Vol. 75, No. 1, 1977

BIOCHEMICAL

AND BIQPHYSICAL RESEARCH COMMUNICATIONS

PROTEIN TURNOVER IN INTESTINAL

MUCOSAL VILLUS

AND CRYPT BRUSH BORDER MEMBRANES David Washington

University

St. Received

January

H. Alpers,

Louis,

M.D.

School

of Medicine

Missouri

63110

21,1977

SUMMARY. Relative turnover rates of intestinal brush border proteins havebeen studied by double labelled technique. Brush borders were isolated from cells at all levels along the villus, and from the crypts. Proteins with large molecular weight (950,000) demonstrated more rapid turnover compared with other brush border proteins at all levels along the villus. This rapid turnover was not seen in crypt brush borders. These findings support the concept of protein turnover in intestinal brush borders, and demonstrate differences between the proteins in rapidly growing crypt cells and non growing villus cells.

INTRODUCTION. membrane

The large

are

of the whole

degraded

and/or

membrane

(1).

can be accounted

for

95% pancreatectomy border the

in

brush

location. from

large

proteins

tease Finally, (lo-15

over

border

those

have

although hours)

action the

of protein

in villus

than

cells

of the

For this access brubk cell

border migration

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

it

lower

It

cells

Crypt

and villus

in the

properties

as the

protein (24-36

the

turnover hours),

anatomical

membranes

clear

presumably along

However,

in their

is not

as well

since

among brush

in the crypt

(3).

that

turnover

(2).

tissue.

of course,

to happen,

to the

rapid

properties,

present

than

turnover

restores

and,

border

proteases,

differential

a homogenous

membranes,

faster

of pancreatic

feeding

not

brush

some of this

biochemical

in cells

rapidly.

should

is

intrinsic

The types

differ

turn

Copyright All rights

mucosa

differ

of their

by the

intestinal

at a rate

At least

and elastase

intestinal

of the

replaced

abolishes

proteins,

cells

proteins

whether

upper

villus

pancreatic entire

pro-

villus.

seems more rapid it

is

possible

130 ISSN

0006291

X

Vol. 75, No. 1, 1977

that

BIOCHEMICAL

migration

from crypt

accounts

for

labelled

technique.

the

lower

turnover

to villus

some of the

issues

we have

teins

in the rat

cell

(>270

intestine

obtained were

with

sacrificed

to allow

method

crypt

were

cells

were

markedly 7.

Thus,

fractions Brush

overnight

3H leucine

(30intestinal

isotopes

were Animals

Mass.).

before into

intestinal

pro-

14C leucine

Both

sacrifice

brush

is

border

mucosa

and adding pooled

crypt.

separation

borders

nine

membranes.

in the rat

This

fractions

of Weiser

(l),

washes

villus,

activity

as previously

of villus

fractions

des-

The collected

almost

Sucrase began

to

Cells

villus,

7 and 8 = upper

removed

propria.

tip

(5).

1 and 2 = upper

procedure

lamina kinase

from villus

wash of 15 minutes.

5 and 6 = lower

and thymidine

(7-10)

using

as follows:

the underlying

the

a tenth

sequentially

by the method

in 10 fractions

9 and 10 = lower from

for

prepared

EDTA washes

3 and 4 = mid villus,

cells

fasted

(Boston,

of label

these

of membrane

the entire

delay

half

in the mouse (4).

collected (5)

The 10 hour

outer

tip.

instances

Corp.

favor

To clarify

of u.1.

3.0 ml).

