Vol.

90,

No.

September

2, 1979 27,

BIOCHEMICAL

AND

RESEARCH

BIOPHYSICAL

COMMUNICATIONS

Pages 425-430

1979

ANALYSIS OF THE NUCLEAR ENVELOPE POLYPEPTIDES BY ISOELECTRIC Keith

FOCUSING AND ELECTROPHORESIS

R. Shelton

and Patsy

M. Egle

Department of Biochemistry and the MCV/VCU Cancer Virginia Commonwealth University, Richmond, Virginia

Received

July

Center, 23298

16,1979

Summary: The more insoluble polypeptides of the avian erythrocyte nuclear envelope have been characterized by a two-dimensional electrophoretic procedure. Most of the polypeptides occur in two classes with isoelectric points of approximately 6.4 and 5.7 respectively. The more acidic class contains two polypeptides, P71 and one which contributes to an electrophoretic band previously identified as P55. The more basic class includes P75, P68, P61 and two or more polypeptides from the P55 band. There are four to six isoelectric point variants of each polypeptide in the more basic class, and the relative stain intensities for the variants are similar for the different polypeptides. These similarities in ionic properties suggest a chemical relationship between the polypeptides. These results are discussed in relation to the in vitro conversion of P75 to polypeptides of the same molecular weight as P68, P61 and P55.

Introduction Electrophoretic weight ing

separation

and isoelectric power.

ficulty

However,

dimensional

preparation

been

characterized

electrophoresis

by these sodium

denaturing

of molecular

methodology

polypeptides

utilizing under

by the properties

a standard

envelope

technique

of great

are dissolved

procedures. dodecyl

with

dif-

We have combined

sulfate

conditions

resolv-

(2)

(1) with

in order

two-

to study

polypeptides. The polypeptides

studies

utilizing

Two polypeptides, thousands), cyte,

has become

the nuclear

and have not

a sample

these

point

of polypeptides

is

of this sodium

P68 and two other when P75 is boiled

dodecyl

have

(3,4).

species minor

been partially

sulfate-polyacrylamide

P75 and P71 (identified

predominate a major

organelle

P68,

abundant

in the same fraction species,

in mild

acid

P61 and P55, (6).

gel

by approximate a less

Peptide

polypeptide liver

apparent

mapping

in

electrophoresis.

molecular

from rat are

characterized

weight in

in

the erythro-

and HeLa cells cleavage

studies,

(5).

products

performed

in

0006-291X/79/180425-06$01.00/0 422

Copyright A ff rights

@ 1979

by Accrdemic Press, (IK. in anyform reserved.

ofreproduction

Vol. 90, No. 2, 1979

sodium

dodecyl

mary

sulfate

sequences

difficulty

on polypeptide

identification.

solution,

AND BIOPHYSICAL

indicate

that

P75,

RESEARCH COMMUNICATIONS

P71 and P61 share

some pri-

(7).

A continuing to rely

BIOCHEMICAL

in these

molecular

The present

studies,

weight

study

however,

as the basis

provides

has been for

both

a new characterization

the necessity

separation

and

of these

polypeptides. Materials

and Methods

The chicken erythrocyte nuclear protein fraction studied in this paper was prepared as follows (6). Cells were harvested from the blood of mature chickens and the nuclei released by nitrogen cavitation. The nuclei were freed of deoxyribonucleohistone by pancreatic DNAse digestion and 1.0 M NaCl extraction. The resulting envelope fraction was washed with Triton X-100 and the DNAse and NaCl treatments were repeated. The final protein pellet was dissolved in 4% sodium dodecyl sulfate, heated for 3-4 minutes in boiling water and dialyzed overnight against 10 mM sodium phosphate (pH 7.2)/0.1% sodium dodecyl sulfate/20 mM sodium azide. This material, the sample protein, was stored at -2O'C and thawed prior to each use. The two-dimensional polyacrylamide electrophoresis system of O'Farrell (2) was used for separating the chicken erythrocyte nuclear fraction for further study. The following modifications were made. Ampholines were obtained from Pharmacia instead of LRB. The cylindrical gels were not prefocused and sample was applied at the anode. The gradient slab gels did not contain glycerol. The proteins in the first dimension were focused at 500 volts, constant voltage for 3 hours. The slab gels were electrophoresed at 60 mA per slab, constant current for 2 hours or until tracking dye reached the end of the slab. Before the protein was focused, it was precipitated from its sodium dodecyl sulfate solution (1) and then redissolved in an 8 M urea solution containing 5% NP-40, 1% BME and Pharmalytes (pH 3-10 or 5-8) in a 1:16 dilution. Briefly, the precipitation step involved combining the sample protein in a 1 to 5 volume ratio with ice cold acetone-NH40H (5.3:0.3). The mixture was stirred vigorously and centrifuged at 12,000 g for 2 minutes in a Sorvall. The supernatant was discarded and the pellet was subjected to a stream of nitrogen to eliminate traces of the acetone-NH40H solution. A small volume (50 pl or 100 ~1) of the 8 M-urea solution mentioned above was used to redissolve the pellet. The resuspended sample protein was centrifuged in a Beckman air-driven centrifuge at a setting of 20 (approx. 70,000-80,000 9) for 10 minutes just previous to focusing. Carbamylated (8) conalbumin served as an internal standard for the twodimensional electrophoresis system of O'Farrell. The conalbumin (2 mg/ml) was boiled in a urea solution at 100°C with aliquots removed at 2 minute intervals over a range of O-18 minutes and then recombined. Results

and Discussion Several

in

procedures

for

this

study.

