Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.     

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.     

Purification and preliminary characterization of phosphoglycerate mutase from Schizosaccharomyces pombe

Phosphoglycerate mutase could be purified to over 95% homogeneity by a single step procedure involving elution from Cibacron Blue-Sepharose by a pulse of cofactor 2,3-bisphosphoglycerate. Although the enzyme has been isolated in only small quantities (c. 100 micrograms), gel filtration and sodium dodecylsulphate polyacrylamide gel electrophoresis indicated that it is monomeric with Mr approximately 23,000, an extremely low value for this enzyme. Preliminary investigations of the kinetic characteristics and the nature of important amino acid side chains have been undertaken.     

Purification and properties of alanine aminotransferase from maize (Zea mays L.) leaves

Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg protein)⁻¹ at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated the presence of arginine residue as anionic binding site in the active centre of AlaAT. This work was supported by the State Committee for Scientific Research, a grant No. 5PO6A00510     

Purification and characterization of phosphoglycerate mutase isozymes from pig heart

The three isozymes of phosphoglycerate mutase from pig heart have been purified to homogeneity. The isozymes have a molecular weight of 57000 as determined by gel-filtration chromatography. Discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate yields a single band with a molecular weight of 29000, indicating that the isozymes are dimers composed of subunits of similar mass. Hybridization experiments show that the three isozymes result from homodimeric and heterodimeric combinations of two different subunits. The two types of subunit differ in their heat lability and in the presence of -SH groups essential for enzymatic activity. No remarkable differences exist in the kinetic constants of the purified isozymes. The kinetic pattern is consistent with a 'ping-pong' mechanism. The homogeneous preparations of the three isozymes show intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity which can be stimulated by glycolate-2-P.     

Purification and properties of the phosphoglycerate mutase isozymes from the mouse

The two homodimeric isozymes of phosphoglycerate mutase have been purified from murine kidney and muscle. No differences were observed in the Michaelis-Menten constant for the substrate 2-phospho-D-glycerate, in molecular weight, temperature and pH optima, when the purified isozymes were compared. The isozymes differ in their inhibition constants for phosphoenolpyruvate, in their Michaelis constants for 3-phospho-D-glycerate and 2,3-bisphospho-D-glycerate, their thermal and pH lability and in their sensitivity towards mercury ions.     

Purification and characterization of a novel antimicrobial peptide from maize (Zea mays L.) kernels.

Several small, acid-soluble, basic peptides with anti-microbial properties have been isolated from maize (inbred B73) kernels. One of these peptides (MBP-1) has been purified to homogeneity and characterized. The peptide has a molecular weight of 4127.08 as determined by plasma desorption mass spectroscopy, has no free cysteines, and is predominantly alpha-helical as determined by circular dichroism. The primary sequence of the peptide (33 residues) has been determined by Edman degradation and shows no homology to the thionins, a group of cysteine-rich peptides found in some cereals including wheat, barley, and sorghum, as well as several dicot species. Like the thionins, however, MBP-1 has been found to have antimicrobial properties in vitro. MBP-1 inhibits spore germination or hyphal elongation of several plant pathogenic fungi, including two seed pathogens of maize (Fusarium moniliforme Sheld. and Fusarium graminearum (Gibberella zeae (Schw.) Petsch)), and several bacteria, including a bacterial pathogen of maize (Clavibacter michiganense ssp. nebraskense). A synthetic MBP-1 peptide, air-oxidized and purified by reverse phase chromatography, was equally antifungal as compared with the naturally occurring peptide.     

Purification and characterization of a galactose-specific lectin from corn (Zea mays) coleoptyle

We purified and characterized a lectin from the corn coleoptyle (Zea mays). The lectin (CCL) was purified by affinity chromatography on a Lactosyl–Sepharose 4B column. It is a glycoprotein of 88.7 kDa, composed mainly by glutamic, aspartic, glycine, and Ser residues; in a minor proportion, it contained methionine and cysteine residues. Carbohydrates that constituted 12% of the total weight comprised galactose, mannose, and N-acetyl-D-glucosamine. The lectin contained the blocked amino-terminus. Analysis of the lectin, determined from peptides obtained after trypsin digestion by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight), indicated that CCL has 18% homology with a putative calcium-dependent Ser/Thr protein kinase, from Arabidopsis thaliana, and 39% homology with a NADPH-dependent reductase from Z. mays. The lectin showed hemagglutinating activity toward several erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that the lectin interacts specifically with the OH on C4 from galactose residues. OH- on C1 plays a relevant role in the interaction with CCL, since β-galactose residues are better recognized than those from the anomeric α-galactose. Lack of lectin activity was observed in corn extracts; the highest specific activity was obtained from coleoptyle obtained at the 7th day after seeding.     

Induction of acid phosphatase activity during germination of maize (Zea mays) seeds.

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize (Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5-5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 degrees C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.     

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization

The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.     

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of (32)P- and (14)C-labelled substrates at chemical equilibrium. 3. With (14)C-labelled substrates no induced transport was found over a wide concentration range, and with (32)P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. (14)C-labelled substrates exchange at twice the rate of (32)P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4x10(6)s(-1). The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the K(m) for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.     

