Vol.
68, No.
BIOCHEMICAL
1, 1976
DEMONSTRATION
AND
OF HYDROLASE-RELATED
MEMBRANE
VESICLES K.
Department
GLUCOSE
PREPARED
Ramaswamy,
FROM
P.
GUINEA
Malathi
PIG
and
College
of
of
New
Rutgers
Medical
November
New
K.
COMMUNICATIONS
IN
SMALL
R.
Physiology, Jersey,
RESEARCH
TRANSPORT
of
Piscataway,
Received
BIOPHYSICAL
BRUSH
BORDER
INTESTINE
Crane
Medicine
and
Dentistry
School
08854
Jersey
4,1975
Summary Osmotically active brush border-membrane vesicles were prepared from guinea pig small intestine. Transport into these vesicles of glucose released by the action of the membrane enzymes, sucrase and alkaline phosphatase, was measured. At comparable hydrolytic rates, several fold more glucose was transferred into the intravesicular space from sucrose than from G-l-P, thus confirming the existence of hydrolase-related transport in the brush border membrane.
Introduction vitro
Hydrolase-related
by
finding
dependent brush
to
cell
In
glucose
and
susceptible been
enzymes
a direct
it
sugars
in
and
complex
into
Following vesicles are
preserved
and
used
from
artificial
to
transported
the
The
the
incorporation
been
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
of
intestine
the
be
is
phenomenon of
of
HRT
as
has
glucose free
glycosides (2,3).
viz:
purified
rat, been
HRT mouse,
reported
sucrase-isomaltase
(5).
membranes
(6)
Kaback in
with
which
purified
membrane
transport
from
intestinal
brush
border
studying
events
occurring
in
162
free to
enzymes far,
(1,2). across
available
presented
so
used
from
not
Na+-
various
hydrolysis
distinguished it
to
were of
- in
addition of
membrane
of
identified
in
products
preparations
prepared
a means
the
tested
work
similar
small
border
(4).
first
substrates
easily
species
pioneering
are
must
brush
was uptake
Na+,
black-lipid
bacteria,
provide
is require
several
by
have
of
by
rabbit
on
transfer
not
be
glucose
hamster
does
the
reconstituted
of
of
pathway
to
(HRT)
that
This
hydrolysis
Found pig
be
that
to
guinea to
be
component disaccharides
membrane.
transport
has
when
membrane
HRT appears the
a major
uptake border
transport
properties membranes the
membrane
Vol. 68, No. 1, 1976
exclusive
of
events
contributions
(7).
HRT
in
Along
purified
membranes
BIOCHEMICAL
these membrane
other
lines
we
cellular have
vesicles
organelles
taken
or
steps
prepared
from
to
cytoplasmic
further
guinea
characterize
pig
brush
border
.
Materials
and
Methods
[14C] Sucrose-([U-‘4C] New England Nuclear All other chemicals Adult male Perfection
from
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
14
D-[UCorp. were
C]
glucose,
n-D-[U’4C]-glucose-1-phosphate,
glucose) and D-[1-3H] Mannitol Radioactivity was measured from commercial sources.
guinea pigs Breeders,
(Perfection Hartley, Inc., Douglassville,
were purchased in a Beckman
300-350 Pennsylvania.
g.)
were
from LS counter.
obtained
Purified brush border membrane vesicles were prepared according to Sigrist-Nelson (8) and suspended in a final medium containing --et al. 60 mM mannitol-20 mM KCl-1 mM Hepes-Tris pH 7.5. Uptake of the compounds tested was measured essentially as described previously by Hopfer --et al. fi 1 ters (HAWP 02500) were obtained from the Mi 1 Ii pore (7) * Membrane Corp. Protein was assayed by the method of Lowry et al. (9). R,asults
and
because
in
this
dependent
by
vesicles. as
well
even
in
the
of for
the
Purified this
is, icles
and
equilibration glucose
diffusion
is
the
tissue by
to
be
Na+rate
Na+
to
important
have
Na + by
of
the when
reduction working
is activated
maximal
addition
(4).
and
sucrase
intestinal
possible
animal
preparations
activated
of
test
inactivate on
vitro
out
pig
the
to
effect
--in
absence
of
free
glucose
vesicles
by
from the
dissipation glucose nonspecifically
into
guinea
the
pig
characteristics
Hopfer
Na+-gradient
of diffuses
little
turned
it
Thus,
as
order
are
omission
exhibited
the
in
with
guinea
membrane
imposed
had
complete
described
an
Na+
chosen
by
sucrase
of
activity
relatively
small
k.
