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 (

Demonstration of hydrolase-related glucose transport in brush border membrane vesicles prepared from guinea pig small intestine.

Vol. 68, No. BIOCHEMICAL 1, 1976 DEMONSTRATION AND OF HYDROLASE-RELATED MEMBRANE VESICLES K. Department GLUCOSE PREPARED Ramaswamy, FROM...
323KB Sizes 0 Downloads 0 Views