Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species.

Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydration-responsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show that B. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulated Bn115 gene, an ortholog of the Arabidopsis CBF-targeted COR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivum L. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate.

Detection of metabolites of the Entner-Doudoroff pathway by HPLC with pulsed amperometry: application to assays for pathway enzymes.

Three major metabolites in the Entner-Doudoroff pathway, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, and pyruvate can be detected and quantified by HPLC with pulsed amperometric detection. Resolution is achieved by ion-exchange chromatography at alkaline pH with isocratic elution in 5 to 10 min. Detection limits are in the subnanomolar range, and detector response is linear over 3-4 orders of magnitude. This method can be employed for the assay of the enzymes of the pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14), eliminating the need for coupling enzymes as in the previously employed spectrophotometric assays. The lag in pyruvate production seen in the coupled enzyme spectrophotometric assay for 6-phosphogluconate dehydratase is absent in the HPLC/pulsed amperometric detection assay. This lag represents an artifact of a slow tautomerism of 2-keto-3-deoxy-6-phosphogluconate which must precede its utilization by the coupling enzyme, 2-keto-3-deoxy-6-phosphogluconate aldolase. Kinetic data on the approach to equilibrium of 2-keto-3-deoxy-6-phosphogluconate aldolase-catalyzed interconversion of 2-keto-3-deoxy-6-phosphogluconate, pyruvate, and glyceral-dehyde-3-phosphate can be also accurately quantified by HPLC with pulsed amperometric detection.

Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage.

2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14) has been purified in two chromatographic steps to 99% purity in 73% overall yield from Azotobacter vinelandii. The pure enzyme is a 70 kD trimeric Class I aldolase, inhibitable by bromopyruvate or pyruvate plus sodium borohydride, with a specific activity of 625 mumol per min per mg protein and a Km of 38 microM for 2-keto-3-deoxy-6-phosphogluconate. The enzyme also has 2-keto-4-hydroxy glutarate aldolase (E.C. 4.1.3.16) activity, with a specific activity of 4.8 mumol per min per mg protein and a Km of 39 microM. 2-keto-4-hydroxy glutarate inhibits the 2-keto-3-deoxy-6-phosphogluconate aldolase activity of the enzyme with an apparent Ki of 0.17 mM. Slow steps following formation of the Schiff base intermediate between KHG and the enzyme are responsible for both the slower turnover of this substrate and for its inhibitory effect.

Detection of glycinamide ribonucleotide by HPLC with pulsed amperometry: application to the assay for glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14).

An assay method is described for measuring the catalytic activity of glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14), the first enzyme in the de novo purine biosynthetic pathway. Phosphoribosylamine, the unstable product of the enzymatic reaction, is trapped by glycinamide ribonucleotide synthetase (EC 6.3.4.13), the second enzyme in the pathway, to form the stable product glycinamide ribonucleotide. Glycinamide ribonucleotide is resolved by ion-exchange HPLC at alkaline pH and quantified by the use of a pulsed amperometric detector. The assay is specific, reproducible, rapid (15 min chromatography, including column recycling and stabilization), convenient (no radioactive materials used), linear at a wide range of glycinamide ribonucleotide concentrations, and sensitive at subnanomole levels even with crude extracts. Both the glutamine- and ammonia-dependent activity of glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase can be accurately quantified.

Thioltransferases.

A family of small molecular weight proteins with thiol-disulfide exchange activity have been discovered, widely distributed from E. coli to mammalian systems, called thioltransferases or glutaredoxins. There are no substantiated reports of thioltransferases-glutaredoxins in plants; however, partially purified dehydroascorbate reductase from peas had thiol-disulfide exchange catalytic activity using glutathione as reductant and S-sulfocysteine as thiosulfate cosubstrate (unpublished data). Thus, this class of proteins is universally distributed. Based on mutagenesis studies, a sequence of Cys-Pro-Tyr(Phe)-Cys- followed by Arg-Lys- or Lys alone is critical for both the thiol-disulfide exchange reaction and the dehydroascorbate reductase activity. The dithiol-disulfide loop represented by this structure is unique since the cystine closer to the N-terminus has a highly acidic thiol pKa (3.8 as determined for the pig liver enzyme) that contributes to the protein's high S- nucleophilicity. Compared with the microbial enzyme, the mammalian thioltransferases (glutaredoxins) are extended at both N and C termini by 10-12 amino acid residues, including a second pair of cysteines toward the C-terminus with no known special function. Yeast thioltransferase is more like mammalian enzymes in length (106 amino acids) but more like E. coli glutaredoxin in being unblocked at the N-terminus and having only one set of cysteines; that is, at the active center. The three mammalian enzymes, for which sequences are available, are blocked at the N-terminus by an acetyl group linked to alanine with no known special function other than possibly to impart greater cellular turnover stability. A report of carbohydrate (8.6%) content in rat liver thioltransferase has not been verified by more sensitive methods of carbohydrate analysis, nor has carbohydrate been identified in samples of purified glutaredoxin from any source. Thiol transferase and glutaredoxin are two names for the same protein based on similarity of amino acid sequence, immunochemical cross-reactivity, and other enzyme properties. The inability of thioltransferase from some mammalian sources to act as an electron carrier in ribonucleotide reductase systems, whether homologous or heterologous in origin, remains to be explained in future studies.

