RESUMEN
Chinese medicine (CM) is a discipline with its own distinct methodologies and philosophical principles. The main method of treatment in CM is to use herbal prescriptions. Typically, a number of herbs are combined to form a formula and different formulae are prescribed for different patients. Regularities in the mixture of herbs in the prescriptions are important for both clinical treatment and novel patent medicine development. In this study, we analyze CM formula data using latent tree (LT) models. Interesting regularities are discovered. Those regularities are of interest to students of CM as well as pharmaceutical companies that manufacture medicine using Chinese herbs.
Asunto(s)
Química Farmacéutica/normas , Medicamentos Herbarios Chinos/uso terapéutico , Medicina Tradicional China , Medicamentos bajo Prescripción/uso terapéutico , Composición de Medicamentos/normas , Humanos , Modelos BiológicosRESUMEN
O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked beta-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63-342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K(m) value and catalytic efficiency (k(cat)/K(m)) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K(m) value of 0.26 mM and the highest catalytic efficiency of 3.51.10(3). These results reveal that the N-terminal region (a.a. 1-350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.
Asunto(s)
Antígenos de Neoplasias/química , Dominio Catalítico , Histona Acetiltransferasas/química , Hialuronoglucosaminidasa/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biocatálisis , Estabilidad de Enzimas , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N(cyt)-C(peri) topology with 11 transmembrane segments for the STT3 family proteins.
