03-09-2012, 11:21 AM
CHARACTERIZATION AND KINETICS OF fl-D-GLUCO/FUCO/GALACTOSIDASE FROM SHEEP LIVER
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Introduction
These three activities are catalyzed by the same
enzyme in two different active sites (see abovementioned
references) in some cases. However, in
barley and in the limpet (Conchie et al., 1967)
fl-D-fucosidase and fl-D-glucosidase activities are associated
in the same enzyme, and both are separated
from fl-D-galactosidase activity, that is catalyzed by
a different enzyme.
/%Galactosidase and fl-glucosidase were classified
in 1961 as independent enzymes by the Commission
on Enzymes of the International Union of Biochemistry
(IUB, 1961). In this first official report on
enzyme nomenclature ~-fucosidase was not present,
and its activity was considered to be a side activity
of fl-galactosidases. However, fl-fucosidase was
classified in 1964 as an independent enzyme (IUB,
1965); it was later deleted in 1972 (IUB, 1973), to be
finally reclassified in 1978 in the corresponding
official Recommendations on Enzyme Nomenclature
(IUB, 1979). Consequently, it seems interesting to
study enzymes showing a well defined fl-D-fucosidase
activity.
MATERIALS AND METHODS
Materials
The livers were removed immediately after killing, perfused
with isotonic saline, and were stored at -20°C before
use.
pNPh-Fucoside*, pNPh-glucoside, pNPh-galactoside,
D-fucose, L-fucose, glucose, galactose, mannose, N-acetylglucosamine,
6-B-gluconolactone, ~,-D-galactonolactone,
lactose, maltose, sucrose, ~-methyl-D-mannoside, bovine
serum albumin, sodium azide and DEAE-cellulose (medium
mesh) were obtained from Sigma Chemical Co., St. Louis,
MO. Sephadex G-200 and Concanavalin A-Sepharose were
purchased from Pharmacia, Uppsala. Ampholines were
from LKB Produkter A.B., Bromma. General laboratory
chemicals were obtained from Probus, Spain. All products
were of analytical grade.
Enzyme preparation
The livers were homogenized with distilled water (1:15,
w/v) in a Sorvall omnimixer blender at top speed for 90 sec.
In ammonium sulphate fractionation the enzyme precipitated
from 30 to 65~o saturation. Sephadex G-200 was
equilibrated and eluted with 50 mM potassium phosphate
buffer, pH 6.1. DEAE-cellulose chromatography was carried
out in 10mM potassium phosphate buffer, pH 6.1; the
enzyme was eluted with a linear gradient (0-500 mM NaC1).
Concanavalin A-Sepharose chromatography was carried
out in 10mM potassium phosphate buffer, pH 5.5; no
enzyme activity was detected by elution with 1 M NaCI and
0.5 M ~t-methyl-D-mannoside. Preparative isoelectric focusing
was performed by the method of Vesterberg (1971) as
previously described (Calvo et al., 1978). The ampholyte
solution, at pH ranging from 4.0 to 5.0, gave high resolution.
The run was performed at 500 V for 1 day.
DISCUSSION
This enzyme shows the highest Vm,x/Km value for
the fl-D-glucoside, and the lowest one for the
fl-D-galactoside. Therefore, it must be considered as
a fl-D-gluco/fucosidase with secondary fl-D-galactosidase
activity. It is not a fl-D-galactosidase with
secondary fl-D-fucosidase activity (Wallenfels and
Malhotra, 1961; Tomino and Meisler, 1975); in these
cases, the fl-D-fucosides are very poor substrates. On
the other hand, typical fl-D-galactosidases do not
significantly hydrolyze fl-D-glucosides (Wallenfels
and Malhotra, 1961; Wallenfels and Weil, 1972).