Interactions of platinum complexes with thioltransferase(glutaredoxin), in vitro.

Under anaerobic conditions, recombinant pig liver thioltransferase (glutaredoxin)(TT, GRX) (EC 1.8.4.1) was strongly inhibited by cis and carbo-platin and somewhat less sensitive to trans-platin, in vitro. By extrapolation to total inhibition, the ratio of platinum drug/thioltransferase was approximately 1.0 for cis and carbo-platin, but greater than 1.0 for trans-platin. When thioltransferase was not reduced, inhibition by preincubation with the platinum complexes required molar excesses of 1,300 and 675 to one for cis-platin and trans-platin, respectively or 400-500 microM for 50% inhibition. The inhibition of thioltransferase at high drug concentrations in the presence of oxygen was associated with cross-linking of monomers into dimers within 5 min and, in the case of cis-platin treatment, to trimers in 20 min incubation.

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.

Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction.

The effect of salts on the catalytic activity of the molybdenum-containing nitrogenase complex from Azotobacter vinelandii has been investigated. NaCl was found to inhibit the reduction of the substrates, protons, acetylene, and dinitrogen by a common mechanism. The pattern of inhibition is sigmoidal, indicating a highly cooperative interaction involving multiple inhibitor sites. Sixteen other salts that were investigated also exhibited this pattern of inhibition. NaCl functions as a dead-end inhibitor without altering the number of MgATP hydrolyzed/electron transferred to substrate. The level of expressed inhibition is sensitive to MgATP concentration, the molar ratio of the MoFe-protein (Av1) to the Fe-protein (Av2), and total protein concentration. In addition, NaCl is an inhibitor of the MgATP-dependent, iron chelation of Av2. Although the inhibition is exhibited over the same salt concentration range as that for inhibition of substrate reduction, the pattern of inhibition is hyperbolic. A model based upon simple equilibrium interactions among the enzyme species, nucleotides, and inhibitor has been developed which quantitatively accounts for the observed effects of salt. In this model, the formation of the active complex between Av1 and Av2 is abolished by salts. Likewise, the apparent affinity of Av2 for MgATP is reduced. An additional prediction based upon the model is that the affinity between Av2 and Av1 is independent of nucleotide binding.

Kinetics of MgATP-dependent iron chelation from the Fe-protein of the Azotobacter vinelandii nitrogenase complex. Evidence for two states.

Chelation of Fe from the Fe-protein component (Av2) of Azotobacter vinelandii nitrogenase has been investigated. The chelation, which requires MgATP binding by Av2, is best described as a two-exponential process. The rates for the two phases differed by approximately 10-fold and increased as the concentration of MgATP was increased. The rates for both phases were 50% of maximum at approximately 1.5 mM MgATP. At MgATP concentrations greater than 100 microM, the more rapid phase represented approximately 25% of the total Fe chelated from Av2. However, below 100 microM MgATP, the proportion of the faster phase decreased until at 20 microM MgATP, only a single phase could be detected. The properties of Av2 were studied at various stages of Fe chelation. The partially chelated protein was isolated from the reaction by gel filtration and was subjected to a second MgATP-dependent Fe chelation. Material isolated after the completion of the first phase regained biphasic kinetics in subsequent chelation reactions. However, if MgATP was present during the isolation of Av2, then only a single phase was observed in the subsequent chelation studies. In addition, the enzymatic activity of Av2 decreased concomitantly with total Fe chelation. To account for these observations, a model is presented in which Av2 exists in two conformers. Fe chelation is proposed to occur from either conformer but only when two MgATP are bound. Both conformers bind MgATP with the same affinity but are distinguished by a 10-fold difference in chelation rate. The two conformers are in equilibrium and can interconvert only in the absence of MgATP. That is, MgATP binding prevents the conversion of the two conformational states.

Conformational control of ovoperoxidase catalysis in the sea urchin fertilization membrane.