Nuclear

same in would

even in the

100 pc of 4,5

incorporation

base using

cribed

50 pc

the this

(160 gm) were

(about

has been validated

Intestinal

cells

fluid

at 9 AM.

villus,

to villus

In both

at 11 PM (1).

and confirmed

were

with

from New England

necessary This

rats

were

turnover

from crypt

tip

by the double

occurring.

relative

at 1 PM, and with

lumen was filled

upper

is

the

intrajejunally

50 Ci/mmole)

measured

of turnover

extrusion

investigated

mCi/mmole)

at the villus

on the membrane

MATERIAL AND METHODS. Wistar and injected

turnover

as in the

in situ

where

extrusion

the pattern

membranes

occurring

of the villus

protein

If

villus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to rise (l-6)

all

crypt,

epithelial

activity

fell

by fraction from crypt

was confirmed. were

prepared

from each group

131

of cells

according

Vol. 75, No. 1, 1977

BIOCHEMICAL

to the method borders

of Schmitz

as the

border

fraction

there

was less

pellet

final

(7).

derived

than

animals

was 1 mm thick

and used a tris

phoresis,

gel

blade

were

transferred

(New England England

holder.

Nuclear

Nuclear

37O.

Corp.,

The vials

2000

liquid

were

rejected.

reported

were

Recovery

for

all

control

later.

cells.

along

in the

After

with

wells

(New

overnight

in a Packard than

slice,

a multi-

3% Protosol

twice

was 70 - 80%.

each gel

using

0.4% Omnifluor

and incubated

less

slab

electro-

to four

containing

samples

into

On the other

found

The pattern

the villus,

with

proteins

(left

larger

experiments

simultaneously

The gel

5 mm pieces

and counted

each pooled

1% SDS and 1% 2-

corresponding

1 shows the results

from villus

occurring

for

and 320-

at Model

background

The data

was

and represent

the mean

experiments.

Figure

levels

into

Mass.)

of counts

of cells

electrophoresis.

wells

(8).

covered

chilled

pro-

3H counts

in

system

in toluene

for

by SDS gel

to application.

vials,

All

as 3H/14C ratios

RESULTS. proteins

then

for

from each group

boiled

and cut

Boston,

spectrometer.

of two separate

were

The pieces

Corp.)

and analyzed

successive

buffer

to counting

antL

The microvillus

940-1400

prior

was rinsed

in 1:he brush

250 pg.

to four

3 minutes

brush

the homogenate,

DNA.

separated

Samples

for

razor

with

containing

applied

of the villus.

ple

over

protein

then

protein

were

this

fold

was about

were

pg of membrane

mercaptoethanol

11-13

unfragmented

activity

in 0.2 ml of buffer

proteins

580 l4 C counts

the

Sucrase

of membrane

three

Membrane

using

3% contamination

The yield

fraction

(6),

was increased

from

Fifty

et al.

preparation.

was resuspended

tein

AND BIOPHYSICAL RESEARCV COMM’w’r\“CATIO”\‘S

were the

performed jejunum

hand,

the

in the brush of turnover

the most rapid side

by injecting and sacrificing

proteins

132

border

was similar turnover

of each gel'). both

isotopes

the animals

of the crypt

The

cell

10 hours brush

Vol. 75, No. 1, 1977

BIOCHEMICAL

I 2 3 4 5 6 7 8 9 10 DISTANCE FROM ORIGIN (cm1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

II

I 2 3 4 5 6 DISTANCE FROM

7 8 9 10 1, ORIGIN (cm)

Figure 1. Relative turnover of membrane proteins along the villus. The dot and lines on the right of the graph refer to the mean _+ 1 standard error of the mean for an experiment in which both isotopes, 3H and 14C, were administered to the animal simultaneously. The vertical lines at the bottom of the graph depict the Coomassie blue stain of a fixed and unsliced gel of membrane proteins run simultaneously. These patterns were rather similar at all three levels of the villus. Figure crypt.

border

2. Relative Symbols are

(Figure

2) showed

the variation standard both though crypt were

amino

no differences

acids

origin,

administered

weight

large did

not

of similar

differ

proteins brush

proteins,

size

were

the relatively

133

since

in which

simultaneously. not

borders,

in villus

whatever,

experiment

(MW >150,000),

display

intestinal

from the mean + one

of a control

were

in the 1.

in turnover

not

in the villus

These

in proteins

on right)

molecular

were

present.

cm from the found

(shown

the large as they

of membrane proteins to those in Figure

in 3H/14C rat' 10 did

error

labelled

turnover identical

cells.