laboratory
transport
its
(10).
necessary
concentrations Control
Na+
was
generally
Fortunately, as
of
measured
sucrases
activity
pig
transport
HRT as
, intestinal
sucrase
K’
by
guinea
omission
glucose
uptake
However
with
The
species
free
glucose
of
Discussion
et
al.
enhanced of across
the
for
the
entry
sodium the into
small of
(7)
rat
these
163
intestine
of
membrane.
prepared
glucose vesicles;
glucose with
vesicles,
as
Nat-dependent membrane
gradient
vesicular
vesicles:
into time
that the
ves-
coincided However,
whether
with some
from
rat
in
BIOCHEMICAL
Vol. 68, No. 1, 1976
or
guinea
In
studies
total
pig, with
The that
may
available
always
within
the
the
almost
this
of
have
the
enzyme
turns
alkaline
menbrane
to fold
of
glucose
released
However G-I-F
, this as
Control
for Since
glucose,
it
sucrose
and
and
be
used
the
to
higher
that
G-l-P
as
pig
was
necessary
of brush
for
Measurement
.
With
sucrase
location
and or
at
to
from the
sucrose entry
of
sucrose glucose
and
was
passive
diffusion
action
is
not
would
the
same
rate
to
HRT.
As and
be by
it
can
(11). pig
determine
at
tissue
this
guinea
even
transferred
glucose
brush the
experiments. and
by
With
(G-l-P)
border
substrate
Alkaline 1 mM G-l-P at
10 mM
glucose
phosphatase the
rate
(Table
II).
released
serves
to
from
increase
the
diffusion.
sucrose:
membrane
determine of
is
properties
of
medium
vesicles
nothing
required
means
distinguish
free
by
unhydrolyzed border
to
+
this
to
membrane
from
tissue
suspending
the
favors
a control
the
for
that
in
(2,3).
diffusion.
compared
than
fIRT
control
sucrase
released
diffusion
Na
transport
than
differential
guinea
G-l-P.
the
the
by
activities and
but
which
glucose-l-phosphate
for
active
of
the
sucrase
added
terms
because
1 ittle
measured
is
rate
of
same
has
from
more
compared
reliability
the
the
contributes
were
several
at
not
glucose
enters
absence
enters.
possible
problem
a proper
at
phosphatase
is
the
in
of
which
that
simplest
not
portion
total
extracellularly the
is
the
exceeds
accumulate it
that
released
rapidly
concentrations
that
phosphatase
vesicles
to and
of
which
giucose The
found
course,
always
In
RESEARCH COMMUNICATIONS
of
appears
a serious
released
a membrane
release
be
essence
glucose
out,
vesicles.
in
of
action
in
not
glucose
is,
amount
from
excluded
and
component
HRT
was
entirely case,
sucrase
vesicles,
by
component
glucose
methods
vesicles
preparations
to
by
analytical
into
the
free
expected
will
as
a considerable
a substantial
be
glucose well
is
released and
same
it
sucrose,
glucose
preparations
as
and
AND BIOPHYSICAL
vesicles
whether sucrose
164
they and
G-l-P
are may
‘leaky& be
diffusion
to
leaky
free
also was
done
to
BIOCHEMICAL
Vol. 68, No. 1, 1976
as
described
of
Tris
in
+
.
use
end
of
and
transferred
at
Table
1.
is
a strong
competitive
to
identify
the
Tris+
its
can
help
incubation,
the to
O-5’.
The
the
The
solvent were
calculate
the
measured
due
to
glucose
the
presence
and of
proportions
of
space.
The
to
plates
Table
1.
rest
glucose
and
with
G-l-P
of
the
Uptake
were
Fol lowing
Sucrose
2 min. Sucrose
and
Tr is
used
the
absence the
of
vesicles With
to
less These
portion
Tris+, was
sucrose
than
40%
results of
the
entry
of
to
in and
the
show vesicular the
1 Sucrose with
1 min.
obtained
with
and
(6~2)
acid
data
detectable
and
Time
~1.)
glucose.
in
some no
Incubation
100
from
reversed.
occupy
Glucose
overnight (