Investigation of the acylation mechanism of class C beta-lactamase: pKa calculation, molecular dynamics simulation and quantum mechanical calculation

β-Lactamases are bacterial enzymes that act as a bacterial defense system against β-lactam antibiotics. β-Lactamase cleaves the β-lactam ring of the antibiotic by a two step mechanism involving acylation and deacylation steps. Although class C β-lactamases have been investigated extensively, the details of their mechanism of action are not well understood at the molecular level. In this study, we investigated the mechanism of the acylation step of class C β-lactamase using pKa calculations, molecular dynamics (MD) simulations and quantum mechanical (QM) calculations. Serine64 (Ser64) is an active site residue that attacks the β-lactam ring. In this study, we considered three possible scenarios for activation of the nucleophile Ser64, where the activation base is (1) Tyrosine150 (Tyr150), (2) Lysine67 (Lys67), or (3) substrate. From the pKa calculation, we found that Tyr150 and Lys67 are likely to remain in their protonated states in the pre-covalent complex between the enzyme and substrate, although their role as activator would require them to be in the deprotonated state. It was found that the carboxylate group of the substrate remained close to Ser64 for most of the simulation. The energy barrier for hydrogen abstraction from Ser64 by the substrate was calculated quantum mechanically using a large truncated model of the enzyme active site and found to be close to the experimental energy barrier, which suggests that the substrate can initiate the acylation mechanism in class C β-lactamase.     

Purification and properties of a second fructan exohydrolase from the roots of Cichorium intybus

A 1-FEH II (1-fructan exohydrolase, EC 3.2.1.80) was purified from forced chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A (Con A) affinity chromatography and anion and cation exchange chromatography. This protocol produced a 70-fold purification and a specific activity of 52 nkat mg⁻¹ protein. The apparent size of the enzyme was 60 kDa as estimated by gel filtration and 64 kDa on SDS-PAGE. Optimal activity was found between pH 5.0 and 5.5. The temperature optimum was around 35°C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent K_m of 58 m M for 1-kestose (Kes) and 64 m M for 1,1-nystose (Nys). The purified 1-FEH II hydrolyzed the β (2↠1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the β (2↠6) linkages in levan oligosaccharides and levanbiose. Fructose did not affect the 1-FEH II activity but sucrose (Suc) was a strong inhibitor of this 1-FEH II (K_i=5.9 m M ). The enzyme was partially inhibited by Na-EDTA and CaCl₂ (1 m M ).     

Purification and characterization of β-amylase from leaves of potato (Solanum tuberosum)

Amylolytic activity is widely distributed in plants. In potato leaves ( Solanum tuberosum L.) the abundant amylolytic activity was found to be β-amylase (EC 3.2.1.2, a-1,4-D-glucan maltohydrolase). β-Amylase from potato leaves was purified to homogeneity for study of enzyme characteristics. The purification steps included ammonium sulphate precipitation, anion exchange chromatography, affinity chromatography and gel filtration. The end product of α-1,4-glucan degradation was maltose. The protein is a 111-kDa homo-dimer with a subunit molecular mass of 56 kDa and a pl of 5.6. The pH-optimum is 6.5 using p -nitrophenylmaltopentaoside (PNPG5) as substrate. The optimal temperature for hydrolysis is at 40°C. The enzyme is unstable at temperatures above 40°C. The K_nt-value for PNPG5 is 0.73 m M and the activity is inhibited by cyclodextrins. At a concentration of 1 m M , β-cyclodextrin is a stronger inhibitor than α-cyclodextrin (68 and 20% inhibition, respectively). Branched glucans (e.g. starch and amylopectin) are superior substrates as compared to long, essentially unbranched glucans (e.g. amylose). This study of the catalytic properties of β-amylase from potato leaves indicates the importance of β-amylase as a starch degrading enzyme.     

Purification and partial characterization of β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min, 21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58).     

