Role of the redox state of nicoptinamide adenine dinucleotides in the in vivo regulation of inositol biosynthesis in Neurospora crassa

A possible _vivo role of pyridine nucleotides and their oxidized/reduced ratio on the regulation of inositol biosynthesis in Neurospora crassa was studied.A direct correlation was obtained when the values of of the water-soluble free inositol pool from intact N. crassa mycelia were plotted agains their NAD⁺/NADH or NAD⁺ + NADP⁺/NADH + NADPH ratios. Higher values in this inositol pool coincided with higher values in the chosen ratios.In long-term experiments (48 h), where the mold was grown without shaking, lower values for the inositol pool, the in vitro activity of D-glucose-6-phosphate cycloaldolase (glucocycloaldolase) and the myo-inositol (inositol) in phospholipids were found than those for cells grown with vigorous shaking.In short-time experiements (20 min), using N. crassa cells depleted of endogenous substrates, the in vivo synthesis of inositol was higher in cells incubated with vigorous shaking than in cells incubated without shaking. Nevertheless, in these experiments the in vitro activity of glucocycloaldolase was not affected by the earation conditions.     

Developmental regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa.

The formation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase [NAD(P)ase; EC 3.2.2.6] in Neurospora crassa was found to be both spatially and temporally programmed. Ascospores were devoid of the enzyme. Vegetative hyphae contained little or no NADase activity. During the differentiation of aerial cell types (aerial hyphae and macroconidia), the specific activity of the enzyme increased by at least three orders of magnitude. Although transiently associated with young aerial hyphae, the enzyme became an integral and stable part of the mature macroconidia. NAD(P)ase could also be “derepressed” under conditions that permitted aerialogenesis in the absence of conidiation. The increase in the specific activity of NAD(P)ase during cell differentiation required concomitant RNA and protein synthesis; in vitro mixing experiments revealed no cell-specific activators or inhibitors of enzyme activity. The temperature-critical period for the in vitro inactivation of a temperature-sensitive enzyme variant was restricted to the period of actual enzyme expression.The data reported in this paper combined with data reported in a previous paper (Nelson et al., 1975b) underscore an important distinction in studies of development, namely, developmental regulation of a macromolecule versus regulation of development by a macromolecule. This paper provides evidence that NAD(P)ase is developmentally regulated. The previous paper provides evidence that the appearance of this enzyme need not regulate development.     

In vivo regulation of intermediate reactions in the pathway of tryptophan biosynthesis in Neurospora crassa

The in vivo regulation of intermediate reactions in the pathway of tryptophan synthesis in Neurospora crassa was examined in a double mutant (tr-2, tr-3) which lacks the functions of the first and last enzymes in the pathway from chorismic acid to tryptophan. The double mutant can convert anthranilic acid to indole and indole-3-glycerol, and the production of these indolyl compounds by germinated conidia was used to estimate the activity of the intermediate enzymes in the pathway. Indole-synthesizing activity was maximal in germinated conidia obtained from cultures in which the levels of l-tryptophan were growth-limiting; the formation of this activity was markedly repressed when the levels of l-tryptophan exceeded those required for maximal growth. d-, 5-methyl-dl-, and 6-methyl-dl-tryptophan were less effective than l-tryptophan, and 4-methyl-dl-tryptophan, tryptamine, and indole-3-acetic acid were ineffective in repressing the formation of indole-synthesizing activity; anthranilic acid stimulated the formation of indole-synthesizing activity. Preformed indole-synthesizing activity was strongly and specifically inhibited by low levels of l-tryptophan; several related compounds were ineffective as inhibitors. These results suggest that, in addition to repression, an end product feedback inhibition mechanism is operative on an intermediate enzyme(s) in tryptophan biosynthesis. The relation of these results to other in vivo and in vitro studies and to general aspects of the regulation of tryptophan biosynthesis in N. crassa are discussed.     

Carbon source regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa: induction and repression of enzyme synthesis

Synthesis and release of NAD(P)ase by Neurospora crassa wild type was studied in experiments in which mycelia grown in Vogel minimal medium were transferred to media containing protein as the only carbon source. Several results are presented suggesting that the NAD(P)ase may be induced by the presence of protein in the culture medium. Low concentrations of sucrose or glucose (0.1%), Casamino acids or some amino acids such as methionine, cysteine, phenylalanine and tryptophan strongly repressed the enzyme synthesis. Under induction conditions NAD(P)ase and alkaline protease appeared together in the culture medium. It would appear that NAD(P)ase and alkaline protease are coordinately regulated by a common control mechanism related to carbon catabolism.     

Developmental-stage-dependent adenine transport in Neurospora crassa.

