RESUMEN
Site-directed mutagenesis was utilized to identify binding sites for UDP-galactose in galactosyltransferase (EC 2.4.1.22). Mutant cDNAs were generated by a procedure based on PCR, and the mutated enzymes were expressed in E.coli cells. The mutant enzymes were purified by Ni-NTA Sephadex, and the degree of purification was judged by SDS-PAGE. Purified mutant GTs, F305L, P306V, N307S, N308S, showed dramatic decreases in activities in comparison with the activity of the wild-type GT. Enzyme kinetic analysis revealed that the Km values of F305L, P306V, N307S and N308S for UDP-galactose were, respectively, 9-, 11-, 50- and 20-fold higher than the Km of wild-type GT, but the Km values for manganese were not significantly different from that of the wild-type GT. The quartet mutant F305L/P306V/N307S/N308S showed no activity. From the results of this study it is concluded that amino acids, Phe-305, Pro-306, Asn-307 and Asn-308, in GT are most probably involved in GT catalysis or are located close to the UDP-galactose binding region but are not involved in the binding of manganese.
Asunto(s)
Lactosa Sintasa/metabolismo , Uridina Difosfato Galactosa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Cartilla de ADN , ADN Complementario , Escherichia coli , Cinética , Lactosa Sintasa/química , Lactosa Sintasa/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Membrane-bound 4-beta-galactosyltransferase (lactose synthase; UDP galactose: D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) was purified 1500-fold to near homogeneity from pig thyroid microsomes with about 30% yield. The purified enzyme behaved as a lipophilic protein, rapidly losing activity and aggregating if not supplemented with either Triton X-100 or serum albumin (both of these were equally effective for long-term stabilization). The enzyme preparation showed an absolute requirement for Mn2+, which could not be replaced by other cations. Catalytic properties were very similar to those reported for soluble forms of the enzyme in biological fluids. The purified galactosyltransferase showed a major protein band of approx. 74,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity was eluted at approx. 70,000 daltons. It is concluded that the membrane-bound thyroid galactosyltransferase is a monomeric protein significantly larger than the soluble forms of this enzyme described earlier; but it resembles recently reported galactosyltransferases from sheep mammary Golgi membranes and liver microsomes.
Asunto(s)
Lactosa Sintasa/aislamiento & purificación , Glándula Tiroides/enzimología , Animales , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Membranas Intracelulares/enzimología , Manganeso , Microsomas/enzimología , PorcinosRESUMEN
The galactosyltransferase (Uridine diphosphate-D-galactose: D-glucose 1-galactosyltransferase EC 2.4.1.22) was purified from human colostrum by chromatography on DEAE-cellulose, cellulose phosphate, sephadex G-100, and hydroxylapatite after removal of caseins by centrifugation. The final preparation showed two forms of protein on polyacrylamide disc gel electrophoresis, and both of them exhibited galactosyltransferase activity. The molecular weights of the two forms of the protein were estimated as 44,000 to 45,000 and 55,000 to 57,000 by polyacrylamide disc gel electrophoresis containing sodium dodecyl sulfate. General properties of galactosyltransferase were investigated.