RESUMO
PFK1 and PFK2 coding for the subunits of 6-phosphofructokinase from Saccharomyces cerevisiae were cloned into plasmids suitable for runoff transcription. In vitro translation products of both kinds of subunit were obtained using rabbit reticulocyte lysate as the synthesis and folding system. They were monitored by chemiluminescent Western-blot analysis. Folding and assembly of the alpha-subunit and beta-subunit of 6-phosphofructokinase were found to occur in the cell-free system resulting in an enzymatically active protein. The in vitro generated enzyme exhibits a folding state that is similar to that of the heterooctameric form of 6-phosphofructokinase in the presence of fructose 6-phosphate, ATP and ammonium sulfate, as demonstrated by size-exclusion HPLC followed by ELISA.
Assuntos
Fosfofrutoquinase-1/biossíntese , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sulfato de Amônio/metabolismo , Animais , Western Blotting , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Frutosefosfatos/metabolismo , Medições Luminescentes , Fosfofrutoquinase-1/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Coelhos , Reticulócitos/metabolismo , Transcrição GênicaRESUMO
Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, alpha-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the alpha-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody alpha-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC50 = 1.5 microM) than the peptide A-P-W-H-D-S-F (IC50 = 33.3 microM). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 microM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody alpha-F3.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Proteínas Fúngicas/imunologia , Biblioteca de Peptídeos , Fosfofrutoquinase-1/imunologia , Saccharomyces cerevisiae/imunologia , Bacteriófagos/genética , Ligação Competitiva , Sequência Consenso , Ensaio de Imunoadsorção Enzimática , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Yeast phosphofructokinase is an oligomeric enzyme whose detectable activity in vitro depends on its hetero-octameric structure. Here we provide data demonstrating that an alanine residue at positions 874 (for the PFK1-encoded alpha-subunit) or 868 (for the PFK2-encoded beta-subunit) is crucial to achieve this structure. Thus subunits carrying substitutions by either aspartate or lysine of this residue cause a lack of phosphofructokinase activity in vitro and signals of the subunits are poorly detectable in Western blots. Size-exclusion HPLC in conjunction with ELISA detection of the enzyme protein confirmed that no functional octamer is produced in such mutants. Our data suggest that the mutant subunits, not being assembled, tend to aggregate and subsequently become degraded. Substitution of the alanine by valine in either subunit leads to a reduction in specific activities, as expected from a conservative exchange. The kinetic data of the latter mutant revealed a higher affinity to the substrate fructose 6-phosphate, a lower extent of ATP inhibition and a lower degree of activation by fructose 2,6-bisphosphate. In addition, the affinity of mutants carrying a valine instead of an alanine in either the alpha- or the beta-subunit to fructose 2, 6-bisphosphate was increased. As no X-ray data on eukaryotic phosphofructokinases are available yet, our data provide the first evidence that a non-charge amino acid at position 874 or 868 is essential for the formation of the functional oligomer. This conclusion is substantiated by comparison with the structure of the well-known prokaryotic enzyme.
Assuntos
Mutação , Fosfofrutoquinase-1/química , Saccharomyces cerevisiae/enzimologia , Alanina/genética , Alelos , Sequência de Aminoácidos , Estabilidade Enzimática , Escherichia coli/enzimologia , Genes Fúngicos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutagênese , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/metabolismo , Ligação Proteica , Conformação Proteica , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Valina/genéticaRESUMO
Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, alpha and beta, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption. Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the beta-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed. The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits. Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo.
Assuntos
Fosfofrutoquinase-1/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Deleção de Sequência , Western Blotting , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Reativadores Enzimáticos , Ensaio de Imunoadsorção Enzimática , Genes Fúngicos , Glucose/metabolismo , Peso Molecular , Fosfofrutoquinase-1/química , Fosfofrutoquinase-1/genética , Protoplastos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Lactate dehydrogenase (LDH), an ubiquitous enzyme among vertebrates, invertebrates, plants and microbes was discovered in the early period of enzymology. The enzyme has been dissolved in several distinguishable molecular forms. In mammals, three types of subunits encoded by the genes Ldh-A, Ldh-B and Ldh-C give rise to a selected number of tetrameric isoenzymes. LDH-A4, LDH-B4 and the mixed hybrid forms of the A- and B-subunits are present in many tissues but with certain distribution patterns. LDH-C4 is confined in mammals to testes and sperm. Numerous techniques have been employed to purify, characterize and separate the different forms of the enzyme. This report deals with the main protocols and procedures of purification of LDH and its isoenzymes including chromatographic and electrophoretic methods, partitioning in aqueous two-phase systems and precipitation approaches. In particular, affinity separation techniques based on natural and pseudo-biospecific ligands are described in detail. In addition, basic physico-chemical and kinetic properties of the enzyme from different sources are summarized in a second part, the clinical significance of the determination of LDH in diverse body fluids in respect to the total activity and the isoenzyme distribution in different organs is discussed.