The ovoperoxidase-catalyzed oxidation of iodide has been investigated as a function of pH for the homogeneous enzyme and for ovoperoxidase incorporated into several forms of the egg fertilization membrane. The pH dependent hysteresis previously observed in purified ovoperoxidase (Deits, T. L., Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888) is entirely absent in ovoperoxidase incorporated into the mature fertilization membrane, where the enzyme is bound noncovalently in vivo. The pH activity profile of ovoperoxidase incorporated into the mature fertilization membrane closely resembles the profile observed only transiently in purified ovoperoxidase subjected to a rapid downward pH shift. These observations can be accounted for by our previously presented mechanism for ovoperoxidase hysteresis (ibid.). We hypothesize that ovoperoxidase, upon incorporation into the fertilization membrane, is restricted to a limited subset of the conformational states available to the purified enzyme. This matrix-dependent conformational restriction is a novel control mechanism that serves to enhance the catalytic activity of ovoperoxidase upon its assembly into the fertilization membrane and thereby modulates ovoperoxidase catalysis in the vicinity of the developing egg.

pH-induced hysteretic transitions of ovoperoxidase.

Ovoperoxidase, the enzyme that catalyzes the dityrosine cross-linking of fertilization membranes of eggs from the sea urchin Stronglyocentrotus purpuratus, exhibits slow changes in catalytic activity upon alterations of pH, with attendant changes in spectral properties. For ovoperoxidase pre-equilibratated at pH 8, abrupt decreases in pH are accompanied by a slow loss in activity that is temporally associated with a change in absorbance at the Soret band. With enzyme pre-equilibrated at pH 4.5 and then shifted to higher pH, there was a slow increase in catalytic activity following a rapid change in the Soret band absorbance. These changes were reversible and led to the same equilibrium state, regardless of the direction of pH shift. The rate of approach to the equilibrium state of ovoperoxidase was independent of enzyme concentration, the presence of substrates, or temperature (from 6.5 to 39.7 degrees C). The pH-induced interconversions of catalytic and spectral properties indicate that ovoperoxidase undergoes hysteretic transitions, in which alterations in the heme environment accompany, but are not sufficient for, the expression of catalytic activity. We present a kinetic mechanism for the hysteretic relaxations and suggest how these transitions may have relevance to the assembly of the fertilization membrane in vivo.

Purification and properties of ovoperoxidase, the enzyme responsible for hardening the fertilization membrane of the sea urchin egg.

The ovoperoxidase from the egg of the sea urchin, Strongylocentrotus purpuratus, has been purified to apparent homogeneity. Ovoperoxidase is secreted from the egg at fertilization and is responsible, in vivo, for hardening of the fertilization membrane by forming cross-links between protein tyrosyl residues. Purification was accomplished by activation of cortical granule exocytosis with acetic acid, followed by NH4SO4 precipitation, DEAE-Sephacel chromatography in the absence of divalent cations, and CM-Sephadex chromatography. The purified enzyme is a glycoprotein of Mr 70,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibits a UV-visible spectrum typical of heme peroxidases (epsilon 412 = 1.19 X 10(5) M-1 cm-1). Ovoperoxidase catalyzes the oxidation of tyrosine, guaiacol, iodide, and bromide, but not chloride, and can employ either H2O2 or, with 8% relative efficiency, ethyl peroxide as an oxidative substrate. Phenylhydrazine, 3-amino-1,2,4-triazole, azide, and sulfite all inhibit purified ovoperoxidase at concentrations similar to those that inhibit hardening in vivo. Inhibition by 3-amino-1,2,4-triazole is reversible, requires H2O2, and is slow relative to substrate turnover. The purified enzyme is sensitive to protease cleavage in the native state, yielding an active product of Mr approximately 50,000 which varies slightly depending upon the protease employed. Ovoperoxidase should provide a useful tool for the study of fertilization membrane formation as a paradigm of macromolecular assembly and modification.

Fluorescent inhibitors of thymidylate synthetase.

Two fluorescent derivatives of 2'-deoxy-5-fluorouridine 5'-p-aminophenyl phosphate were prepared by treatment of this compound with fluorescein isothiocyanate in dimethyl sulfoxide or 5-(dimethylamino)naphthalene sulfonyl chloride in pyridine. The products of the reactions were isolated by diethylaminoethylcellulose chromatography and were shown to be homogeneous by polyacrylamide electrophoresis and TLC. Confirmation of the structure was provided by elemental analysis, absorption and fluorescence spectra, PMR measurements, and liberation of nucleotide upon hydrolysis with snake venom phosphodiesterase. The fluorescent derivatives are good competitive inhibitors (Ki approximately10(-6) M) of thymidylate synthetase from a methotrexate-resistant strain of Lactobacillus casei.