Purification and characterization of four beta-expansins (Zea m 1 isoforms) from maize pollen

Four proteins with wall extension activity on grass cell walls were purified from maize (Zea mays) pollen by conventional column chromatography and high-performance liquid chromatography. Each is a basic glycoprotein (isoelectric point = 9.1-9.5) of approximately 28 kD and was identified by immunoblot analysis as an isoform of Zea m 1, the major group 1 allergen of maize pollen and member of the beta-expansin family. Four distinctive cDNAs for Zea m 1 were identified by cDNA library screening and by GenBank analysis. One pair (GenBank accession nos. AY104999 and AY104125) was much closer in sequence to well-characterized allergens such as Lol p 1 and Phl p 1 from ryegrass (Lolium perenne) and Phleum pretense, whereas a second pair was much more divergent. The N-terminal sequence and mass spectrometry fingerprint of the most abundant isoform (Zea m 1d) matched that predicted for AY197353, whereas N-terminal sequences of the other isoforms matched or nearly matched AY104999 and AY104125. Highly purified Zea m 1d induced extension of a variety of grass walls but not dicot walls. Wall extension activity of Zea m 1d was biphasic with respect to protein concentration, had a broad pH optimum between 5 and 6, required more than 50 micro g mL(-1) for high activity, and led to cell wall breakage after only approximately 10% extension. These characteristics differ from those of alpha-expansins. Some of the distinctive properties of Zea m 1 may not be typical of beta-expansins as a class but may relate to the specialized function of this beta-expansin in pollen function.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Purification and properties of the manganese-dependent phosphoglycerate mutase of Bacillus subtilis.

Phosphoglycerate mutase of Bacillus subtilis was purified to apparent homogeneity. It specifically required manganese ions for stability and activity, but it does not need 2,3-diphosphoglycerate as cofactor; the Km for Mn2+ is about 4.5 micrometer. Enzyme activity was inhibited by heavy-metal ions, 2,3-butanedione, and sulfhydryl agents. The mutase has a molecular weight of about 74,000 as shown by Sephadex gel filtration and by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; it consisted of one polypeptide.     

Hybrid forms of phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase-phosphatase

Purified phosphoglycerate mutase from pig skeletal muscle and 2,3-bisphosphoglycerate synthase-phosphatase from pig erythrocytes were hybridized “in vitro”. The hybrid showed a behaviour on electrophoresis and on ion-exchange chromatography similar to that of a naturally occurring enzyme with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities present in pig skeletal and heart muscle. Both the hybrid and the muscle enzyme possess similar activities ratio. From these and previous data it is suggested that the six enzymatic forms with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities detected in mammalian tissues (Carreras et al. 1981, Comp. Biochem. Physiol. 70B, 477–485) result from combination of three subunits (types M, B and E).     

Biochemical characterization of maize (Zea mays L.) for salt tolerance

The inherent differences for salt tolerance in two maize cultivars (Agatti-2002 and Sahiwal-2002) were evaluated in pot experiments. Plants were grown in half-strength of Hoagland nutrient solution added with 0, 80, 100, 120, 140 and 160 mM of NaCl. Salt stress markedly reduced the shoot and root lengths and fresh and dry masses. Reduction in growth attributes was more pronounced in cv. Agatti-2002 than cv. Sahiwal-2002. Both maize cultivars exhibited significant perturbations in important biochemical attributes being employed for screening the crops for salt tolerance. Cultivar Sahiwal-2002 was found salt tolerant as compared to cv. Agatti-2002 because it exhibited lower levels of H₂O₂, malondialdehyde (MDA) and higher activities of antioxidant enzymes. In addition, cultivar Sahiwal-2002 exhibited less salt-induced degradation of photosynthetic pigments, lower levels of toxic Na⁺ and Cl^?  and higher endogenous levels of K⁺ and K⁺/Na⁺ ratio. The results indicate that salt stress induced a marked increase in MDA, H₂O₂, relative membrane permeability, total soluble proteins and activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase andascorbate peroxidase). Moreover, increase in endogenous levels of Na⁺ and Cl^?  and decrease in K⁺ and K⁺/Na⁺ ratio and photosynthetic pigments were recorded in plants grown under salinity regimes.     

Purification and properties of a pyruvate carboligase from Zea mays cultured cells

An enzyme able to catalyze the synthesis of acetoin (3-hydroxy-2-butanon) from either pyruvate or acetaldehyde was isolated, partially purified and characterized from maize (Zea mays L. cv Black Mexican Sweet) cultured cells. It exhibited a maximal rate at neutral pH values, and strictly required thiamine pyrophosphate and a divalent cation for activity; on the contrary, unlike bacterial pyruvate oxidases, flavin was not required. Apparent Michaelis constants were 260±20 mM for pyruvate and 24±7 mM for acetaldehyde. Both substrate affinity and specificity were notably higher than those of pyruvate decarboxylase, an enzyme that also synthesizes acetoin as by-product. The partially purified protein was unable to catalyze the formation of other possible products of pyruvate decarboxylation, thus carboligase appears to be its main activity. Results suggest that acetoin synthesis may be of physiological significance in plants.     

Purification and characterization of benzoyl-L-arginine p-nitroanilide hydrolase from etiolated leaves of Zea mays

Benzoyl-L-arginine p-nitroanilide hydrolase in the etiolated leaves of Zea mays L. has been purified 1,266-fold by a combination of gel filtration, ion exchange, and hydrophobic chromatography with a recovery of 13%. The specific activity of the purified enzyme is 5.7 units/mg protein. The enzyme is an acidic protein with a pI value of 4.6 and optimum pH of 8.2. The molecular weight of the enzyme was estimated to be 59,000. Substrate inhibition was observed at a concentration higher than 30 microM BAPA and the apparent Km for BAPA was 29 microM at pH 8.0. The enzyme activity was inhibited by sulfhydryl reagents, leupeptin, antipain, and N-tosyl-L-lysine chloromethyl ketone. The inhibitor study suggests that the enzyme belongs to the class of the sulfhydryl proteases.     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.