Expression of β-galactosidase multiple forms during barley (Hordeum vulgare) seed germination. Separation and characterization of enzyme isoforms

β-Galactosidase (EC 3.2.1.23) activity in barley ( Hordeum vulgare ) seedlings increases moderately during the first stages of germination. The level of activity in the whole seedling is the result of increasing activity of β-galactosidase in the roots and shoots and of declining enzyme activity in the grain. β-Galactosidase was purified during different developmental stages and from various parts of the barley seedling using affinity chromatography and was resolved into multiple forms by isoelectric focusing on polyacrylamide gels. The expression of the isoforms was shown to be under temporal and tissue-specific control. Four sets of isozymes were separated by DEAE-cellulose chromatography and were shown to be functionally similar. β-Galactosidase isoforms also exhibit size microheterogeneity, the more acidic entities having higher molecular masses. The differences in molecular weight are mainly restricted to the size of the small subunit. Multiplicity can not be attributed to glycosylation, since treatment of the enzyme preparation with N- or O-glycanase did not alter the isoelectric points or the molecular weights of the isoforms.     

Purification and characterization of extracellular invertase from the hypocotyls of mung bean (Phaseolus radiatus L.)

The extracellular invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by ammonium sulfate fractionation and sequential chromatography over diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A-Sepharose 4B affinity and Sephadex G-200. The overall purification was about 77-fold with a recovery of about 11%. The finally purified enzyme exhibited a specific activity of about 113 μmol of glucose produced mg^-1 protein min^-1 at pH 5.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The enzyme had the native molecular mass of 134 kD and subunit molecular weight of 67 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of homodimeric proteins. On the other hand, the enzyme appeared to be a glycoprotein containing mannosyl residues on the basis of its ability to interact specifically with the immobilized Con A and the separability of invertase-Con A complex by methyl-α-D-mannopyranoside. The enzyme had a K_m for sucrose of 3.4 mM and its pH optimum of 4.0. The enzyme showed highest enzyme activity with sucrose as substrate. Raffinose and cellobiose were hydrolyzed at a low rate, maltose and lactose were not cleaved by the enzyme. These results indicate the extracellular invertase is a β-fructofuranosidase.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Purification and properties of a BT-glucosidase of Hanseniaspora vineae Van der Walt and Tscheuschner with the view to its utilization in fruit aroma liberation

The β-glucosidase of Hanseniaspora vineae was purified by ion-exchange chromatography and gel filtration. Its molecular weight was 295000 PT 15000, its optimum pH was between 6 and 6–5, and its optimum temperature was 55°C. The enzyme was active against different soluble glucosides with β(1–2), β(1–3), β(1–4), β(1–6) and even aP(1–4) configurations. A glucosyltransferase activity appeared in the presence of ethanol. The enzyme was constitutive but its synthesis was repressed by glucose.     

Isolation, characterization and significance of papaya β‐galactanases to cell wall modification and fruit softening during ripening

β‐Galactosidases (EC 3.2.1.23) from ripe papaya ( Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel‐filtration chromatography into three isoforms, viz., β‐galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS‐PAGE of about 31 and 33 kDa for β‐galactosidases I and III, respectively, and of 67 kDa for β‐galactosidase II. The β‐galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β‐galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β‐Galactosidase I seemed to be an important softening enzyme; its activity increased dramatically (4‐ to 8‐fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β‐Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali‐soluble hemicellulose fraction of the cell wall. The physiological significance of both β‐galactosidase isoforms may partly be attributed to their functional capacity as β‐(1,4)‐galactanases.     

Human plasma immunoreactive beta-lipotropin: correlation with basal and stimulated plasma ACTH concentrations.

A sensitive radioimmunoassay for human β-lipotropin (LPH) has been developed utilizing an N-terminal antibody which exhibits no cross-reactivity with β_h-MSH and appears to be species specific, with less than 10% crossreactivity with rat, ovine or bovine LPH. 0800-0900 mean plasma LPH concentrations were 47.9±5.7 pg/ml (5 normal subects), 100.5±13.2 pg/ml (Cushing's Disease (CD) n=6), 769.3±390.4 pg/ml (Nelson's Syndrome (NS) n=5). Mean plasma ACTH/plasma LPH ratios were: 1.96±0.13 (normal subjects), 1.69±0.11 (CD) and 1.16±0.07 (NS) Plasma ACTH and LPH rose in parallel in response to insulin-induced hypoglycemia in 4 normal subjects. There was a 375% increase in plasma ACTH concentration, a 474% increase in plasma LPH concentration. Plasma ACTH/LPH ratios in specimens obtained following attainment of peak concentrations were significantly lower than those in either control or peak specimens.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Carbohydrate binding specificities of several poly-N-acetyllactosamine-binding lectins

