Showing papers on "Affinity chromatography published in 1998"
TL;DR: Affinity labeling and binding experiments showed that the 110-kDa NT3 receptor can be partly transformed into a higher affinity by cotransfection with furin, which is the first transmembrane neuropeptide receptor that does not belong to the superfamily of G-protein-coupled receptors.
396 citations
TL;DR: The identification of BBK32 as a receptor for fibronectin binding may enhance the understanding of the pathogenesis and chronic nature of Lyme disease.
Abstract: The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.
261 citations
TL;DR: A unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein) and a small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification.
Abstract: A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.
232 citations
TL;DR: Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major, suggesting that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.
Abstract: Vaccination of BALB/c mice with Leishmania major promastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species of Leishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. major promastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.
182 citations
Journal Article•
TL;DR: SIL-6R of human plasma must be regarded as an antagonistic molecule that enhances the inhibitory activity of sgp130, a promising candidate for the development of IL-6 antagonists.
Abstract: Soluble receptors for several cytokines have been detected in body fluids and are believed to modulate the cytokine response by binding the ligand and thereby reducing its bioavailability. In the case of IL-6, the situation is more complex. The receptor consists of two components, including a ligand-binding alpha-subunit (IL-6R, gp80, or CD126), which in its soluble (s) form (sIL-6R) acts agonistically by making the ligand accessible to the second subunit, the signal transducer gp130 (CD130). Soluble forms of both receptor subunits are present in human blood. Gel filtration of iodinated IL-6 that had been incubated with human serum revealed that IL-6 is partially trapped in IL-6/sIL-6R/sgp130 ternary complexes. sgp130 from human plasma was enriched by immunoaffinity chromatography and identified as a 100-kDa protein. Functionally equivalent rsgp130 was produced in baculovirus-infected insect cells to study its antagonistic potential on four different cell types. It was found that in situations in which cells lacking membrane-bound IL-6R were stimulated with IL-6/sIL-6R complexes, sgp130 was a much more potent antagonist than it was on IL-6R-positive cells stimulated with IL-6 alone. In the latter case, the neutralizing activity of sgp130 could be markedly enhanced by addition of sIL-6R. As a consequence of these findings, sIL-6R of human plasma must be regarded as an antagonistic molecule that enhances the inhibitory activity of sgp130. Furthermore, in combination with sIL-6R, sgp130 is a promising candidate for the development of IL-6 antagonists.
175 citations
Journal Article•
TL;DR: Rat macrophage galectin was purified from activated macrophages by a single-step affinity chromatography on a lactosyl-Sepharose matrix and showed a definitive acidic pattern, and internal amino acid sequencing of selected peptides obtained by proteolytic cleavage revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galECTin-1 subfamily.
Abstract: Galectins, a family of closely related beta-galactoside-binding proteins, show specific immunomodulatory properties We have recently identified the presence of a galectin-like protein in rat peritoneal macrophages by means of a cross-reactivity with a polyclonal Ab raised against a galectin purified from adult chicken liver Galectin expression was up-regulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells In an attempt to further explore its functional significance, rat macrophage galectin was purified from activated macrophages by a single-step affinity chromatography on a lactosyl-Sepharose matrix The eluted fraction was resolved as a single protein band of approximately 15,000 Da by SDS-PAGE that immunoreacted strongly with the anti-chicken galectin serum Gel filtration studies revealed that the protein behaved like a dimer under native conditions, and saccharides bearing a beta-D-galactoside configuration were able to inhibit the hemagglutinating activity displayed by the purified galectin In agreement with its isoelectric point of approximately 48, the amino acid analysis showed a definitive acidic pattern Internal amino acid sequencing of selected peptides obtained by proteolytic cleavage revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin-1 subfamily Finally, biochemical and ultrastructural evidence, obtained by genomic DNA fragmentation and transmission electron microscopy, are also provided to show its potential implications in the apoptotic program of T cells This effect was quantified by using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling assay and was found to be associated to the specific carbohydrate-binding properties of galectin
173 citations
TL;DR: To isolate and characterize cDNA clones encoding this proteinase, two degenerate primer approaches were used to amplify part of the coding region using polymerase chain-reaction (PCR) and yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library.
Abstract: The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed ...
170 citations
TL;DR: Two ribosome inactivating proteins designated alpha- and beta-pisavins were isolated from seeds of the garden pea Pisum sativum var.
