A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Expression of spinach nitrite reductase in Escherichia coli: site-directed mutagenesis of predicted active site amino acids.

Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein. E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite of E. coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic and biochemical characterization of corn inbred lines tolerant to the sulfonylurea herbicide primisulfuron.

Inbred lines of corn (Zea mays L.) have been characterized, which exhibit differential sensitivity to the sulfonylurea herbicide primisulfuron (2-[3-(4,6-bis(di-fluoromethoxy) pyrimidin-2-yl)-ureidosulfonyl]-benzoic acid methylester). When treated postemergence with 160 g a.i. per hectare, inbred 4CO exhibited complete tolerance while inbred 4N5 was killed. The F1 hybrid 4C0 x 4N5 was uniformly tolerant indicating dominance of the tolerance trait. The field observations correlated with laboratory tests in which seedling root growth was measured. Based on IC50, inbred 4CO was more than ten times more tolerant than inbred 4N5. In the F2 and F3 generations, a 3∶1 segregation of tolerant and sensitive individuals was observed, consistent with tolerance being inherited as a single dominant trait. Backcrosses of heterozygous F1 plants with the sensitive parent (4N5) yielded progeny that segreated at the expected 1∶1 ratio. Backcrosses with 4C0 yielded tolerant offspring only. Inhibition characteristics of acetohydroxyacid synthase (AHAS; E.C. 4.1.3.18) were determined. The enzymes from both inbreds and their F1 hybrid were equally sensitive and strongly inhibited by primisulfuron (IC50: 7 nM). The fate of (14)C-labeled primisulfuron in seedling tissues of inbred 4C0 and the hybrid, 4C0 x 4N5, indicated rapid metabolism with a half-life (t 1/2) of approximately 3 h. On the other hand, the herbicide-sensitive inbred 4N5 was considerably slower to metabolize primisulfuron (t 1/2 >24 h). These data indicate that differential metabolism is the mechanism of tolerance to the sulfonylurea herbicide primisulfuron in tolerant corn.

Transient Accumulation of Nitrite Reductase mRNA in Maize following the Addition of Nitrate.

Expression of the gene coding for nitrite reductase (NiR) is induced upon the addition of nitrate. We have analyzed this induction process in hydroponically grown maize (Zea mays L.) seedlings where the level of nitrate in the medium can be easily manipulated. There is a rapid induction of NiR mRNA upon addition of nitrate, increasing first in the roots and then in the leaves. The rapidity of the response depends on the nitrate concentration and the growth medium. However, the general pattern of expression is the same: the mRNA level increases, reaches a maximum, and then decreases, despite the fact that the nitrate concentration in the medium remains constant. This decline in mRNA level can be quite rapid, particularly in root tissue. If the nitrate is given as a pulse, the mRNA levels decrease even more rapidly. It is clear that the NiR mRNA is short-lived, with a half-life in the roots of less than 30 minutes. The NiR protein level, on the other hand, increases gradually somewhat after the increase in mRNA and remains at high levels at least for 24 hours after the addition of nitrate.

Nitrate effects on nitrate reductase activity and nitrite reductase mRNA levels in maize suspension cultures.

Nitrate reductase (NR) activity and nitrite reductase (NiR) mRNA levels were monitored in Black Mexican Sweet maize (Zea mays L.) suspension cultures after the addition of nitrate. Maximal induction occurred with 20 millimolar nitrate and within 2 hours. Both NR and NiR mRNA were transiently induced with levels decreasing after the 2 hours despite the continued presence of nitrate in the medium. Neither ammonia nor chlorate prevented the induction of NR. Furthermore, removal of nitrate, followed by its readdition 22 to 48 hours later, did not result in reinduction of activity or message. NR was synthesized de novo, since cycloheximide completely blocked its induction. Cycloheximide had no effect on the induction of NiR mRNA or on the transient nature of its induction. These results are similar to those reported previously for maize seedlings.

Radiolabeling of a wound-inducible pyridoxal phosphate-utilizing enzyme: evidence for its identification as ACC synthase.

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase, a pyridoxal phosphate-utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate-limiting step in the biosynthesis of the plant hormone ethylene. We report the partial purification (400-fold) of ACC synthase from wounded pink tomato pericarp. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase-enriched fraction was achieved by reduction using tritiated sodium borohydride. Evidence that this radiolabeled protein is ACC synthase is presented.

Interactions between spinach ferredoxin-nitrite reductase and its substrates. Evidence for the specificity of ferredoxin.

