RESUMEN
In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the KM and VMAX values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze-thaw cycles, and salts on the activity of the cytosolic enzymes. We also present data on the protein-to-lipid ratio and lipid composition dependence of the antiporter.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Sistemas de Transporte de Aminoácidos/metabolismo , Antiportadores/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/metabolismo , Hidrolasas/metabolismo , Lactococcus lactis/enzimología , Ornitina Carbamoiltransferasa/metabolismo , Fosfotransferasas (aceptor de Grupo Carboxilo)/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Amoníaco/metabolismo , Antiportadores/genética , Proteínas Bacterianas/genética , Dióxido de Carbono/metabolismo , Metabolismo Energético/genética , Regulación Bacteriana de la Expresión Génica , Hidrolasas/genética , Cinética , Lactococcus lactis/genética , Liposomas/química , Liposomas/metabolismo , Ornitina/metabolismo , Ornitina Carbamoiltransferasa/genética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The N-linked glycoprofile of bovine whey is the combined result of individual protein glycoprofiles. In this work, we provide in-depth structural information on the glycan structures of known whey glycoproteins, namely, lactoferrin, lactoperoxidase, α-lactalbumin, immunoglobulin-G (IgG), and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1, PP3). The majority (â¼95%) of N-glycans present in the overall whey glycoprofile were attributed to three proteins: lactoferrin, IgG, and GlyCAM-1. We identified specific signature glycans for these main proteins; lactoferrin contributes oligomannose-type glycans, while IgG carries fucosylated di-antennary glycans with Gal-ß(1,4)-GlcNAc (LacNAc) motifs. GlyCAM-1 is the sole whey glycoprotein carrying tri- and tetra-antennary structures, with a high degree of fucosylation and sialylation. Signature glycans can be used to recognize individual proteins in the overall whey glycoprofile as well as for protein concentration estimations. Application of the whey glycoprofile analysis to colostrum samples revealed dynamic protein concentration changes for IgG, lactoferrin, and GlyCAM-1 over time.