Biomedical subjects

A Oaks

Publications and source records attributed to A Oaks.

At least 19 recordsLinked to original sources

PubMed1993

Characterization of a maize root proteinase.

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.

Amino Acid Sequence↗

PubMed1991

Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots.

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO(3) (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.

Journal Article↗

PubMed1991

Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus.

Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance.

Journal Article↗

PubMed1990

Stabilization of nitrate reductase in maize roots by chymostatin.

Nitrate reductase (NR) in maize (Zea mays cv W64A x W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO(-) (2) produced per hour per gram fresh weight was measured at 4 days).

Journal Article↗

PubMed1988

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves.

Corn seedlings (Zea mays cv W64A x W182E) were grown hydroponically, in the presence or absence of NO(3) (-), with or without light and with NH(4)Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO(3) (-). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO(3) (-) was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO(3) (-). Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO(3) (-) was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH(4)Cl(-) was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO(3) (-) or by some other unidentified factor, and that higher levels of NO(3) (-) are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO(3) (-) to NO(2) (-).

Journal Article↗

PubMed1987

Influence of light on the ferredoxin-dependent glutamate synthase in maize leaves.

The ferredoxin (Fd)-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) activities are carried out by two immunochemically distinct enzyme proteins in maize leaves (Zea mays W64A and W182E). Continuous irradiation of etiolated tissue at 75 micro einsteins per square meter per second for 24 hours resulted in a 3-fold increase on a fresh weight basis in the activity of the Fd-dependent glutamate synthase and a slight decrease in the activity of the NADH-dependent enzyme. There was also a significant increase of the Fd-glutamate synthase protein during greening of etiolated tissue.

Journal Article↗

PubMed1987

Characterization of Nitrate Reductases from Corn Leaves (Zea mays cv W64AxW182E) and Chlorella vulgaris: Sensitivity to a Proteinase Extracted from Corn Roots.

The sensitivity of the two forms of nitrate reductase, NR(I) and NR(II), obtained from the primary leaf of corn, to a limited action corn root proteinase has been examined. The corn inactivating protein (CIP) inhibited the overall reaction (NADH-NR) and the two partial reactions, cytochrome c reductase and reduced methyl viologen NR (MV-NR) of both forms of NR. NADH-cytochrome c reductase was more sensitive to the protease than MV-NR. NR(II) was less sensitive to inactivation than NR(I). When NR(I) and NR(II) were inactivated and then subjected to native gel electrophoresis the protein bands associated with MV-NR activity shifted from an R(m) value of 0.32 to 0.61 for NR(I) and from an R(m) of 0.28 to 0.60 for NR(II). For Chlorella NR these values are 0.32 and 0.70. The initial cleavage of the 116 kilodalton subunit of NR(I) yielded fragments of 84 and 80 kilodaltons after a 5 minute incubation with CIP. With longer incubation times smaller fragments were also identified. For the Chlorella NR the initial cleavage products are approximately 68 and 25 kilodaltons. Longer incubation times also led to smaller fragments. The products of hydrolysis by this limited action protease are quite different for the corn and Chlorella NRs.

Journal Article↗

PubMed1986

Functional domains of assimilatory NADH:nitrate reductase from Chlorella.

Assimilatory nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and molybdenum per subunit. Besides the reduction of nitrate by NADH, nitrate reductase also catalyzes the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase. Incubation of native nitrate reductase with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in a loss of NADH:nitrate reductase and NADH:cytochrome c reductase activities but no loss of reduced methyl viologen:nitrate reductase activity. Incubation of nitrate reductase with V8 protease or corn inactivator protease resulted in two different products, each of which retained a different partial activity. Reduced methyl viologen:nitrate reductase activity was associated with a homotetrameric fragment of about 260 kDa which contained heme and molybdenum but no FAD. The molecular mass of native nitrate reductase determined under the same conditions was 375 kDa. NADH:ferricyanide reductase activity was associated with a monomeric species of approximately 30 kDa which contained FAD and the NADH-binding site. These results are consistent with a structure-function model of nitrate reductase which has the following features: FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, and the quaternary structure maintained via association sites on the heme/molybdenum domain.

Affinity Labels↗

PubMed1986

Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots.

