RESUMEN
Removal of the N-glycan from the concanavalin A (Con A) glycoprotein precursor is a key step in its conversion into an active lectin. N-Glycanase (EC 3.5.1.52), the enzyme from jackbean catalysing this process, has been purified to homogeneity as judged by native PAGE. One of the purification steps is binding of the enzymic activity to Con A-Sepharose and its elution by methyl alpha-mannoside. On SDS/PAGE the principal components were found to be 78 kDa, 74 kDa, 54 kDa, 32 kDa and 30 kDa polypeptides. These did not react with Con A on an affinity blot. Cleveland mapping indicated that some of these polypeptides had related primary structures. The enzyme has a broad pH optimum in the region of 5.0.
Asunto(s)
Amidohidrolasas/aislamiento & purificación , Concanavalina A/biosíntesis , Fabaceae/enzimología , Plantas Medicinales , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Cromatografía de Afinidad , Glicoproteínas/química , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Peso Molecular , Mapeo Peptídico , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Lectinas de Plantas , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Unión ProteicaRESUMEN
Post-translational processing of concanavalin A (Con A) is complex, involving deglycosylation, proteolytic cleavage on the carboxy group side of asparagine residues and formation of a peptide bond de novo. This has been studied with the 125I-labelled Con A glycoprotein precursor as a substrate for processing in vitro. Extracts of immature jackbean cotyledons and the commercially available purified preparation of asparaginylendo-peptidase were able to catalyse the above processes. The processing resulted in the conversion of the 33.5 kDa inactive glycoprotein precursor into an active lectin. Processing activity was maximal at approx. pH 5.5. Evidence to support processing at authentic sites was obtained by observation of the release of 125I at positions in the sequence where tyrosine residues were present.
Asunto(s)
Concanavalina A/metabolismo , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Secuencia de Aminoácidos , Cromatografía de Afinidad , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Fabaceae/enzimología , Fabaceae/metabolismo , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Lectinas/biosíntesis , Datos de Secuencia Molecular , Compuestos Organofosforados/metabolismo , Lectinas de Plantas , Proteínas de Plantas/metabolismo , Plantas MedicinalesRESUMEN
Methods for the purification and separation of peptidyl prolyl cis-trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appears as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).
Asunto(s)
Isomerasas de Aminoácido/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Zea mays/enzimología , Isomerasas de Aminoácido/antagonistas & inhibidores , Isomerasas de Aminoácido/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Ciclosporina/farmacología , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Humanos , Inmunoquímica , Cinética , Microsomas/enzimología , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos/química , Isomerasa de Peptidilprolil , Fenilglioxal/farmacología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por SustratoRESUMEN
3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
Asunto(s)
Oxidorreductasas de Alcohol/química , Brassica/enzimología , Ácido Graso Sintasas/química , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Catálisis , Electroforesis en Gel de Poliacrilamida , Ácido Graso Sintasas/metabolismo , Inmunohistoquímica , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Alineación de SecuenciaRESUMEN
We have previously shown that concanavalin A is synthesized as a glycoprotein precursor that is unable to bind to sugars and is processed through six intermediate forms before assembly of the mature active lectin. Since processing involves removal of the N-glycan, four proteolytic steps and a religation, the precise event that leads to carbohydrate binding activity was not known. We have now purified the glycoprotein precursor from microsomal membranes and show that deglycosylation in vitro is sufficient alone to convert the precursor to an active carbohydrate binding protein. This is the first demonstration of a novel role for N-glycans and N-glycanases in the regulation of protein activity.
Asunto(s)
Amidohidrolasas/metabolismo , Concanavalina A/metabolismo , Glicoproteínas/metabolismo , Precursores de Proteínas/metabolismo , Cromatografía de Afinidad/métodos , Concanavalina A/genética , Concanavalina A/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Fabaceae/metabolismo , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Lectinas de Plantas , Plantas Medicinales , Precursores de Proteínas/genética , Precursores de Proteínas/aislamiento & purificación , Procesamiento Proteico-PostraduccionalRESUMEN
cDNA clones encoding the fatty-acid- biosynthetic enzyme NADPH-linked 3-oxoacyl-(acyl carrier protein) (ACP) reductase were isolated from a Brassica napus (rape) developing seed library and from an Arabidopsis thaliana (thale cress) leaf library. The N-terminal end of the coding region shows features typical of a stromal-targeting plastid-transit peptide. The deduced amino acid sequences have 41% and 55% identity respectively with the nodG-gene product of Rhizobium meliloti, one of the host-specific genes that restrict infectivity of this bacterium to a small range of host plants. The probability that the nodG-gene product is a oxoreductase strengthens the hypothesis that some of the host-specific nod-gene products are enzymes which synthesize polyketides that uniquely modify the Rhizobium nodulation signal molecule.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Brassica/genética , Genes Bacterianos , Plantas/genética , Sinorhizobium meliloti/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Brassica/enzimología , Clonación Molecular/métodos , Biblioteca de Genes , Datos de Secuencia Molecular , Mapeo Peptídico , Plantas/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana). The enzyme is inactivated by low ionic strength and low temperature. On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa. Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric. The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH. Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate. It exhibits a broad pH optimum around neutrality. Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH. Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy. It is localized in plastids. N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f. Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp.