyll. The rate of O₂uptake linked to NADH₂ oxidation was higher in the neutral-to-acidicpH range than in the alkaline range, whereas that linked tosuccinate oxidation was higher in the alkaline range. The O₂ uptake linked to NADH₂ oxidation was inhibited by rotenone,amytal, antimycin A, KCN and NaN₃, while that linked to succinateoxidation was inhibited by antimycin A, KCN and NaN₃. Malate,citrate, pyruvate, acetate, -ketoglutarate, NADPH₂ and thiosulfatedid not serve as substrates for the O₂ uptake of isolated chromatophores. The rates of the O₂ uptake linked to both NADH₂ and succinateoxidation were not stimulated by adding uncouplers or underphosphorylating conditions. Little or no ATP was synthesizedin the dark, coupled to either NADH₂ or succinate oxidation,in spite of a high activity of photophosphorylation in Chromatiumchromatophores. (Received February 26, 1980; )     

Studies on the metabolism of a sulfur-oxidizing bacterium VI1. Fractionation and reconstitution of the elementary sulfur-oxidizing system of Thiobacillus thiooxidans

The sulfur-oxidizing system of a strain of Thiobacillus thiooxidanswas obtained in cell-free state. The system is resolved intothree fractions and can be reconstituted from these fractions.Both the soluble and particulate fractions are required forthe oxidation of elementary sulfur. The soluble fraction wasfurther separated into two fractions, the collodion membrane-permeable(S-P)and the impermeable(S-IP). S-P contains a low molecular weight,relatively heat stable substance(s) which is indispensable forthe reconstitution of the sulfur-oxidizing system and was identifiedas a pyridine nucleotide. The function of S-P can be replacedby NAD or NADP, but not by cysteine nor GSH. Oxidation of NADH₂ and NADPH₂ is catalyzed by the particulatefraction. Oxidation of the latter is much more rapid than thatof the former. Oxidation of NADPH₂ as well as sulfur oxidationis inhibited by cyanide, pCMB and CO, the CO-inhibition beingphoto-irreversible. However, strong inhibitors of sulfur oxidationsuch as DDC, 8-hydroxyquinoline and salicylaldoxime have noeffect on the oxidation of NADPH₂. The optimum pH values for sulfur and sulfite oxidations by thecell-free extract are shifted to the neutral side in comparisonwith pH values by intact cells. ¹V = References(I). ²Partly supported by a grant from the Ministry of Education. (Received April 3, 1969; )     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

Screening of Microorganisms for Catechol Production from Benzene

Pseudomonas strain BE-81, capable of growing significantly on benzene as a sole carbon source, was isolated from enrichment culture. Strain BE-81 is suggested to metabolize benzene by the oxidation system via benzene glycol as an intermediate, and to be suitable for the continuous production of catechol without addition or regeneration of NADH₂.

The catechol accumulating mutant, strain 136R-3, was developed from strain BE-81 by complete blockage of catechol catabolisms including the meta and ortho pathways, using double mutagenesis induced by NTG, and enrichment by non-selective growth with p-hydroxy-benzoate, suicide treatment with halogenated compounds and antibiotics lysises in the presence of benzene and benzoate.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

The regulation of glutamate dehydrogenase,nitrite reductase,and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO₂ ^- concentrations between these enzymes. NH₄ ⁺ depressed the induction of nitrite reductase. NADH₂ dependent glutamate dehydrogenase activity was enhanced by those NO₂-concentrations in the medium at which unmetabolized NO₂ ^- occurred in the roots. NADPH₂ and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH₂ dependent GDH activity.     

Sul Controllo Della Glicolisi Nel Lievito

Abstract

On the control of carbohydrate utilization in yeast. — The results of a previous investigation showed that in higher plants the stimulating action of 2,4 dinitrophenol (DNP) on oxygen uptake and glycolysis is accompained by a fall of the level of reducing sugars, due to an increase of their respiratory utilization, and thus — according to every evidence — of the rate of hexose phosphate synthesis.

In the present work, the occurrence of a similar phenomenon in yeast (where the inhibiting effect of DNP on glucose uptake is not so much marked as in higher plant tissue) was investigated.

Here again DNP, at a 10^-4M concentration, induced a rapid decrease of the disaccaride trehalose and of glycogen, such as to account for the increased rate of respiration and of fermentation. The ratio between the contributions to CO₂ of Carbons 1 and respectively 6 of glucose was not significantly changed by DNP, which suggests that at least part of the DNP induced increase of glycolysis was mediated by the Embden Meyerhof pathway, and thus that a larger amount of fructose diphosphate was formed in the presence of the uncoupler.

In other experiments the effects of DNP on the dissimilation of C¹⁴ labeled glucose, glycerol and pyruvate to CO₂ and ethanol, and on the incorporation of the radioactive isotope into various fractions, 15 minutes after feeding the labeled substrates, was investigated. It was found that:

1) Glucose and glycerol uptake is not markedly inhibited by DNP at the concentration employed (^10–4M).

2) In the absence of DNP, a considerable portion of the radioactivity fed as glucose or glycerol and taken up by the yeast cells is recovered in the glycogen and trehalose fractions. (35% of the glucose, and 22% of the glycerol taken up). This is also observed for carbons 2 and 3, but not for carbon 1 of pyruvate. This indicates a reversibility of the glycolitic processes comprehended in the region between phospho-enol pyruvate andpolysac-carides; while the pyruvate kinase reaction appears to represent a sharp barrier at the « lower » end of glycolysis.

3) DNP almost completely inhibited the incorporation of C¹⁴ from glucose and glycerol into glycogen and trehalose, although it increased the rate of its dissimilation to CO₂ and ethanol. The total amount of glucose and glycerol transformed in the various metabolites (and thus — according to every evidence — phosphorylated) was somewhat lowered and proteins synthesis severely depressed. These effects are interpreted as due to the uncoupling action of DNP at the mitochondrial level, and to the consequent general decrease of the ATP and UTP levels required for protein and for polysaccharide synthesis.