Characterization of an [125I]-Insulin binding plasma membrane fraction from rat liver

BIOCHEMICAL

Vol. 41, No. 3, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

125 I -INSULIN BINDING OF AN c I MEMBRANE FRACTION FROM RAT LIVER

PLASMA

CHARACTERIZATION

P.D.R.

House

and M.J.

Weidemann

Department of Biochemistry, School of General Studies, Australian National University, Canberra, Australia. Received September 18, 1970 Summary A procedure for the isolation of three different plasma membrane fractions from rat liver is described. The heavy subfraction (PLl) has a high specific activity in alkaline while the lightest subfraction (PL3) contains a phosphatase, high specific activity in 5' -Nucleotidase, ATPase and phosphodiesterase. The major part of the plasma membrane recovered is found in PL3. A specific binding of p254 -Insulin is shown to occur in PL3, with a ten-fold enhancement over that of any other plasma membrane fraction. The action at

least

one,

suggested

of if

that

results

in

The main

not the

in

the

it

has been

shown that

is

not

the

Any attempted a careful

membrane

fragments.

basis

an enzymatic

capacity

hepatic

three

the

effect

of

the

the

membrane

membrane

are possibly (2,3).

level

of cyclic

insulin

receptor

However,

fractions

transport

site(s) the

tried,

membrane with

about

AMP (4).

of

work we have

(1).

and

brought

on glucose

parameters

541

has been

gluconeogenesis

of plasma

membrane

requires

plasma

of the

of insulin

present

membrane

different

the

characterization

analysis

It

AMP levels

intracellular

In

with

structure

cell

cyclic

preliminary

recognizable in

the

insulin

the

surface

sites.

on glycolysis,

analysis

requires

correlate

in

membrane

receptor

of

intracellular

to

plasma

specific

change

by lowering

of

two,

of insulin

metabolism

related

at the

interaction

a physical

effects

protein

insulin

various on the

fragments, insulin

from

rat

binding liver.

to

Vol. 41, No. 3,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Methods Rat of

the

Male

liver

plasma

methods rats

removed

of Coleman

(200-250g) after

clearance minced

were

pH 7.4 liver

after

pellet

material

of the

following

loml;

homogenate once

was loaded

after

90 mins

onto

(Spinco

washed

and centrifuged

10 mins.

A second

discontinuous

until

out

and the

sucrose

and 5mM to

10 mins

at

for

10 mins,

and the

sucrose

gradients

in

and the

Medium

gradient

collected

1.195-5ml;

1.184-

were centrifuged

SW 25.2)

at

fractions

H at

were

10,OOOxg

similar

fractions

and

The combined

1.21-5ml; These

35Oxg,

for

to the

were

stored

first at -2OOC

assayed. The supernatants

fuged

for

10 mins

mitochondrial 75,000xg layered 5ml;

the

was adjusted

discontinuous

1.30-5ml;

collected,

was carried

for

1,500xg

and 1.157-1Oml.

RPM for

livers

to hand-homogenize in 0.25M

(6).

(radial

rehomogenization.

at

densities:

1.171-10ml

25,000

and the

homogenizer

was spun

were centrifuged

pellet

18 hours

The homogenate

H).

and Kamat

filtration.

was washed

supernatants

for

was used

(Medium

by a modification

(5) and Wallach

up and down strokes)

The filtered the

al

starved

in)

(7-8

were prepared

A Teflon-glass

0.005-0.007

Tris-HCl,

et

stunning.

livers

lOml/g

membranes

lO,OOOxg,

from

1 hour.

The pellet

onto

a Ficoll

gradient

and the

1,500xg pellet

of the

following

SW 25.1)

in Medium

were

the at

medium

H and

densities: were

and the

H and stored

centri-

was designated

up into

The gradients

(Spinco

90 mins.

spin

was centrifuged

was taken

and 1.036-10ml.

were washed

the

The supernatant

for

RPM for

fractions

at

fraction.

1.055-1Oml

25,000

obtained

1.104-

spun at

collected

at -2OOC.

Results In

the

three

plasma

membrane 542

fractions

(Table

1)

(PL1-1.18

Vol. 41, No. 3,197O

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNlCATlONS

TABLE I Enzyme

Activities

of Plasma Subcellular

Membrane Fractions Fractions.

and Other

Homog.

pL1

pL2

pL3

-SER

RER -

Mito --

Sol.

