Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Effects of norflurazon (San 9789) on light-increased extractable nitrate reductase activity in soybean [Glycine max (L.) Merr.]seedlings

Abstract. The effects of norflurazon (San 9789) on light-increased extractable NADH nitrate reductase activity (NRA) in soybean seedlings were studied. Continuous white light (W) increased NRA steadily in root and cotyledonary tissues over a 5 d period. Morflurazon, a pyridazinone herbicide which causes chlorophyll bleaching in W, reduced the initial NRA induction rate in roots and cotyledons. However, in cotyledons of norfiurazon-treated plants NRA increased at a more rapid rate than in the control after 24 h of W, with activity levels reaching three times those of control seedlings after 5 d. NRA induced by W in control and norflurazon-treated cotyledons was fluence-rate dependent. Continuous FR induced equal amounts of NRA in control and norflurazontreated tissues, suggesting that the superinduceable NRA of norflurazon-treated plants under W is not phytochrome induced. The FR-induced NRA of control and norflurazon-treated cotyledons had pH optima of 6.6, but during development under W the pH optimum of control cotyledons changed from 6.3 to between 6.6 and 7.1. The pH optimum of the norflurazon-induced NRA of the cotyledon under W was about 7.5. The NADH/NADPH NRA ratio after 4 d of W was 1.3 in control and 2.5 in norflurazontreated cotyledons. These data indicate that photosynthelic pigments are involved only secondarily in light-induction of NRA in this system.     

Immunological approach to structural comparisons of assimilatory nitrate reductases

Homogeneous squash cotyledon reduced nicotinamide-adenine dinucleotide (NADH):nitrate reductase (NR) was isolated using blue-Sepharose and polyacrylamide gel electrophoresis. Gel slices containing NR were pulverized and injected into a previously unimmunized rabbit. This process was repeated weekly and antiserum to NR was obtained after four weeks. Analysis of the antiserum by Ouchterlony double diffusion using a blue-Sepharose preparation of NR resulted in a single precipitin band while immunoelectrophoresis revealed two minor contaminants. The antiserum was found to inhibit the NR reaction and the partial reactions to different degrees. When the NADH:NR and the reduced methyl viologen:NR activities were inhibited 90% by specifically diluted antiserum, the reduction of cytochrome c was inhibited 50%, and the reduction of ferricyanide was inhibited only 30%. Antiserum was also used to compare the cross reactivities of NR from squash cotyledons, spinach, corn, and soybean leaves, Chlorella vulgaris, and Neurospora crassa. These tests revealed a high degree of similarity between NADH:NR from the squash and spinach, while NADH:NR from corn and soybean and the NAD(P)H:NR from soybean were less closely related to the squash NADH:NR. The green algal (C. vulgaris) NADH:NR and the fungal (N. crassa) NADPH:NR were very low in cross reactivity and are apparently quite different from squash NADH:NR in antigenicity. Antiserum to N. crassa NADPH:NR failed to give a positive Ouchterlony result with higher plant or C. vulgaris NADH:NR, but this antiserum did inhibit the activity of squash NR. Thus, it can be concluded from these immunological comparisons that all seven forms of assimilatory NR studied here have antigenic determinants in common and are probably derived from a common ancestor. Although these assimilatory NR have similar catalytic characteristics, they appear to have diverged to a great degree in their structural features.     

Histochemical localization of nitrate reductase

Summary NADH-dependent nitrate reductase (E.C. 1.6.6.1) was ultrastructurally localized in norflurazon-treated and control soybean cotyledons [Glycine max (L.) Merr.] by a method based upon the increase in osmiophilia due to the formation of an azo dye. The reaction product was observed in small vesicles throughout the cytoplasm. An apparent transport of nitrite to the plastid, the site of nitrite reduction, may occur through fusion of the nitrite-containing vesicles with the chloroplast envelope. Plants grown in tungstate lacked nitrate reductase activity as measured by standard assay procedures, and showed no increase in osmiophilia, suggesting a degree of specificity of this cytochemical procedure.     

Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr₁ ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr₁ soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH₂-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH₂-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH₂ as a reductant source. Nitrite reductase activity was normal in the nr₁ plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH₂ as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO₃⁻ and HCO₃⁻, compared with inducible NR from NO₃⁻-grown nr₁ plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 degrees C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 degrees C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 degrees C, respectively, with only 33 and 23% of maximal activities being retained at 10 degrees C. In presence of 2 microM flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 degrees C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 degrees C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 degrees C (Q10) and apparent activation energy (E(a)) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 degrees C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.     

