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The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Publisher Summary The chapter analyzes the results available in the process of nitrate assimilation as it occurs in two filamentous ascomycetes— Aspergillus nidulans and Neurospora crassa (N. crassa). Not only are these results in general representative of observations made on other fungi and yeasts, but they provide for the most concise and comprehensive description of nitrate assimilation and its regulation. It is a vagary of scientific fortune that the genetics of nitrate assimilation is best understood for A. nidulans, whereas the enzymology of nitrate assimilation is better characterized in N. crassa , the organism more often subjected to biochemical investigation. The assimilatory reduction of nitrate to ammonia is achieved by a metabolic pathway composed of just two enzymes—namely, nitrate reductase and nitrite reductase, which act in this sequence. Recently both have been purified to homogeneity and detailed characterizations of each are being made. Regulation of nitrate assimilation in fungi is eminently simple and straightforward. Ammonia, the end product of the nitrate assimilatory pathway, strongly represses its expression. This phenomenon is the dominant aspect in regulation of nitrate assimilation.

Rat liver xanthine oxidase activity is regulated in response to dietary protein and iron. To investigate whether the change in activity was mediated by a change in the rate of protein degradation, we measured the fractional turnover rate using the double-isotope technique with (/sup 3/H)- and (/sup 14/C)leucine and calculated the apparent half-life of xanthine oxidase in rats fed diets containing either 20 or 5% casein with either 35 or 5 mg iron/kg diet. Under control conditions, xanthine oxidase had an apparent half-life of 4.8 d and approximately 65% of the enzyme subunits were active. Rats fed diets with low dietary protein had lower xanthine oxidase activity, but the enzyme had a slower fractional turnover rate, resulting in an apparent half-life of 6.4 d, and only 15-20% of the enzyme was active. The apparent half-life of xanthine oxidase increased to 7.5 d in rats fed diets with low dietary iron, but dietary iron did not affect the specific activity of the enzyme or the percentage of active subunits. These results suggest that the loss of enzyme activity is not due to loss of enzyme protein by increased degradation, but rather to inactivation of the enzyme.

THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000.

Highly selective and sensitive methods for determining nitrate reductase levels independent of any expression of its enzymatic activity have been developed, based on immunoelectrophoretic procedures. Specific antisera raised against Neurospora crassa NADPH-nitrate reductase inhibited the overall NADPH-nitrate reductase activity of this enzyme as well as partial activities of the electron transfer process associated with the nitrate reductase reaction. Three immunological techniques were employed to detect the presence of nitrate reductase-related protein in various Neurospora extracts: (i) a protection against inhibition assay, (ii) rocket immunoelectrophoresis, and (iii) crossed immunoelectrophoresis. These three methods were then applied to an analysis of nitrate reductase gene expression in the nit mutants of N. crassa .

Mo is an essential trace element for virtually all organisms since it is required for the activities of nitrate reductase, formate dehydrogenase, nitrogenase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and a variety of other enzymes. All molybdoenzymes, with the exception of nitro­genase, have molybdenum incorporated into a pterin-containing Mo-cofactor (1) and the structure of this cofactor has been conserved throughout evolution from bacterial, plant and animal sources. Therefore, an understanding of Molybdenum-cofactor metabolism in one organism will have far-reaching impli­cations for the general understanding of molybdenum metabolism.

Children born at very low birth weights (VLBW) (less than or equal to 1500 g) who were beneficiaries of modern neonatal intensive care are reaching middle childhood, and their school achievement can be evaluated. We compared 65 9-year-old children born in 1976, who were very low birth weight and who were free of neurological impairment, with 65 children of normal birth weight who had been matched for race, sex, age, and social class on measures of IQ, cognitive, visuo-motor, and fine motor abilities, and academic achievement. VLBW children scored significantly lower than controls on the WISC-R, Bender-Gestalt, Purdue Pegboard, subtests from the Woodcock Johnson Cognitive Abilities Battery, and reading and mathematics (math) achievement. Exploratory analysis of a subset of 43 VLBW and matched controls with IQ scores greater than or equal to 85 yielded a similar trend, except that, on achievement tests, differences were significant only in math. Further analyses revealed that the differential in math achievement between VLBW and control children is not fully attributable to differences in IQ.

The purpose of this study was to determine whether iron-deficient anemic infants show affective and attentional disturbances during play. The behavior of 21 iron-deficient anemic and 21 nonanemic 6- to 24-month-old Guatemalan infants and their mothers was analyzed during a videotaped 8-minute free-play session. There were no statistically significant differences between the two groups in measures of infant irritability, distractibility, or apathy. There were differences, however, in measures of spatial relations. In 71% of the anemic infants, the duration of child-initiated body contact with their mothers was high, compared with a high level of contact in only 26% of the nonanemic babies (p = 0.01). Mothers of anemic infants spent less time at a distance from them, were less likely to break close contact, and were more likely to reestablish close contact if the baby moved away (p less than 0.03). The increase in body contact was interpreted as a reflection of fearfulness, hesitance, or inactivity. The results suggest that the specific behavioral manifestations of iron deficiency anemia in infancy may vary with the context, differing in free play and structured developmental testing.