168 citations
TL;DR: An enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycosphingolipids in mammals, is purified over 61,000-fold to near homogeneity with a 29.7% yield from rat brain membrane fractions.
167 citations
TL;DR: Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1beta (ChIL-1 beta), and northern blot analysis showed that ChIL- 1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment.
Abstract: Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC-32. We used a cDNA expression cloning strategy in COS cells to characterize this activity. The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N-terminal domain that could serve as secretory signal. Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1 beta (ChIL-1 beta). Northern blot analysis showed that ChIL-1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment. To test for biological activity of putative mature ChIL-1 beta, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N-terminus for easy purification by nickel chelate affinity chromatography. Purified His-ChIL-1 beta potently induced CXC chemokine RNA synthesis in CEC-32 cells. When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.
163 citations
TL;DR: It is demonstrated by affinity chromatography that hepatocyte growth factor/scatter factor (HGF/SF) binds strongly to dermatan sulfate (DS), with a similar ionic strength dependence to that previously seen with heparan sulfATE (HS).
TL;DR: A unique interaction between pro-MMP-9 and α2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network is suggested.
TL;DR: Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes, which mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.
Abstract: Monocyte-dependent as well as direct inhibitory effects of antimalarial antibodies point toward antigens accessible at the time of merozoite release as targets for biologically active antibodies capable of mediating protection against Plasmodium falciparum. The glutamate-rich protein (GLURP), being an antigen associated with mature schizont-infected erythrocytes, was therefore the object of the present investigation, in which we analyzed whether anti-GLURP antibodies can either interfere directly with merozoite invasion or act indirectly by promoting a monocyte-dependent growth inhibition, antibody-dependent cellular inhibition. GLURP-specific human immunoglobulin G (IgG) antibodies, from pooled IgG of healthy Liberian adults who were clinically immune to malaria, were purified by affinity chromatography on columns containing R0 (N-terminal nonrepetitive region of GLURP) or R2 (C-terminal repetitive region of GLURP) recombinant protein or synthetic peptides as ligands. Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes. One epitope was specific for R0, two were specific for R2, and the fourth displayed cross-reactivity between R0 and R2. None of the purified IgG antibodies had direct invasion-inhibitory effects, even at high concentrations. In contrast, when allowed to cooperate with monocytes, all anti-GLURP IgG preparations mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.
TL;DR: Results indicate that apoA-I is the mainLL-37 binding protein in human plasma and may work as a scavenger of LL-37, thus suggesting a novel mechanism involved in the regulation of a cathelicidin peptide.
TL;DR: In one run the binding constants Kd for all the active components of a ligand library at sub-microgram quantities can be determined.
Abstract: In one run the binding constants Kd for all the active components of a ligand library at sub-microgram quantities can be determined. A mixture of ligands is continuously infused through a column of immobilized receptor, and the eluent analyzed by electrospray mass spectrometry. From the affinity chromatogram produced (see picture) the breakthrough volume of a single compound and hence its Kd value can be determined.
TL;DR: RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate, and highly specific binding of INH to CWI could be shown by affinity chromatography.
Abstract: Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.
TL;DR: Potato tubers have been successfully used for high-level production of a recombinant single-chain Fv (ScFv) antibody and its specific activity did not decrease during tuber storage.
Abstract: Potato tubers have been successfully used for high-level production of a recombinant single-chain Fv (ScFv) antibody. Ubiquitous high-level expression was achieved under control of the CaMV 35S promoter through retention of the scFv protein in the endoplasmic reticulum. Recombinant antibodies accumulated up to 2% of total soluble tuber protein. After 1.5 years of tuber storage at 4 °C still half of the amount of scFv present in freshly harvested tubers was detectable. Its specific activity did not decrease during tuber storage. Recombinant protein could be efficiently purified from crude extracts by affinity chromatography.
TL;DR: The hypothesis that ACAT is an allosteric enzyme regulated by cholesterol is supported, as the cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal.
TL;DR: Validation of antibody affinity purification processes for therapeutic use is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands.