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50 581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide.A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.     

Protein synthesis in aging soybean cotyledons. Loss in translational capacity

Ribosomes and supernatant fractions from soybean cotyledons of different ages were prepared to study the Poly U (polyuridylic acid)-directed phenylalanine incorporation. Ribosomes from younger cotyledons were more effective in phenylalanine incorporation compared to ribosomes from older cotyledons. Similarly, the supernatant fractions from younger cotyledons were more efficient, resulting in enhanced incorporation, than the older cotyledons. Substitution of wheat embryo supernatant fraction for soybean cotyledon supernatant fraction resulted in a several fold increase in amino acid incorporation activity, in ribosomes from all ages of soybean cotyledons. Such increase in activity was particularly significant in the older cotyledons. From these experiments it is concluded that in aging soybean cotyledons there is a loss in translational capacity.     

Species and Organ Diversity in the Effects of Hydrogen Peroxide on Superoxide Dismutase Activity In Vitro

Superoxlde dlsmutase （SOD） is ubiquitous in aerobic organisms and constitutes the first link In the enzyme scavenging system of reactive oxygen species. In the present study, species and organ diversity of SOD activity In a solution and In an in-gel assay system, as well as the effects of hydrogen peroxide （H202） on SOD activity, were Investigated. In a solution assay system, SOD activity of jackfruIt root, shoot, leaves, axes, and cotyledons, of maize embryos and endosperms, of mung bean leaves and seeds, of sacred lotus axes and cotyledons, and of rice and wheat leaves was Increased by 1-15 mmol/L H2O2. However, SOD activity In rice root and seeds, maize roots and leaves, mung bean roots and shoots, and wheat seeds was decreased by 1-15 mmol/L H2O2. The SOD activity of wheat root and soybean roots, leaves, axes, and cotyledons was Increased by 1-4 mmol/L H2O2, but was decreased by concentrations of H2O2 〉4 mmol/L. The SOD activity of soybean shoots was not affected by 1-15 mmol/L H2O2. The SOD activity In crude mltochondrla of jackfruIt, maize, and upas seeds, as well as In purified mitochondria of jackfruIt, was also Increased by 1-15 mmol/L H2O2. In the In-gel assay system, the SOD In jackfruIt cotyledons was comprised of Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the crude mltochondria of jackfruit seeds and maizes embryo was comprised of Mn-SOD and Cu/ Zn-SOD, and the crude mltochondria of maize seeds was comprised of Mn-SOD only. In the present study, H2O2 markedly Inhibited Cu/Zn-SOD and Fe-SOD activity.     

Comparative effectiveness of cattle manure, poultry manure, phosphocompost and fertilizer-NPK on three cropping systems in vertisols of semi-arid tropics. II. Dry matter yield, nodulation, chlorophyll content and enzyme activity

A field experiment was conducted on a deep Vertisol of Bhopal, India to compare root and shoot biomass, chlorophyll content, enzyme activity and nodulation in three cropping systems at three combinations of organic manure and inorganic-fertilizer: 75%NPK + 5 t farmyard manure (FYM), 75%NPK + 1.5 t poultry manure (PM), and 75%NPK + 5 t phosphocompost (PC) vis-a-vis 0%, 75% and 100% of fertilizer-NPK. In general, nodule number and its mass were lower in intercrop soybean than sole soybean. Also there was decrease in the nodule number with higher NPK dose. The FYM treated plots recorded 22.0% and 7.6% higher nodule mass than poultry manure and phosphocompost plots, respectively. Also, the total chlorophyll content was higher in organically treated plots than that in 100% NPK particularly at 30 days after sowing (DAS, pre-flowering). In sorghum the peak nitrate reductase (NR) activity was recorded at 60 DAS while in soybean it was at 30 DAS. The NR activity was higher in intercrop sorghum than that in sole sorghum. Maximum NR activity was observed in 100% NPK. Soybean/sorghum intercropping system recorded significantly higher root and shoot biomass than sole soybean and sorghum. The crop growth rates were relatively rapid during 30-60 DAS and followed the order; intercropping > sole sorghum > sole soybean. With the increase in NPK dose from 0% to 100% there was significant improvement in the dry matter (DM) production in sole sorghum and soybean/sorghum intercropping system. Soybean as preceding crop recorded the highest DM, chlorophyll content, NR activity in wheat while these values were the lowest in sorghum-wheat system.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.     

