Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). II. Localization of a putative auxin receptor

With the aid of affinity chromatography on auxin-binding protein-Sepharose (ABP-Sepharose) monospecific IgGanti-ABP from rabbit antisera were isolated as judged by immuno-double diffusion test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this IgGanti-ABP the ABP is localized within the outer epidermal cells of coleoptiles using indirect immunofluorescence labeling. Auxin-induced growth of coleoptile segments can be inhibited by IgGanti-ABP, and the auxin response of split coleoptile sections is also strongly reduced by IgGanti-ABP. The ABP, therefore, is referred to as an auxin receptor. This auxin receptor is localized at the plasmalemma of the outer epidermal cells of the coleoptile.     

Characterization and partial purification of multiple electron transport activities in plasma membranes from maize (Zea mays) roots

The plasma membrane of eukaryotic cells contains endogenous, integral electron transport proteins. In the maize ( Zea mays L. cv. Golden Cross Bantam) root plasma membrane, these activities include NAD(P)H-ferricyanide reductase. NAD(P)H-duroquinone reductase (1.6.5.1) and NAD(P)H-ascorbate free-radical reductase (EC 1.6.5.4). Differences in degree of stimulation upon vesicle rupture with detergent and in specificities for pyridine nucleotides suggest that these activities constitute distinct components in the membranes. Solubilization of reductase activities was examined using Triton X-100 over a wide range of retergent-to-protein ratios. The Triton-solubilized enzymes were purified using dye-ligand affinity chromatography on Cibacron blue 3G-A agarose utilizing biospecific elution with NADH. Resolution of the redox activities was accomplished upon differential elution with 0.1.1.0 and 10 m M NADH. The distinctive characteristics of the enzymes and the differential chromatographic behavior of the respective activities provided evidence for the presence of separate enzymatic redox components in maize root plasma membranes with implications for an electron transfer chain.     

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.     

Interaction of maize (Zea mays) protein phosphatase 2A with tubulin

Immunological and biochemical evidence has been obtained for an interaction of maize protein phosphatase 2A (PP2A) holoenzyme with tubulin. Tubulin co-purifies with maize seedling PP2A. Affinity chromatography of the maize PP2A preparation on immobilized tubulin revealed two peaks of phosphorylase alpha phosphatase activity. In one of the peaks, the catalytic (C) and constant regulatory (A) subunits of PP2A were identified by Western blotting. The subunits (C and A) of PP2A were co-immunoprecipitated from maize seedlings homogenate by an anti-alpha-tubulin antibody. The interaction of plant PP2A with tubulin indicates a possible role of reversible protein phosphorylation in the dynamic structure of plant cytoskeleton.     

Partial purification and reconstitution of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes.

Benzenemethane Sulfonylfluoride (329-98-6) is an irreversible inactivator of many esterases including mammalian acetylcholinesterases. However, previous reports indicated that acetylcholinesterase from the electric eel, Electrophorus electricus (EC 3.1.1.7) failed to react with benzenemethane sulfonylfluoride at measurable rates. We report here that eel acetylcholinesterase reacts with this inactivator at a low rate. Hydrolysis of the sulfonylating agent is so much faster than enzyme inactivation that, under most conditions, there will be only slight inactivation. Like the reaction of other active site acylating agents with this enzyme, inactivation can be accelerated in the presence of certain organic cations. We introduce a rate equation for enzyme sulfonylation which incorporates both the hydrolysis of the inactivator and the complication that fluoride resulting from hydrolysis of the inactivator is a potent competitive inhibitor of this enzyme. This rate equation accurately describes the time course of enzyme inactivation.     

Biochemical characterization of a truncated penta-EF-hand Ca2+ binding protein from maize

Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.     

Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.     

Phosphorylation of a stromal enzyme protein in maize (Zea mays) mesophyll chloroplasts.

When intact maize (Zea mays) mesophyll chloroplasts were illuminated in the presence of [32P]orthophosphate and subsequently subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a major polypeptide species of Mr 100000 was found to be heavily labelled. This polypeptide was not found in maize mesophyll thylakoid or cytoplasmic fractions, but was localized solely in the chloroplast stroma. No phosphorylation of polypeptides in the 100000-Mr region was observed in the mesophyll chloroplasts from C3 species (where the primary product of CO2 fixation is a 3-carbon compound), suggesting that this polypeptide arises from a protein associated with C4 metabolism (where the first product of CO2 fixation is a 4-carbon compound). The 100kDa polypeptide was major component of the maize mesophyll chloroplast, comprising 10-15% of the total protein, which banded in an identical position to the apoprotein of the enzyme pyruvate, orthophosphate dikinase, which catalyses a reaction of the C4 cycle [Edwards & Walker (1983) C3, C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis, Blackwell Scientific Publications, Oxford and London]. Phosphorylation in the 100kDa species was prohibited by treatment of lysed chloroplasts with antibody to pyruvate, orthophosphate dikinase (EC 2.7.9.1). These data suggest that the phosphorylated polypeptide observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is the monomeric form of this enzyme. The 100kDa polypeptide was partially phosphorylated in darkness, but a significant increase in the degree of phosphorylation was found on illumination. This polypeptide was found to be dephosphorylated only slowly when the chloroplasts were returned to darkness. Maximum phosphorylation was observed in the presence of pyruvate or dihydroxyacetone phosphate, which also caused maximum activation of pyruvate, orthophosphate dikinase. Phosphorylation of the 100kDa polypeptide did not coincide with deactivation of pyruvate, orthophosphate dikinase, but maximum phosphorylation occurred under conditions that promoted maximum activity of the enzyme, at which time one phosphate group was associated with each enzyme molecule. Protein phosphorylation did not appear to arise from the reaction mechanism of the enzyme.     