Assuntos
Cromatografia Líquida/métodos , L-Lactato Desidrogenase , Animais , Corantes/química , Corantes/metabolismo , Humanos , L-Lactato Desidrogenase/isolamento & purificação , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/fisiologia , LigantesRESUMO
Aqueous two-phase systems consisting of dextran, polyethylene glycol and dye-liganded polyethylene glycol were employed to investigate the affinity partitioning behaviour of isoenzymes of human alkaline phosphatase. Whereas in the system without a dye ligand the partition coefficients of the isoenzymes from human intestine and placenta were identical, the isoenzyme from human liver showed a significantly lower partition coefficient under the same conditions. After addition of dye-liganded polyethylene glycol two groups of dyes possessing substantial affinities to the isoenzymes were found. One, represented by Procion Yellow HE-3G, interacts specifically with the active centre of the isoenzymes. Differences in the affinity of the isoenzymes towards the individual dye ligands are caused only by the carbohydrate content, especially by the terminal sialic acid residues. The other group of dye ligands, represented by Procion Navy MX-RB, binds obviously in a more complex fashion involving other binding sites, which are only present in alkaline phosphatase of human liver. Procion Navy MX-RB was found to function as a suitable affinity ligand for the separation of human liver alkaline phosphatase from the other isoenzymes. Differences in the primary structure of two allelic forms of human placental alkaline phosphatase [(SS) and (F)] are not recognized in aqueous two-phase systems with or without dye-liganded polyethylene glycol.
Assuntos
Fosfatase Alcalina/metabolismo , Corantes/metabolismo , Isoenzimas/metabolismo , Triazinas/metabolismo , Cromatografia de Afinidade , Humanos , Fígado/enzimologia , Neuraminidase/metabolismo , Análise EspectralRESUMO
Aqueous two-phase systems consisting of dextran and polyethylene glycol (PEG) were used to study the partition behaviour of isoenzymes of lactate dehydrogenase (LDH; E.C. 1.1.1.27) from rabbit tissues in the presence and absence of a series of triazine dyes covalently coupled to PEG. The variations in the primary structures of LDH1(H4) and LDH5(M4) are reflected by significantly different partition coefficients. A class of dyes exhibiting defined structural elements is able to distinguish between both of these isoenzymes. This may be based on differences in the binding affinity to the catalytic site of the enzyme. The difference in the relative affinities of LDH1 and LDH5 to Procion Blue H-5R, as estimated by affinity partitioning, were corroborated by chromatographic experiments. Affinity partitioning in aqueous two-phase systems can be used to predict and to optimize conditions for the fast and simple chromatographic separation of isoenzymes.
Assuntos
Cromatografia de Afinidade/métodos , Corantes , L-Lactato Desidrogenase/isolamento & purificação , Triazinas , Animais , Dextranos , Isoenzimas , Músculos/enzimologia , Miocárdio/enzimologia , Polietilenoglicóis , CoelhosRESUMO
Difference spectroscopy and enzyme kinetics were employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle with the azo-dye Procion Red HE-3B and two of its structural variants in order to follow the significance of the sulphonated terminal rings for the strength and specificity of binding. Procion Red HE-3B possesses a significantly higher affinity to LDH compared to the dye Cibacron Blue F3G-A, a well characterized pseudo-biospecific ligand of dehydrogenases. Moreover, Procion Red HE-3B showed competition towards the cofactor NAD+/NADH. The enzyme-dye complex is mainly stabilized by hydrophobic interactions, but other binding forces cannot be excluded. LDH possesses one dye-binding site per subunit. As a binding region the active center of LDH, preferentially the hydrophobic nicotinamide pocket is involved. Removal of the negatively charged sulphonic acid group from the terminal rings of Procion Red HE-3B decreases the affinity to LDH significantly but does not change the type of binding. Addition of an anilino group to the terminal rings of Procion Red HE-3B does not affect the affinity to the active site significantly but enables the binding on other sites with lower affinity in dependence on the dye concentration.