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [(15)N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [(15)N] H(4) (+) or [(15)N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [(15)N]H(4) (+) was provided, mitochondria isolated from light-grown pea shoots synthesized [(15)N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [(15)N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [(15)N]glycine was used, pea mitochondria synthesized [(15)N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [(15)N]glycine and synthesis of [(15)N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [(15)N]H(4) (+) by the [(14)N]H(4) (+) pool in the mitochondria. The majority of the [(15)N]H(4) (+) released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [(15)N]glycine and little synthesis of [(15)N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [(15)N]glutatmate was synthesized at a rate of 7% of that of [(15)N]serine synthesis by corn mitochondria.

Journal Article↗

PubMed1986

Hydrolysis of storage proteins in barley endosperms : analysis of soluble products.

Soluble products, released by the hydrolysis of hordeins into the media of barley (Hordeum vulgare cv. Perth) half-seeds were analyzed. Large polypeptide fragments (methanol-insoluble) were identified using the Western immunoblot technique with the antibodies prepared against B and C polypeptides of hordein. A number of hordein IgG-reacting bands were noted in the samples from dry kernels. In samples incubated in the absence of gibberellic acid, polypeptide fragments in the size range of 25 to 30 kilodaltons appeared within 24 hours, and those in the size range of 40 kilodaltons became more prominent. In samples incubated in the presence of gibberellic acid, polypeptide fragments in the size range of 45 to 67 kilodaltons were less apparent and those in the size range of less than 15 kilodaltons were more pronounced. The hordein-related polypeptide fragments were present in low amounts after 72 hours in the presence of gibberellic acid. Methanol-soluble peptides were fractionated, on the basis of size, into two broad peaks. In the absence of gibberellic acid, there was no significant change in their profile over a 72 hour incubation period. In the presence of this growth substance, however, there was a decrease in the proportion of large size peptides (50-70 amino acid residues in length), and an increase in the levels of small peptides (15-35 amino acid residues in length) and amino acids. Our interpretation of the results is that the release of the initial large polypeptide fragments from hordein proteins is mediated by a protease(s) whose appearance is not dependent on the exogenously added gibberellic acid. Further hydrolysis is, however, mediated by proteases induced in the presence of this growth substance.

Journal Article↗

PubMed1985

Glutamine metabolism in corn kernels cultured in vitro.

The fate of glutamine, the major source of nitrogen supplied to the developing maize endosperm, has been examined in endosperm tissues of corn caryopsis grown under sterile conditions. In the culture system, [U-(14)C]glutamine was included in the medium or was injected directly into the endosperm. Samples were harvested at intervals up to 168 hours. Protein and starch fractions were then separated and analyzed for their (14)C content. At 168 hours, 31% of the total label incorporated in the endosperm was in zein, 15% in glutelin, and 24% in starch. When individual amino acids and sugars in the endosperm powders were analyzed, the (14)C still remaining in the glutamine accounted for only 12 to 14% of the total radioactivity.

Journal Article↗

PubMed1985

An electron transport system in maize roots for reactions of glutamate synthase and nitrite reductase : physiological and immunochemical properties of the electron carrier and pyridine nucleotide reductase.

A non-heme iron containing protein which bears an antigenic similarity to ferredoxin from spinach leaves (Spinacia oleracea L.) has been identified in extracts prepared from young roots of maize (Zea mays L., hybrid W64A x W182E). The ferredoxin-like root electron carrier could substitute for ferredoxin in a cytochrome c reduction system in which pyridine nucleotide (NADPH) reduces the root electron carrier in a reaction catalyzed by ferredoxin-NADP(+) reductase (EC 1.6.7.1) from spinach leaves. However, the root electron carrier did not mediate the photoreduction of NADP(+) in an illuminated reconstituted chloroplast system.A pyridine nucleotide reductase which shares identical immunological determinants with the ferredoxin-NADP(+) reductase from spinach leaves has also been characterized from maize roots. Root pyridine nucleotide reductase mediated the transfer of electrons from either NADPH or NADH to cytochrome c via ferredoxin or the root electron carrier. Under chemical reducing conditions with sodium dithionite and bicarbonate, the ferredoxin-like root electron carrier served as an electron carrier for the ferredoxin-requiring glutamate synthase (EC 1.4.7.1) and nitrite reductase (EC 1.7.7.1) obtained from maize roots or leaves. In the presence of root pyridine nucleotide reductase and root electron carrier, either NADPH or NADH served as the primary electron donor for glutamate synthesis in extracts from maize roots or leaves. The electron transport system originating with NADH or NADPH, was, however, not able to mediate the reduction of NO(2) (-) to NH(3).