In the most

dodecyl

sulfate

solution,

sample

solubilization

successful

method,

was precipitated

426

from

and application protein,

dissolved

acetone/ammonia

were in

tried sodium

and redissolv-

Vol. 90, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

f

5.7

6.4 PI-I

Fig. 1. Two-dimensional separation of nuclear envelope polypeptides. a Sample protein (3.6 up) was separated by electrophoresis on a 0.5x 8.5-cm, 52 polyacrylamide gel and the stained bands were scanned spectrophotometrically as previously described (7). The more prominent bands are identified by approximate molecular weights in thousands. b Sample protein (160 pg prepared as described under Materials and Methods) was separated in the horizontal dimension by isoelectric focusing (pH 3-10, Pharmalytes) and in the vertical dimension by sodium dodecyl sulfate electrophoresis in a 5-20% exponential gradient gel. The more basic side of the gel which contained almost no protein is not shown. The origin is indicated by an o. The spots are identified by reference to the peaks in la. The pH values for the principal spots are the averages determined from three onedimensional gels.

ed

in

a small

ticularly

volume

appropriate

previous

studies

electric

focusing. In

focusing

Fig. and

and

then

the

polypeptides

in

the

second traces

only

sodium

dodecyl

sulfate

polypeptides.

the

quickly

1,

contained

of

isoelectric-focusing

because

which

tion

of

buffer. sample concentrated

were dimension of

could

Fig.

protein

lb,

prepared

to

the

in

molecular is

electropherogram In

be

separated by

not is

some

427

desired

one

shown.

was

not

parto

volume

dimension The

by

Fig. enter

our

for

iso-

isoelectric

basic

A typical in

did

method according

weight.

included

protein

This

region

one-dimensional la

for the

identificafirst

di-

Vol. 90, No. 2, 1979

Fig.

2.

BIOCHEMICAL

Comparison

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of P75 and P68 with

partially

carbamylated

Chicken erythrocyte nuclear envelope protein (241 ug) and were prepared as described under Materials and Nethods. horizontal dimension was achieved by isoelectric focusing and in the vertical dimension as described under Fig. 1.

partially

mension tified

carbamylated

and produced by 0).

soluble

a streak

This

protein

conalbumin

in

material

produced

are identified

the second

was decreased three

outstanding

conalbumin.

conalbumin

Separation (pH 5-8, The

spots

(100

ug)

in the Pharnalytes) produced

by

by 5.

dimension

from

when less

sample

was applied.

corresponding

to P75,

spots

the

origin

(idenThe P71

and P68. P71 was the most form

as evidenced

polypeptide ing pared

with

in

P75 is

consistent

were

to contain of

striking.

several

by single

third

spots

semblance

charge from

in

and contained

of multiple

spots.

buffer

dimension. with

compared

with

to yield increments

the basic

the distribution

side

are of spot

approximately

to basic

almost

variants.

In

Fig.

the most intensities

amino

intense. for

acids

point the is

(7).

and appear

multiple

(9))

There

remain-

Several

points

2 the

P75 and P68,

soluble

half

completely.

(p1 = 6.8-7.1

In both

least

of P71 as com-

isoelectric

of isoelectric

428

was the

of acidic

conalbumin

(8).

one isoelectric

acidity

dissolved

a series

It

with

P75 and P68 have similar point

only

The greater

the ratio

to P71,

isoelectric

carbamylated

ing

the first

in contrast

P75 and P68 are

partially

three

isoelectric-focusing

origin

P75 and P68, features

of the

by the absence

in the

at the

acidic

forms

which

had been

variants

differ-

second

and

a marked

the two polypeptides.

re-

Vol. 90, No. 2, 1979

P68 is

located

the loss

in the

of one net

Although

point

polypeptide

Those

polypeptides

erties

of P75,

(X)

polypeptides

tion

in which

0.9 g acetic

acid

significant (10)

in mature

but

cells. 2.3

ticular

are

specific

at pH 3.5

peptide

but

might

for

also

of boiling

blood

contain major

protein

and proceed several

some 68 000-

(Shelton,

K.,

Artifactual

deamida-

out

directly

by experiin 8 -M urea/

do not

incubated groups

with which

of a urea-soluble

within the

these

incorporate

[32P]phosphate do not

turn

fraction

P75 yields

both

429

two classes

in vitro

conversion

weights.