Abstract: A synthetic ligand [TG19318], able to mimic protein A in the recognition of the immunoglobulin Fc portion, has been previously identified in our laboratory through the synthesis and screening of multimeric combinatorial peptide libraries. In this study we have fully characterized its applicability in affinity chromatography for the downstream processing of antibodies, examining the specificity and selectivity for polyclonal and monoclonal immunoglobulins derived from different sources. Ligand specificity was broader than protein A, since IgG deriving from human, cow, horse, pig, mouse, rat, rabbit, goat and sheep sera, IgY obtained from egg yolk, and IgM, IgA and IgE were efficiently purified on TG19318 affinity columns. Adsorbed antibodies were conveniently eluted by a buffer change to 0.1 M acetic acid or 0.1 M sodium bicarbonate pH 9, with full retention of immunological properties. Monoclonal antibodies deriving from cell culture supernatants or ascitic fluids were also conveniently purified on TG19318 affinity columns, even from very diluted samples. The affinity constant for the TG19318–IgG interaction was 0.3 µM, as determined by optical biosensor measurements. Under optimized conditions, antibody purity after affinity purification was close to 95%, as determined by densitometric scanning of SDS–PAGE gels of purified fractions, and maximal column capacity reached 25 mg Ig/ml support. In vivo toxicity studies in mice indicated a ligand oral toxicity greater than 2000 mg kg−1 while intravenous toxicity was close to 150 mg kg−1. Validation of antibody affinity purification processes for therapeutic use, a very complex, laborious and costly procedure, is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands. Copyright © 1998 John Wiley & Sons, Ltd.
TL;DR: The results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.
TL;DR: Results with wild-type 3A4 and F304A indicated that at high progesterone concentrations, hydroxylation rates and product ratios are independent of the presence of alpha-NF, suggesting that progester one may be equivalent to alpha- NF as an activator.
TL;DR: It is suggested that SPP or an SPP-like protein contributes to the neuronal survival-promoting activity of serum and is supported more effectively than inorganic selenium.
Abstract: We purified from bovine serum a glycoprotein that promotes the survival of rat embryonic neurons cultured from septum and other brain regions. A 40,000-fold purification was achieved by using a combination of ammonium sulfate precipitation, Zn 2+ affinity chromatography, Cibacron blue 3-GA dye affinity chromatography, ABx ion exchange chromatography, and preparative PAGE. The active protein had an apparent molecular weight of 50–60 kDa. The concentration required for half-maximal survival (EC 50 ) was 12 ng/ml (∼200 pm) for the final fraction. Amino acid sequencing after cyanogen bromide cleavage yielded two sequences that are homologous to regions of deduced sequence of the selenoprotein-P (SPP) family in bovine, rat, and human. Antibodies against a synthetic peptide within the bovine SPP sequence immunoprecipitated and inhibited the survival-promoting activity of a partially purified serum fraction. The purified protein supported neuronal survival more effectively than inorganic selenium. These results suggest that SPP or an SPP-like protein contributes to the neuronal survival-promoting activity of serum.
TL;DR: A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography and the N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.
Abstract: A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced alpha-, beta-keto esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.
TL;DR: The results suggest that rPLD1 is a glycosylated protein and that it is negatively regulated by phosphorylation by PKC in vitro.
TL;DR: Results indicate that the keratinase belongs to the subtilisin-like serine protease family, which is similar to other fungal subtilisins.
Abstract: A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin-agarose and gel filtration. The apparent molecular mass of the enzyme was 31·5 kDa and the pI was 11·8. The enzyme was not glycosylated and its first 15 N-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 whileinactivation of the enzyme was reversible at pH4, but not at pH 11. The enzyme was stable at 37 °C with an apparent optimum temperature around 55 °C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0·37 mm) and physiological efficiency (kcat/Km) were obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide. These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins G prepar...
TL;DR: Results suggest that Lys-gingipain plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction ofperiodontal tissue components and the disruption of normal host defense mechanisms.
Abstract: The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed "Lys-gingipain," was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of Mr=51,000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and alpha1-antichymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.
TL;DR: This method may constitute a very general procedure to purify proteins to near homogeneity without the need to tailor conditions individually, and it may thus be very attractive for high-throughput screening programs and for developing general protocols for clinical grade material.
TL;DR: This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid which produces a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions.
Abstract: Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.
TL;DR: GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.
Abstract: Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca 2+ /calmodulin but not by Ca 2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mmNH 4 Cl, 5 mm NH 4 NO 3 , 5 mm glutamic acid, or 5 mm glutamine as the sole nitrogen source compared with samples from plants treated with 10 mm KNO 3 . The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.
TL;DR: This study effectively illustrates the engineering of separation media which combine several desired properties for the development of a new branch of metal affinity-based bioseparation.