Journal Article↗

PubMed1984

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase.

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 A, compared to a value of 81 A for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1-2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a Mr 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of Mr 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.

Chemical Phenomena↗

PubMed1984

Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme.

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N(2) and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0 degrees C and for several months at -80 degrees C. The enzyme was purified using (NH(4))(2)SO(4) fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO(3). About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO(2) (-) per minute per milligram protein). A sequential elution with NADH followed by KNO(3) (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO(3) (-)-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.

Journal Article↗

PubMed1984

Comparisons of Peptide hydrolase activities in cereals.

Carboxypeptidase activity (hydrolysis of N-carbobenzoxy-l-phenylalanyl-l-alanine) is high in a number of temperate zone cereals, originating in Asia Minor (wheat, barley, oats, wild oats, rye, triticale) compared to other cereals originating in central America or Asia (maize, sorghum, rice). However, endopeptidase activity (hydrolysis of azocasein or hemoglobin) is relatively much higher in the latter group. Comparison of trichloroacetic acid (TCA)-soluble products derived from the hydrolysis of hemoglobin showed that carboxyterminal amino acids (histidine, arginine, and tyrosine), are released when extracts from wheat and barley endosperms are used. With extracts from corn endosperms, much more TCA-soluble ultraviolet- absorbing material is released, but very little is released as free amino acids within the first 2 hours and the expected C-terminal amino acids of hemoglobin are not detected in significant amounts. These results suggest that the method of hydrolysis of the storage proteins may be significantly different in these two classes of cereals.

Journal Article↗

PubMed1984

Stimulation of Mitochondrial Calcium Uptake by Light during Growth of Corn Shoots.

Ca(2+) uptake in mitochondrial fractions, isolated on Percoll discontinuous density gradients, from light- and dark-grown corn (Zea mays L. var W64A x W182E) shoots was characterized by dual wavelength spectroscopy and the Ca(2+)-sensitive dye murexide. In light-grown seedlings, the rate of mitochondrial Ca(2+) uptake was about 40 nanomoles per minute per milligram of mitochondrial protein. A portion of the Ca(2+) uptake required an exogenous supply of ATP (65%) while the remaining 35% was the respiratory substrate-dependent reaction. Ruthenium red (2 micromolar) completely inhibited both ATP- and substrate-dependent reactions. There was no detectable Ca(2+) efflux from the mitochondria with the inhibitor. When the mitochondrial fraction was prepared from the dark-grown shoots, the rate of uptake, in particular the ATP-dependent reaction, was greatly reduced. The dark treatment caused a reduction in mitochondrial Ca content which is largely due to the reduction of Ca associated with the mitochondrial membrane rather than to a reduction of Ca in the soluble matrix.

Journal Article↗

PubMed1984

Characteristics of glutamate dehydrogenase in mitochondria prepared from corn shoots.

The amination of alpha-ketoglutarate (alpha-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca(2+). The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca(2+) in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca(2+) had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca(2+).About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca(2+) had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca(2+). In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar alpha-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca(2+), 10 and 50 millimolar NH(4) (+), and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.

Journal Article↗

PubMed1983

Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C(3) and C(4) Leaves : The Relationship between NH(3) Accumulation and Photorespiratory Activity.

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH(3) in seedling leaves of C(3) (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C(4) (corn [Zea mays W6A x W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH(3) accumulation is higher in C(3) (about 17.8 micromoles per gram fresh weight per hour) than in C(4) (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH(3), the rate of NH(3) accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH(3) was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO(3) (-) during either the growth or experimental period. When O(2) levels in the air were reduced to 2%, MSX resulted in some accumulation of NH(3) (6.0 micromoles per gram fresh weight per hour). At these levels of NH(3), there was no significant inhibition of rates of CO(2) fixation. There was also a minor, but significant, accumulation of NH(3) in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH(3) in both C(3) and C(4) leaves. These results support the hypothesis that GS is important in the assimilation of NH(3) in leaves and that the glycine-serine conversion is a major source of that NH(3).

Journal Article↗