Characterization and partial purification of indole-3-butyric acid synthetase from maize (Zea mays)

In previous work it has been shown that the route from indoleacetic acid (IAA) to indolebutyric acid (IBA) is likely to be a two-step process with an unknown intermediate designated ‘product X′. Our objective was to characterize and purify enzyme activities that are involved in these reactions. Indole-3-butyric acid synthetase was isolated and characterized from light-grown maize seedlings (Zea mays L.), which were able to synthesize IBA from indole-3-acetic acid (IAA) with ATP and acetyl-CoA as cofactors. The enzyme activity is most likely located on the membranes of the endoplasmic reticulum, as shown by means of aqueous two-phase partitioning and sucrose density gradient centrifugation, with subsequent marker enzyme analysis. It was possible to solubilize the enzyme from the membranes with a detergent (CHAPS) and high concentrations of NaCl. The molecular mass of solubilized IBA synthetase was ca 31 kDa and its isoelectric point was at pH 4.8. The enzyme forming the reaction intermediate had a molecular mass of only 20 kDa and it seemed to be located on different membranes. Inhibition experiments with reducing agents and sulfhydryl reagents indicated that no sulfhydryl groups or disulfide bridges were present in the active centre of IBA synthetase. KCN inhibited the enzyme activity completely, and sodium azide by about 50%. Substrate analogs. such as 1-IAA, 2,4-dichlorophenoxyacetic acid, phenylacetic acid, and naphthaleneacetic acid, inhibited IBA formation to a high extent. Experiments with tunicamycin gave evidence that the enzyme is not a glycoprotein. These findings were confirmed by affinity chromatography with Concanavalin A. where the enzyme did not bind to the matrix. Further purification of the IBA synthetase on an ATP-affinity column resulted in a more than 1 000-fold purification compared to the microsomal membranes. IBA synthetase activity was also present in other plant families. Our results present further evidence that IBA is synthesized by a two-step mechanism involving two different enzyme activities.     

Modeling the tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin.

The tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin (Hbm) was modeled using computer tools and the known tertiary structure of rice Hb1 as a template. This method was tested by predicting the tertiary structure of soybean leghemoglobin a (Lba) using rice Hb1 as a template. The tertiary structures of the predicted and native Lba were similar, indicating that our computer methods could reliably predict the tertiary structures of plant Hbs. We next predicted the tertiary structure of Hbm. Hbm appears to have a long pre-helix A and a large CD-loop. The positions of the distal and proximal His are identical in Hbm and rice Hb1, which suggests that heme-Fe is hexacoordinate in Hbm and that the kinetic properties of Hbm and rice Hb1 are expected to be very similar, i.e. that Hbm has a high O2-affinity. Thermostability analysis showed that Hbm CD-loop is unstable and may provide mobility to amino acids located at the heme pocket for both ligand binding and stabilization and heme-Fe coordination. Analysis of the C-terminal half of Hbm showed the existence of a pocket-like region (the N/C cavity) where interactions with organic molecules or proteins could be possible. Lys K94 protrudes into the N/C cavity, suggesting that K94 may sense the binding of molecules to the N/C cavity. Thus, it is likely that the instability of the CD-loop and the possibility of binding molecules to the N/C cavity are essential for positioning amino acids in the heme pocket and in regulating Hbm activity and function.     

A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro.

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.     

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.     

Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes.

Rap2b is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a approximately 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.     

Phytoliths as indicators of prehistoric maize (Zea mays subsp.mays,Poaceae) cultivation

Maize (Zea mays L. subsp.mays) has been identified in archaeological contexts by a high proportion of large cross-shaped phytoliths. Given the numerous races of maize, this study was undertaken to determine if differences below the species level could be noted. It was also designed to see if phytoliths differed in various plant parts at various stages of growth. Several races were grown under experimental conditions. No significant differences were found. Furthermore, few phytoliths alleged to be diagnostic of maize were discovered. Systemic studies of maize and analyses of prehistoric cultivation by means of phytoliths seem not to be as promising as some researchers have argued.     

Identification and characterization of a novel adenine phosphoribosyltransferase gene (ZmAPT2) from maize (Zea mays L.).

Adenine phosphoribosyltransferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. It was found that several different forms of APRT gene exist in plants, but no APRT gene in maize has been reported up to now. In this study, a novel maize APRT gene was cloned and characterized through a combination of bioinformatic, RT-PCR and RACE strategies. The full length of APRT cDNA sequence is 1202 nucleotides, with an ORF encoding 214 amino acid residues. Alignment of the deduced protein with that of other plant APRT genes indicates that the new gene is the form 2 of maize APRT, thus it was named ZmAPT2. Through basic local alignment search tool, search in the genomic survey sequence database of MaizeGDB, the putative genomic sequence of ZmAPT2 was obtained. Comparison of the cDNA and genomic sequence of the ZmAPT2 gene revealed that it contained seven exons and six introns. The locations of the introns within the maize ZmAPT2 coding region were consistent with those in the previously isolated APRTs of arabidopsis and rice. RT-PCR analysis showed that ZmAPRT was constitutively expressing in different organs under high temperature and salt stresses. Southern blot analysis indicated that at least three APRT genes existed in maize genome. These results confirmed that the novel maize ZmAPT2 gene was truly identified, and its potential role in maize growth and development was discussed.