The structural requirements for the interaction of the Asn-linked poly-N-acetyllactosamine-type oligosaccharide moieties of glycoproteins with variousN-acetylglucosamine-binding lectins were investigated by means of affinity chromatography on immobilized lectin-Sepharose columns.High molecular weight glycopeptides containing poly-N-acetyllactosamine-type oligosaccharides obtained by Pronase digestion of human erythrocyte ghosts were treated with 0.1 M trifluoroacetic acid at 100°C for 40 min and then several oligosaccharide fragments were purified with an amino-bonded silica column. Among these oligosaccharide fragments, trisaccharide Gal1-4GlcNAc1-6Galol bound to the wheat germ agglutinin (WGA)- and pokeweed mitogen (PWM)-Sepharose columns, and also showed affinity to theDatura stramonium agglutinin (DSA)-,Lycopersicon esculentum (tomato) agglutinin-andSolanum tuberosum (potato) agglutinin-Sepharose columns. Pentasaccharide Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Galol showed weaker affinity to the WGA- and PWM-Sepharose columns, compared to the trisaccharide. Trisaccharide GlcNAc1-3(GlcNAc1-6)Galol showed weak affinity to the WGA-Sepharose column and did not show any affinity to the other lectin-Sepharose columns. Hexasaccharide Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAcol bound only to the DSA-Sepharose column, indicating that only DSA does not require a GlcNAc(1-6)-linkage for interaction.Abbreviations HPLC high performance liquid chromatography - WGA wheat germ agglutinin - PWM pokeweed mitogen - DSA Datura stramonium agglutinin - LEA Lycopersicon esculentum (tomato) agglutinin - STA Solanum tuberosum (potato) agglutinin - EVA Erythrina variegata agglutinin - PBS 10 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl - Galol galactitol - GlcNAcol N-acetylglucosaminitol     

Electrophoretic analysis of DNA-binding proteins during induction of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase in wild type and mutants of Streptomyces hydrogenans

Abstract Treatment of the wild-type strain HY 0 of Streptomyces hydrogenans with estradiol, a specific inducer of 3β,17β-hydroxysteroid dehydrogenase (17β-HSD) formation, caused several soluble proteins to bind to DNA-cellulose with altered affinity. Hydrocortisone which induces biosynthesis of 3α,20β-hydroxysteroid dehydrogenase (20β-HSD), and progesterone which induces production of both 17β- and 20β-HSD, had no effect on DNA-binding properties of the proteins. In mutants with altered activity/inducibility of 17β- and 20β-HSD only one DNA-binding protein (protein 23) still showed an altered DNA affinity in response to estradiol-treatment and this in only one strain. In other mutants the DNA affinity was not altered during induction with estradiol but the DNA affinity of protein 23 varied between low, low-and-high, and high affinity, depending on the strain. In the mutant where DNA affinity was altered by estradiol treatment the change was opposite to that found in the wild type.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterisation

The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M_r 23 kDa) and 218 amino acids (β subunit, M_r 24 kDa) and the NHase activator of 413 amino acids (M_r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.     

Production and purification of Japanese quail ovalbumin as fusion protein with glutathione S-transferase inEscherichia coli

A plasmid encoding a fusion protein interlinked by thrombin recognition sequence between glutathione S-transferase and Japanese quail ovalbumin (without 40 amino acid residues from the 5′-end of the ORF) has been constructed, employing the expression system pGEX-2T. The deglycosylated fusion protein (64 kDa) was purified by affinity chromatography on glutathione agarose beads, analyzed by SDS-polyacrylamide gel electrophoresis, immunochemically detected with antiserum raised against Japanese quail ovalbumin and tested for its stability.     

The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units mg⁻¹ of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline β-naphtylamide among various amino acid β-naphtylamides. An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.     

Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β-GAD and γ-GAD) with the effector ATP were studied by affinity chromatography. A third form, γk-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM P_i In contrast, a high concentration (140 mM) of P_i by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.