Assuntos
Corantes/farmacologia , L-Lactato Desidrogenase/metabolismo , Triazinas/farmacologia , Animais , Ligação Competitiva , Isoenzimas , Cinética , L-Lactato Desidrogenase/antagonistas & inibidores , Músculos/enzimologia , Coelhos , Espectrofotometria/métodosRESUMO
Aqueous two-phase partitioning has been elaborated in order to improve the purification of alkaline phosphatase from calf intestine in larger scale. The laborious precipitation and centrifugation steps for the removal of the enzyme from the cell debris and from the bulk protein were replaced by this technique yielding a high recovery (88%) and a significant lower time requirement. For the preparation of 100.000 units (46 mg) of a homogeneous enzyme 2.0 kg of a system containing 200 g PEG 4000 and only 10 g dextran M 70 is necessary. Affinity partitioning in aqueous two-phase systems was used to screen 41 dyes for selecting a suitable ligand for the dye-ligand chromatography of the enzyme. In the case of alkaline phosphatase the results obtained by affinity partitioning coincide with the experimental requirements for the affinity chromatography of the enzyme. Procion Navy HE-R (Blue 171) exhibits a high affinity, selectivity and binding capacity for the enzyme compared with other dyes investigated. The purification procedure provided the same degree in purity (2200 U/mg) and yield (59%) if mucosa or chyme was applied as starting material. In the range of practical use the purified enzyme contains no detectable activities of DNAses (endonucleases) and DNA-nicking activities. The contamination with phosphodiesterase I (EC. 3.1.4.1.) is less than 0.01%.
Assuntos
Fosfatase Alcalina/isolamento & purificação , Cromatografia de Afinidade/métodos , Intestino Delgado/enzimologia , Fosfatase Alcalina/metabolismo , Animais , Butanóis , Bovinos , Cromatografia DEAE-Celulose , Corantes , Ligantes , SolubilidadeRESUMO
Affinity partitioning in aqueous two-phase systems consisting of dextran and dye-liganded polyethylene glycol was employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle (E.C. 1.1.1.27) with Procion Red HE-3B and four structurally related derivatives of this dye in order to follow the significance of the terminal rings of Procion Red HE-3B for the strength of interaction. The study revealed that the arrangement of the two 1-amino-8-naphthol-3,6-disulphonic acid rings seems to be a prerequisite for the interaction of azonaphthol dyes with LDH. The negatively charged sulfonic acid group at the terminal rings of Procion Red HE-3B enhances the affinity of the ligand for LDH significantly. The removal of this sulphonic acid group or splitting off the complete terminal rings decreases the affinity to LDH and improves the competitive effect of NAD+. The results of affinity partitioning are compared with those of affinity chromatography and kinetic data. The usefulness and the choice of parameters of affinity partitioning as an analytical tool to predict the chromatographic behaviour of dye ligands are discussed.
Assuntos
Corantes/metabolismo , L-Lactato Desidrogenase/metabolismo , Triazinas/metabolismo , Animais , Cromatografia de Afinidade , NAD/farmacologia , Ligação Proteica , Coelhos , SolubilidadeRESUMO
The interaction of alkaline phosphatase (EC 3.1.3.1) from calf intestine with different dyes, especially with Procion Red HE-3B was studied by several methods. From the kinetic analysis a nonlinear noncompetitive type of inhibition with an inhibition constant Ki = 0.03 mM for Procion Red HE-3B and Cibacron Blue F3G-A was estimated. The extent of inhibition of the two dyes at constant substrate and inhibitor concentration is 10 to 20 times higher than that of natural inhibitors like L-phenylalanine and NADH. Difference spectroscopic measurements with Procion Red HE-3B showed that the enzyme dimer possesses two binding sites for the dye. The dissociation constant of the dye-enzyme complex was estimated to be Kd = 0.01 mM. The binding of Procion Red HE-3B to the enzyme is mainly stabilized by electrostatic interactions. Large aromatic parts of a dye molecule like a combination of two naphthol ring systems or an anthraquinone ring flanked by spatially arranged charged substituents are important for the extent of specificity. The elution of the enzyme from the immobilized dye and the quenching of the dye-protein difference spectral signal by the competitive inhibitor phosphate and by substrates suggest the involvement of the active center of the enzyme in the dye binding region.
Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Triazinas/farmacologia , Fosfatase Alcalina/isolamento & purificação , Fosfatase Alcalina/metabolismo , Animais , Sítios de Ligação , Bovinos , Cromatografia de Afinidade , Técnicas In Vitro , Cinética , Espectrofotometria , Triazinas/metabolismoRESUMO
The paper deals with a simple and effective procedure for the isolation of calf intestinal alkaline phosphatase (EC 3.1.3.1) with a yield of 35 per cent by employing immobilized Procion Red HE-3B and Cibacron Blue F3G-A, respectively, as dye-ligands. The resulting enzyme is homogeneous and has a specific activity of about 2500 units per mg of protein. Because dye liganded gels are of low costs and can be used several times without loss of binding properties, the presented method is in particular suited for large scale application.