Subfractionation of the dansylated derivatives of glucosyl galactosyl hydroxylysine by liquid chromatography and its application to a specific alpha-1,2-glucosidase assay.
The dansyl derivative of glucosyl galactosyl hydroxylysine (GGH) was separated into two components, as GP-I (monodansyl GGH) and GP-II (didansyl GGH) by paper chromatography. GP-I was further fractionated into four peaks (a, b, c and d) by reversed-phase liquid chromatography. These peaks corresponded to the dansyl derivatives at the alpha-amino (a and b) and epsilon-amino (c and d) groups of their hydroxylysine residues. There is the possibility that the fractions for b and d are diastereoisomers of a and c, respectively, since the monodansyl derivative from human urine consists of a and c. GP-II was fractionated into two peaks, e and f, which may possibly be diastereoisomers of each other. Treatment of the a, b, c and d fractions with crude chicken liver enzyme resulted in the preferential cleavage of a and b and the production of monodansyl galactosyl hydroxylysine. Components c and d were also cleaved slowly, resulting in the production of monodansyl hydroxylysine by the successive action of beta-galactosidase on dansyl galactosyl hydroxylysine. The detected alpha-glucosidase activity was strongly inhibited by free mannosamine. The method developed using the monodansyl GGH fraction a (or b) and high-performance liquid chromatography facilitated the detection of alpha-1,2-glucosidase, which acts specifically toward GGH even in a crude enzyme preparation.
Iwase
H
H
Ishii
I
I
Kato
Y
Y
Hamazaki
H
H
Hotta
K
K
Umezawa
A
A
Kanzaki
T
T
D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000032:analysis; D006801:Humans; D006901:Hydroxylysine / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification / Q000652:urine; D008099:Liver / Q000201:enzymology; D000520:alpha-Glucosidases / Q000032:analysis*
D003619:Dansyl Compounds; D006901:Hydroxylysine; C001295:glucosylgalactosylhydroxylysine; C042059:glucosidase I; D000520:alpha-Glucosidases