Amino acid sequence of COOH-terminal 20K Da fragment from pig liver microsomal NADPH-cytochrome P-450 reductase

We have determined the complete amino acid sequence of a 20K Da COOH-terminal fragment of porcine NADPH-cytochrome P-450 reductase. The 20K Da fragment is probably produced by a proteolytic cleavage of the intact protein in porcine liver microsomes, and since the cleavage does not affect enzymatic activity, the fragment has been studied as a distinct domain. The sequence comprises 175 amino acids including three cysteine residues, one of which has been previously identified as protected by NADPH from S-carboxymethylation. The NADPH-protected cysteine lies in a stretch of 12 residues with partial homology to glutathione reductase, and is adjacent to a hydrophobic region containing a glycine-rich stretch homologous to other FAD-containing proteins. The predicted secondary structure over this entire region is beta-sheet/beta-turn/beta-sheet/alpha-helix/beta-sheet/beta-turn/alpha-h elix corresponding to hydrophobic residues 21-28/glycine-rich residues 29-33/residues 34-38/residues 39-54/residues 56-61/NADPH-protected cysteine residues 62-78/residues 71-82. It is possible that the 20K Da domain provided a significant portion of the sequence responsible for binding FAD and NADPH in the intact enzyme. This data provides a basis for further active site studies.     

The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley

The effect of cadmium on the biosynthesis of chlorophyll has been investigated in the leaves of dark-grown seedlings of barley ( Hordeum vulture L. cv. Proctor). Cd2+ inhibited the production of chlorophyll by affecting 1) the synthesis of 5-aminolacvulinic acid and 2) the protoehlorophyllide reductase ternary complex with its substrates. Cd2+ had no effect on the constituent enzymes that catalyse the synthesis of free protoehlorophyllide from 5-aminolaevulinic acid. The results obtained are consistent with Cd2+ inhibiting the formation of chlorophyll by reacting with essential thiol groups in both the protochlorophyllide reductase protein and the enzyme(s) involved in the light dependent synthesis of 5-aminolaevulinic acid.     

Distribution and recovery of15N after fertilization of Douglas-fir saplings with different nitrogen sources

Summary Four-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) saplings planted in pots with a sand and peat mix (11) were fertilized at the rate of 200 kg N/ha of (¹⁵NH₂)₂CO (U-15),¹⁵NH₄NO₃ (A-15) and NH₄ ¹⁵NO₃(An-15). They were placed in a shadehouse and watered regularly to maintain soil moisture at field capacity over periods of one and two years. Quantity of¹⁵N in foliage generally increased from old to current growth, irrespective of the nitrogen source. Utilization of¹⁵N fertilizers by saplings after the first and second growing seasons following fertilization was greatest with nitrate labelled ammonium nitrate AN-15, and nearly equal for urea U-15 and ammonium labelled ammonium nitrate A-15. The soil immobilized more fertilizer nitrogen-15 from U-15 and A-15 than from AN-15. Data from the present study, in which leaching losses of fertilizer were minimized, demonstrated that in terms of nitrogen uptake by the saplings the nitrate fertilizer was superior to ammonium fertilizer.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrialbc 1 complex

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrialbc ₁ complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in thebc ₁ complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochromec reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of thebc ₁ complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochromec reductase activity. However, a clear stimulation of the rate of cytochromeb reduction in parallel to an inhibition of cytochromeb oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on thebc ₁ complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.     

The presence and photoregulation of protochlorophyllide reductase in green tissues

Summary The enzyme protochlorophyllide (pchlide) reductase has been identified amongst the peptides, resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), of chloroplast membranes from oat and barley plants. In support of this identification the enzymic activity associated with the enzyme has also been measured in the same preparations. A higher level of enzyme was found in plants which had been darkened prior to extraction. Based on this data, mechanisms for the light regulated diurnal variation of the reductase are discussed.     

Seed germination in Chenopodium album L.: further evidence for the dependence of the effects of growth regulators on nitrate availability

Abstract Chenopodium album L. plants, grown under controlled environmental conditions on different levels of soil nitrate, produced seeds with proportionately different NO^?₃ contents. Regardless of the endogenous NO^?₃ content, few seeds germinated in water or upon treatment with KNO₃. Ethylene promoted germination, and the extent of germination was positively correlated with the endogenous seed NO^?₃ content. Combined application of ethylene and KNO₃ in the dark had a synergistic effect on NO^?₃ -deficient seed. The synergism between ethylene and KNO₃ was attributable to the NO^?₃ moiety of the nitrate salt. Ethylene and light showed moderate synergism in seeds with low or high endogenous nitrate. Addition of nitrate, however, masked the interaction between ethylene and light. Gibberellic acid₄₊₇ (GA₄₊₇) or red light, each alone or combined with KNO₃, had little effect on germination. When applied together in the dark, ethylene and GA₄₊₇ synergistically enhanced the germination of NO^?₃-deficient seed. The combined effects of the two hormones on this seed were further enhanced by the addition of KNO₃. There was no synergism between ethylene and GA₄₊₇ in NO^?₃-rich seed. These interactions among GA₄₊₇, ethylene and KNO₃ were not affected by light. The results confirm and further elaborate our earlier finding that the sensitivity of C. album seeds to ethylene may depend on nitrate availability.     

The nitrogen balance of Raphanus sativus X raphanistrum plants. I. Daily nitrogen use under high nitrate supply

Abstract Growth-chamber cultivated Raphanus plants accumulate nitrate during their vegetative growth. After 25 days of growth at a constant supply to the roots of 1 mol m^?3 (NO^?₃) in a balanced nutrient solution, the oldest leaves (eight-leaf stage) accumulated 2.5% NO^?₃-nitrogen (NO₃-N) in their lamina, and almost 5% NO₃-N in their petioles on a dry weight basis. This is equivalent to approximately 190 and 400 mol^?3 m^?3 concentration of NO^?₃ in the lamina and the petiole, respectively, as calculated on a total tissue water content basis. Measurements were made of root NO^?₃ uptake, NO^?₃ fluxes in the xylem, nitrate uptake by the mesophyll cells, and nitrate reduction as measured by an in vivo test. NO^?₃ uptake by roots and mesophyll cells was greater in the light than in the dark. The NO^?₃ concentration in the xylem fluid was constant with leaf age, but showed a distinct daily variation as a result of the independent fluxes of root uptake, transpiration and mesophyll uptake. NO^?₃ was reduced in the leaf at a higher rate in the light than in the dark. The reduction was inhibited at the high concentrations calculated to exist in the mesophyll vacuoles, but reduction continued at a low rate, even when there was no supply from the incubation medium. Sixty-four per cent of the NO^?₃ influx was turned into organic nitrogen, with the remaining NO^?₃ accumulating in both the light and the dark.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.