Examination of Le and lele Genotypes of Glycine max (L.) Merr. for Membrane-Bound and Buffer-Soluble Soybean Lectin

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.     

Factors affecting nitrate reductase activity in some monocot and dicot species

Activity of nitrate reductase (NR), the first enzyme in the nitrate-assimilation pathway, was estimated in the cotyledons of the sunflower( Helianthus annuus) using a standardized in-vivo method. Seedlings were grown in the light on a nitrate medium. Various factors that affect NR activity were optimized, including the molarity and pH of the reaction buffer, nitrate concentration, and use of a surfactant. We also determined whether NADH was required for nitrate reduction. The surfactant propanol (2%) gave the best results, and no NADH supplement was necessary: In a separate study, we compared the effect of various culturing components on in-vivo NR activity among monocot and dicot species, and found that Triton X-100 was the best surfactant for monocots whereas dicots performed better with n-propanol. Monocot species also required additional NADH as an external energy source. Moreover, specific purification procedures were needed to enhance NR activity in dicotyledons. Finally, we also assessed the efficacy of in-vivo versus in-vitro procedures for assaying monocots versus dicots.     

酸性磷酸酶参与大豆子叶磷转运和利用

本文以磷效率不同的两个大豆品种为材料,研究大豆幼苗期子叶酸性磷酸酶活性和同工酶谱对外源磷有效性的响应,及其参与子叶磷高效转运和利用的过程。结果表明：在幼苗生长前期,子叶酸性磷酸酶活性及其同工酶谱组成变化明显,而且不受外源磷有效性的调控;在幼苗生长的前8天,子叶全磷含量随着酸性磷酸酶的活性增加而显著降低,而且磷高效大豆品种比磷低效大豆品种具有较高的酸性磷酸酶活性和植株全磷含量。以上结果说明在大豆幼苗生长前期,由于大粒种子不仅具有较高的磷含量,而且具有较高子叶酸性磷酸酶活性,促进子叶有机磷的水解和转运是磷高效大豆品种适应低磷胁迫的生理机制之一。     

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.     

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.     

Identification and quantification of highly mutagenic nitroacetoxypyrenes and nitrohydroxypyrenes in diesel-exhaust particles

Heavy-duty diesel-exhaust particles were collected, extracted and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor-induced rat liver (S9 mix). The neutral and acidic fractions showed high mutagenicity with TA98 in the absence of S9 mix, the acidic fraction having the highest specific activity. In the absence of S9 mix, the mutagenicity of crude, neutral and acidic fractions was greater in TA98 than in TA98NR and TA98/1,8-DNP6. Chemically-synthesized nitroacetoxypyrenes and nitrohydroxypyrenes were fractionated into the neutral and acidic fractions, respectively. These nitroarenes were purified by high-performance liquid chromatography and their mutagenicity was measured with the 4 strains. With TA98 in the absence of S9 mix, 1-nitro-3-acetoxypyrene, 1-nitro-6/8-acetoxypyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6/8-hydroxypyrene induced 16 700, 336, 992, 94 His+ revertants per plate per nmole, respectively. In the absence of S9 mix, the level of mutagenicity of these nitroarenes was highest in TA98, lowest in TA98/1,8-DNP6 and intermediate in TA98NR. The neutral and acidic fractions of diesel-exhaust particles were analyzed by gas chromatography-mass spectrometry and gas chromatography-mass fragmentography. The neutral fraction was found to contain nitroacetoxypyrenes, 1-nitropyrene, 1,6-dinitropyrene, while nitrohydroxypyrenes were detected in the acidic fraction. The amounts of 1-nitro-3-acetoxypyrene, 1-nitropyrene, 1,6-dinitropyrene and 1-nitro-3-hydroxypyrene were 6.3, 62, 0.81, and 70 ng per mg of crude extract, and accounted for 12, 3.6, 8.0, and 9.0%, respectively, of mutagenicity of the crude extract in TA98 in the absence of S9 mix.