1.48

1.79

11.8

32.6

6.0

1.6

--

--

AlkalinePase (6)

0.07

1.46

0.46

0.15

0.06

0.06

--

--

PDase

0.07

0.18

0.22

0.31

0.15

--

--

0.07

1.64

2.15

14.1

14.5

1.56

1.15

--

--

2.26

3.10

0.91

0

--

--

20.7

--

2.95

0.16

0.76

0.34

--

11.7

--

--

0.60

0.39

2.61

Enzyme 5' -Nase

(6)

(6)

(Na+ + K+)Mg2+ ATPase (3)

SDH (3) G-6-Pase yield

(3) of pro-

189

Results are expressed in Umoles product per mg protein per hour. All results in each fraction agree to within 20% in all cases. Figures in parenthesis represent the number of preparations. The specific activities are an average of the total number of preparations. Homog. - Homogenate; PL - Plasma membrane; SER - Smooth endoplasmic reticulum; RER - Rough endoplasmic reticulum; Mito. - Mitochondria and Sol. - Soluble fraction. The following enzyme assays were used: 5' -Nucleotidase (5' -Nase) (13); Alkaline phosphatase (Alkaline-Pase) (14); Phosphodiesterase (PDase) (14); ATPase (15); Succinate dehydrogenase (SDH) (16) and Glucose6-Phosphatase (G-6-Pase) (17). Liberated Pi was determined by the method of Ames (18), protein by the method of Lowry et al (19).

sucrose density),

density:

PL2-1.17 PDase

5' -Nase,

sucrose

and ATPase

activity

in PL3 and to a lesser

relative

specific

activity

extent,

Ficoll

found

specific

homogenate.

20 when compared

with

more

than

30% of

5'

the -Nase

543

at high

in PL2.

5'

than

total

were

and PL3-1.036

(RSA) of

more

the

density

-Nase

activity

In

fact,

activity

in

This found

in

the PL3 was

represents the

homogen-

Vol. 41, No. 3, 1970

ate.

Fraction

enzymes

mined

BIOCHEMICAL

but

PLl

contained

did

appear

Contamination

of

from

the

(G-6-Pase)

15% mitochondria, contamination

brane

contamination,

On this

that

microsomal

Preliminary

the

of

active

in Alkaline-Pase

membrane

basis than

PL 2'

PLl

the

above

fractions

2.9% in

SER fraction

mentioned (RSA = 22.4).

was deter-

(SDH) and microsomal contained

approximately

5% and PL3 had none.

as evidenced

Microsomal

PL3 and 1.4% in PLl. contained

more

by 5' -Nase

activity,

It

plasma

mem-

than

PL3

contamination. experiments

1

Figure

plasma

little

of mitochondrial

was 6.5% in

be noted

contained

the

PL2 less

should

very

to be very

activities

enzymes.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

aimed

2 3 ng insulin/

at determining

4 mg protein

5

insulin

binding

6

1.

The Insulin binding is expressed as cpm/mg protein. Insulin binding preparations were kept at -200C for 1 week. was determined with a 50~1 aliquot of each fraction in the presence of 0.5ml Krebs-Ringer phosphate buffer, pH 7.4, P251]-insulin (50-loopc/vg incubated at 370C for 10 mins. insulin) was used and the reaction was stopped with 0.5ml The tubes were centrifuged for 5 mins ice-cold 10% TCA. and the free insulin was extracted with acidic n-butanol. The pellet was homogenized and suspended in 15ml scintillLess than 8% of the p253-insulin used ation cocktail. was soluble in 5% TCA and this is accounted for in the results. 544

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VoL.41, No. 3, 1970

parameters to bind

have

shown that

fraction

insulin

than fraction 125 of c II -insulin*bound

the

amount

the

various

than

the

also

increased

brane

fraction, It

membrane

the

was observed for

binding

occurs

SH-group the

in

that This

less at

than

to observe

the

been

at O'C,

binding

that

protein

of the

plasma

(not

shown).

was resistant

has also

is time

a maximal

to

noted from

allowed

mem-

freezing

that

maximal

which

completed

among

greater

binding/mg

content/mg

2.5 mins

37OC the

indicated

was sufficient

It

in

as lo-fold

20-fold

density

binding

capacity

The difference

than

of insulin in the

up to one week.

be expected quickly.

that

1).

and more

a decrease

as did

(Fig.

can be as great

The amount

with

(-20°C)

PL2,

fractions

homogenate.

PL3 has a much higher

it

might

even more for

the

binding

studies

response.

Discussion The results of at least enzymatic tained

show that

three

pointed

out

that

this

By the have

isolated

1.16-1.19 *

(125

it

membrane

genization,

have

light

plasma

Amersham.

and 1.18

(Lot

77)

fragmented

three

(9).

of this

from

be by

density

of

the

1.17). of

initial these

the homo-

fractions.

investigators

densities

compound

and TCA precipitation. 545

for

several

In none

ob-

must

subfractions

origin

with

and

subfractions

a single

methods

fractions

it

(sucrose

following

was purchased

The purity

chromatography

the

differing

membrane

and heavy

composed

results

though

fraction

shown that

a less

use of widely

I - Insulin

Sephadex

his

is

densities similar

technique,

been obtained

suggesting

membrane

(7) has presented

he obtained

has been

plasma

of different

centrifugation

(8) and 1.16

Center,

liver

of an intermediate

paper

plasma

rat

fractions

Evans

a zonal

rehomogenization In

membrane

activities. with

the

these

of 1.13 cases

(5), have

Radiochemical was checked

by

Vol. 41, No. 3, 1970

Table -rat

BIOCHEMICAL

1.