Reduced ferredoxin can serve as electron donor in the 6-electron reduction of nitrite to ammonia catalyzed by spinach nitrite reductase. We have examined interactions between nitrite reductase and its substrates, ferredoxin and nitrite, with emphasis upon protein-protein interactions between ferredoxin and nitrite reductase. Ferredoxin, of the proteins tested, is the most effective in retarding low ionic strength inactivation of nitrite reductase. The interaction appears to be electrostatic, and the apparent Kd, calculated from the concentration dependence of ferredoxin protection, is about 1 microM in 2 mM Tris. Chemical modification of carboxyl residues of ferredoxin resulting in a change of charge reduces its reactivity with both ferredoxin:NADP+ oxidoreductase and nitrite reductase, indicating the importance of charge-charge interactions. Cross-linking studies provided no evidence for a ternary complex containing the oxidoreductase and nitrite reductase but indicated that the two enzymes will compete for ferredoxin, possibly using the same site (or overlapping sites) on the ferredoxin. A complex containing ferredoxin:NADP+ oxidoreductase, ferredoxin, and cytochrome c was detected, indicating that ferredoxin has different binding sites for cytochrome c and ferredoxin:NADP+ oxidoreductase. Active cross-linked complexes of ferredoxin and nitrite reductase were obtained and were less sensitive to low ionic strength inactivation than free reductase and had decreased ferredoxin-supported nitrite reductase activity. The evidence presented of protein-protein interactions between ferredoxin and nitrite reductase indicates that ferredoxin is indeed the specific physiological electron donor to the reductase.

Effects of D-erythrose on nitrogenase activity in whole filaments of Anabaena sp. strain 7120.

D-Erythrose, which has been shown to enhance nitrogenase activity (acetylene reduction) by isolated heterocysts, was studied for its effects on nitrogenase activity and nitrite uptake by whole filaments of Anabaena sp. strain 7120. D-Erythrose had little effect on acetylene reduction in the light; however, at a concentration of 10 mM, it could restore 3'-(3,4-dichlorophenyl)-1',1'-dimethyl urea-inhibited or dark-limited levels to light-supported levels. Sucrose, glucose, or fructose did not exhibit similar effects. D-Erythrose had little effect on nitrite uptake, an indirect measure of nitrite reductase activity by nitrate-grown whole filaments. It was concluded that erythrose effects were mediated by heterocysts and were therefore specific for nitrogenase.

D-erythrose supports nitrogenase activity in isolated Anabaena sp. strain 7120 heterocysts.

Among organic compounds tested for their ability to support nitrogenase activity in isolated heterocysts of Anabaena sp. strain 7120 under argon, D-erythrose (5 mM) was unique in supporting acetylene reduction at 10 times the control rates. Higher concentrations of D-erythrose exhibited substrate inhibition. At 50 kPa of H2, all concentrations of D-erythrose inhibited H2-supported acetylene reduction. The effects of D-erythrose on nitrogenase activity were explored. Erythrose enhanced 15N2 incorporation by heterocysts, but NADP+ did not enhance erythrose-supported acetylene reduction. H2 protected nitrogenase from O2 inactivation, but erythrose did not; erythrose did not counter protection by H2. Tests with inhibitors of electron transport showed that erythrose-supported acetylene reduction requires electron flow through ferredoxin, a b-type cytochrome, and a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-sensitive transfer agent whose electron flow is not mediated through the plastoquinone and Rieske iron protein.

Permeabilization of isolated heterocysts of Anabaena sp. strain 7120 with detergent.

Heterocysts isolated from Anabaena sp. strain 7120 with lysozyme plus sonication were permeabilized with the cationic detergent cetyltrimethylammonium bromide, and they then exhibited comparable acetylene reduction activity in the light and dark with an ATP-regenerating system plus dithionite. The detergent diminished the effect of H2 in enhancing acetylene reduction.

Adenine nucleotide levels in and nitrogen fixation by the cyanobacterium Anabaena sp. strain 7120.

Adenine nucleotide levels were determined in whole filaments of Anabaena sp. 7120 grown under different N2-fixing or non-N2-fixing conditions. These were compared with levels in isolated heterocysts, Rhodospirillum rubrum, and Azotobacter vinelandii. Adenine nucleotides in whole filaments of Anabaena sp. do not reflect the energetic expense of N2 fixation as they do in R. rubrum and A. vinelandii. However, adenine nucleotide levels in heterocysts were similar to the levels found in N2-fixing R. rubrum, i.e., an ATP:ADP ratio near 1 and an energy charge between 0.5 and 0.7. Nitrogenase activity was only 50% of optimal in permeabilized heterocysts at an exogenous ATP:ADP ratio of 3.33. Hydrogen, which increases acetylene reduction activity, also causes a transient increase (2 to 5 min) in the ATP:ADP ratio. Hydrogen has little effect on energy charge.