Although germinated conidia of Neurospora crassa transport adenine through two different systems, only one of these, namely, the general purine transport system, which transports adenine, hypoxanthine, guanine, and 6-methylpurine, is present in freshly harvested conidia of the wild type. The second system develops during germination. The latter system can transport adenine and 6-methylpurine. Time course and kinetic studies of adenine transport in freshly harvested conidia of an ad-8 mutant indicated that, in contrast to the wild type, the general purine transport activity is very low in this strain and that the second adenine transport system is possibly present in the ungerminated conidia. A study of adenine and hypoxanthine uptake in ad-8 and ad-4 mutants, both of which cannot utilize hypoxanthine for growth, isolated that the two transport systems may be under different metabolic controls.     

Purification and subunit structure of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Neurospora crassa.

Neurospora crassa nicotinamide adenine dinucleotide specific isocitrate dehydrogenase (EC 1.1.1.41) has been purified to homogeneity by the criteria of disc gel electrophoresis and sedimentation equilibrium. Purification of the enzyme is facilitated by the presence of phenylmethanesulfonyl fluoride and by the use of a ribose-linked adenosine 5'-monophosphate affinity column. The enzyme appears to be composed of nonidentical subunits of molecular weights 42 800 and 38 300 as estimated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate. From the intensity of each band and the native molecular weight, it is concluded that the enzyme is composed of either six or eight subunits, three or four of each type, respectively. The availability of pure enzyme will allow clarification of the structure of the enzyme by ligand binding studies.     

Reduction of nicotinamide adenine dinucleotides by sodium cyanoborohydride.

The relatively slow reduction of NAD+ and NADP+ by sodium cyanoborohydride leads to formation of the enzymically active form of NADH and NADPH. This reaction could be useful as a simple procedure to enzymically introduce a specific label into substrates when tritiated or deuterated cynanoborohydride is used for obtaining the reduced nicotinamide adenine dinucleotide.     

Raman spectroscopy of oxidized and reduced nicotinamide adenine dinucleotides

We have measured the Raman spectra of oxidized nicotinamide adenine dinucleotide, NAD+, and its reduced form, NADH, as well as a series of fragments and analogues of NAD+ and NADH. In addition, we have studied the effects of pH as well as deuteration of the exchangeable protons on the Raman spectra of these molecules. In comparing the positions and intensities of the peaks in the fragment and analogue spectra with those of NADH and NAD+, we find that it is useful to consider these large molecules as consisting of component parts, namely, adenine, two ribose groups, two phosphate groups, and nicotinamide, for the purpose of assigning their spectral features. The Raman bands of NADH and NAD+ are found generally to arise from molecular motions in one or another of these molecular moieties, although some peaks are not quite so easily identified in this way. This type of assignment is the first step in a detailed understanding of the Raman spectra of NAD+ and NADH. This is needed to understand the binding properties of NADH and NAD+ acting as coenzymes with the NAD-linked dehydrogenases as deduced recently by using Raman spectroscopy.     

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

High-performance ion-exchange separation of oxidized and reduced nicotinamide adenine dinucleotides

High-performance anion-exchange chromatography of oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) on a Pharmacia Mono Q anion-exchange column is reported. Microgram quantities of all four nucleotides can be separated at pH 7.7 in approximately 20 min. For preparative purposes, greater than 7 mg of NADH can be purified in a single injection, and the peak fractions have an A260 of greater than 80 OD units with an A260/A340 ratio of 2.25.     

Electrostatic and redox potential effects on the rat of electron-transfer reaction of nicotinamide adenine dinucleotides with 1-substituted 5-ethylphenazines

The effects of redox potential and electric charge on the rate of electron-transfer reaction by a two-electron process were investigated. For electron donors, beta-NADH, beta-NADPH and alpha-NADH were used; they have similar structures but different charges and different redox potentials. For electron acceptors, the following 5-ethylphenazine derivatives were used: 1-(3-carboxypropyloxy)-5-ethylphenazine, 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine, and 1-[N-(2-aminoethyl)carbamoylpropyloxy]-5-ethylphenazine. They have similar structures and different charges. Using these donors and acceptors, the potential and the charge effects were estimated separately. In the potential effect, a linear free energy relationship was observed for the change in the redox potential of the donor with a Br?nsted slope of about unity. On the other hand, the slope for the change in the potential of the acceptor was about 0.5. These results show that the potential effect due to electron donors is different from that due to electron acceptors. A linear relationship was also observed between activation free energy and electrostatic force (or potential). The redox potential effect and the electrostatic effect are independent and additive. New theory for the mechanism of electron-transfer reactions is needed to explain these results.