Al-

so abundant

large

in

proteins migrating

l-3

rapid

turnover

It

is unlikely,

Vol. 75, No. 1, 1977

however,

BIOCHEMICAL

that

identical,

the

large

since

of large

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

proteins

of crypt

many enzyme activities

size

are

a unique

and villus

cells

corresponding

property

are

to proteins

of the mature

villus

cell

membrane. DISCUSSION.

During

the

of the experiment, into

the

This

cells

lumen whereas

difference

might

those

from

the

lower

turnover

as those

from

villus

would

fasting

to tip

is

takes

24-36

hours,

cell

during

for

the

border

Thus,

cell

found. the

the double

and transit

loss.

one might is not

this

First,

seems unlikely

by this

experiment

and be extruded

difference

Since

it

protein

large

show the same rapid

10 hours,

migration,

the

do not.

villus.

only

migrate

two reasons.

brush

be much affected

decreases

of migration

for

the upper

of the experiment base

to account

villus

labelled

might

in the crypt

seems unlikely

proteins

villus

of the double

on the villus

be thought

explanation

part

10 hours

labelled

time that

the

Second,

assume that so rapid

from lower

since the rate

as measured

in

fed animals. These in the over

data

support

luminal

membranes

is related

to the

suggest

that

villus

these

as well

that

the concept

levels

along

the villus.

level

of brush

border

synthesis The lack

dividing

crypt

actions

proteins This enzymes

of turnover cells

E. Coli

in logarithmic

and the

level

depends

may relate phase

turnover

If

some of this

to cells

The present

along

cells

shows a low

134

maintains (9)

demonstrate at all a constant

despite

the

found

in the

obtained

from

level

of protein

of growth

lower

con-

(3).

of turnover)

to data

of the

occurring

the villus

low rate

the rate

thus

turn-

the data

findings

is a phenomenon mechanism

occurs

proteases,

available

by villus (or

upon

cells.

villus.

of protein

protein

of pancreatic are

as the upper of large

tinued

of villus

proteases

turnover

that

(lo),

bacteria.

with

catabolism, degradation

Vol. 75, No. 1, 1977

BIOCHEMICAL

increasing

as growth

slows.

comparable

to villus

cells,

further. brush

It

is not

borders

itself

is

surface

of the

heterogeneity

would for

crypt

villus

leaving cells

from This

AM07130

work

proteases that

border

proteins

the mucosal

cell

of the membrane

seen in the crypt

during

of crypt

is clear

the double

increases

the

brush

seen border.

labelled

It

technique

cells

the process

to the

on the villus,

of scraping

the

wall.

was supported

from the National

to Ms. Pam Helms for

it

of brush

to use only behind

turnover

of pancreatic

when using

cells

slow

phase,

breakdown

properties

However,

is not

mucosa,

the muscle

the

intrinsic

rates

the stationary

of protein

whether

cell.

cells

the crypt

enter

of availability

seem reasonable, intestinal

the rate

due to the

in turnover

in mature

When cells

certain

or to the lack

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in part Institutes

secretarial

by Grants of Health.

AM 14038

and

I am grateful

assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Alpers, D.H. (1972) J. Clin. Invest. 51, 2621-2630. Alpers, D.H. and Tedesco, F.J. (1975) Biochim. Biophys. Acta 401, 28-40. Alpers, D.H. (1972) J. Clin. Invest. 51, 167-173. Billington, T. and Nayudu, P.R.V. (1976) J. Memb. Biol. 27, 83-100. Weiser, M.M. (1973) J. Biol, Chem. 248, 2536-2541. Schmitz, J., Preises, H., Maestracci, D., Ghosh, B.K., Cerde, (1973) Biochim. Biophys. Acta 323, 9% J.J. and Crane, R.K. 112. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Neville, D.M. (1971) J. Biol. Chem. 246, 6328-6334. Nordstrom, C., Dahlqvist, A., Josefsson, L. (1968) J. Histochem. Cytochem. 15, 713-721. Goldberg, A.L., Howell, E.M., Li, J.B., Martel, S.B., and Prouty, W.F. (1974) Fed. Proc. 33, 1112-1120.

135