-The effect

adipose

tissue

were

carried

of age on carboxylation mitochondria.

The reaction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mixture

out at 37"

Source

in Figure

1.

Incubations

30 minutes.

Pyruvate

Age

-

carboxylation

-mumoles

Human

of this

boxylation lation

39

54 years

37

6 weeks

1798

6 weeks

1895

10 weeks

350

14 weeks

238

a possible

age of the rat. adipose

effect

was investigated. of pyruvate

tissue

to the aging of pyruvate

markedly

A similar older

phenomenon

of aging

on adipose

As seen in Table

decreases

from

Hl4CO5 fixed/mg

22 years

Rat

view

by human and

-

was as described for

of pyruvate

magnitude animals observed

by human adipose

in adipose

in rat

tissue

mitochondria

with

(14).

tissue (Table

car-

carboxyincreasing

in lipogenesis

observed

adipose

pyruvate

1 mitochondrial

of reduction has been

tissue

tissue

protein

in rat

In contrast

the carboxylation 1) is

not

affected

by age. Acknowledgement: I wish to express my sincerest appreciation to Dr. R.W. -Hanson of Fels Research Institute for his support, encouragement and help in all aspects of this work. I would like to thank Dr. V.H. Auerbach and Dr. A.M. DiGeorge of St. Christopher's Hospital for Children for their support; and Dr. L.I. Goldman and Dr. O.E. Gwen of Temple University School of Medicine for supplying human tissues. This work was supported in part by Grants AM011279, CA-10916, HD-2870, RR-5624 and an institutional grant IN-88 from the American Cancer Society.

553

BIOCHEMICAL

Vol. 41, No. 3, 1970

spite

of

the

transport

readily

demonstrable

adipose

and muscle

is possibly

one of

in

metabolism

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effect

of

tissue, the

insulin

its

on glucose

effect

most

important

suggest

that

on hepatic aspects

of

its

action. These light

observations

subfraction

the

parenchymal

the

action

of

examination ization to reside hepatic fractions various

(PL3)

in

at

problem

least its

from

the

surface

hepatic

is being As this

positive to the

membrane

surface

out

has been

in of

in

liver

the

of for

A further

on the

morphological

resolution

of the

be appropriate

carried

identification

fractions

origin

metabolism.

enzyme

two different

the

sinusoidal

would

affecting

cyclase.

may lead plasma

This

insulin

of adenyl

tissue,

arises

cells.

of this

in

strongly

shown

sites the

source

local(12)

in

present of the

tissue.

References 1. Krahl, M-E., (1961). The Action of Insulin on Cells, Academic Press, New York. 2. Sutherland, E.W., and Robison, G.A., (1969). Diabetes, 18:797. 3. Jefferson, L.S., Exton, J.H., Butcher, R.W., Sutherland, E.W., and Park, C.R., (1968). J. Biol. Chem., 243:1031. 4. Craig, J.W., Rail, T.W., and Larner, J., (1969). Biochim. Biophys. Acta, 177:213. 5. Coleman, R., Michell, R.H., Finean, J.B., and Hawthorne, J.N., (1967). Biochim. Biophys. Acta, 135:573. 6. Wallach, D.F.H., and Kamet, V.B., (1964). Proc. Natl. Acad. Sci., 52:721. 7. Evans, W.H., (1970). Biochem. J., 116:833. 8. Emmelot, P., Box, G.J., Benedetti, E.L., Rumke, P.H., (1964). Biochim. Biophys. Acta, 90:126. 9. Song, C.S., Rubin, W., Rifkine, A.B., and Kappas, A., (1969). J. Cell Biol., 41:124. 10. Graham, J.M., Higgins, J.A., Green, C., (1968). Biochim. Biophys. Acta, 150:303. 11. Mirsky, I.A., (1957). Recent Progs. Hormone Res., 13:429. 12. Reik, L., Petzold, G.L., Higgins, J.A., Greengard, P., and Barrnett, R.J., (1970). Science, 168:382. 13. Ray, T.K., (1970). Biochim. Biophys. Acta, 196:l. 14. Bosman, H.B., Hagopian, A., and Eylar, E.H., (1968). Arch. Biochem. Biophys., 128:51. 15. McKeel, D.W., and Jarett, L., (1970). J. Cell Biol., 44:417. 547

Vol. 41, No. 3,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16. Veegar, C., Der Vartanian, D.V., Zeylamaker, W.P., (1969). Methods of Enzymology, Vol. XIII, p.83. 17. Harper, A.E., (1965). Methods of Enzymatic Analysis, H-U Bergmeyer (Ed)., Academic Press, N.Y. p.788. 18. Ames, B.N., (1966). Methods of Enzymology Vol. VIII, p.115 19. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., (1951). J. Biol. Chem., 193:265.

548