Further,

mild

conditions.

unexpectedly

and 55 OOO-dalton

at pH 11 converts

explanation

acid-Schiff

method

polypeptides

molecular

under

minutes,

re-

over con-

(10).

observed

of corresponding

a likely

periodic

dissolved

phosphate

fraction

acetylation,

has been ruled

is

components

we have

with

These

of the polypeptides because

P71 to polypeptides

shown).

lb).

polypeptides

publication).

are

the

the prop-

of this

is not

step

includes

reflect

hand,

peroxidase

for

when peripheral

pg phosphorousjmg

interest

sions

not

They are

The similarities

radish

lb).

as P71.

deamidation,

either

(Fig.

P75 or P71.

Glycosylation to stain

the same

properties

the P75-like

include

polypeptides

(results

they

either

precipitation

envelope

phosphate

in vitro

taining

the

to

and P61 (Fig.

of the polypeptides

for

submitted

by the acetone/ammonia

ments

most

A-horse

(X)

and solubility

differences

L.,

variants probably

between

resembling

fail

and Higgins,

point

on the other

Possibilities

or by a conconavalin C.,

those

and P61,

and glycosylation.

these

P61 has nearly

electrophoresis,

Thus,

charge

to be determined.

White,

of isoelectric

two classes,

of the

P75 and P68,

has the same ionic

between

phosphorylation

utes

(X)

into

The basis

stain

than

and also

P68 and P61.

can be divided

ing

smaller

by (X)

at

corresponding

charge.

and distribution

indicated

RESEARCH COMMUNICATIONS

from Pi'5 a distance

by one dimensional

The polypeptide

because

AND BIOPHYSICAL

direction

positive

as identified

mains

acidic

considerably

isoelectric P55,

BIOCHEMICAL

primarily material

P75 and PJl

are

of P75 and these

conver-

Upon heat-

a 61 OOO-dalton (6).

of par-

Several

to 55 OOO-dalton

polymin-

polypep-

Vol. 90, No. 2, 1979

tides

(7).

gest

Together

that

ducts

BIOCHEMICAL

with

the smaller

P68 and P61.

the HeLa cell

each class

Additional

evidence

supports

yield

by polypeptide

proof

for

established

increasing.

Such a model

might

an artifact

an integral

On the

part

other

of forming

upon

upon enzymatic

the

indirect

the nuclear

Chicken

heating

in

erythrocyte

(7).

for

this

(6).

Thus,

between

these model

significance,

is re-

or an inconsequential

of the polypeptides

envelope

of

at pH 3.5

digestion

physiological

processing

pro-

interactions

evidence

procedures

sug-

relationship

relationship

be of little

hand,

close

(5).

polypeptide

of our preparative

breakdown.

the

results

be cleavage

polypeptide

a product-precursor

has not been

these

could

crosslinking

a 61 OOO-dalton

polypeptides

vivo

similar

some common fragments

definitive

properties,

within

as revealed

P75 and P61 yield

flecting

of ionic

P75 and P68 have very

P75 and P68 both

although

the analysis

polypeptides

of P75 and P71.

P75,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

might

be

structure.

Acknowledgements This Cancer

study

was supported

Institute,

secretarial

Department

skill

by Grant

No. CA 15923

of Health,

of Judy Watts

Education

awarded

by the National The excellent

and Welfare.

is appreciated.

References 1.

Cabral,

F. and Gottesman,

2.

O'Farrell,

3.

Shelton, Biochem.

4. 5.

R.C. (1976) Biochemistry 15, 5652-5656. Jackson, Cobbs, C.S. and Shelton, K.R. (1978) Arch. Biochem.

6.

Shelton,

K.R.

7.

Cochran, Supramol.

D.L., Yeoman, L.C., Egle, Strut. lo, in press.

8.

Steinberg, R.A., lo, 381-391.

O'Farrell,

9.

Young, Stotz,

E. (1963) pp. l-55,

in Comprehensive Biochemistry Elsevier, New York.

10.

Cobbs,

C.S.

11. 12.

Gerace, L., Krohne, G., Is, 22-38,

P.H.

(1975)

M.M.

(1978)

.I. Biol.

Anal.

Biochem.

Chem. 250,

4007-4021.

K.R., Cobbs, C.S., Povlishock, Biophys. 174, 177-186.

(1978)

Biochem.

and Shelton,

Biophys.

P.H.,

K.R.

J.T.

P.M.

Arch.

548-556.

and Burkat,

R.K.

Biophys.

Commun. 2,

and Shelton,

Friedich,

(1975)

Blum, A. and Blobel, Franke, W., Ely, S.,

Res.

91,

U. and Coffino,

Biochem.

189,

Arch.

323-335.

1333-1338.

K.R.

7, eds.

(1976)

(1979)

P. (1977)

M. Florkin

Biophys.

J.

170,

Cell

and E. 468-475.

G. (1978) J. Cell Biol. 79, 546-566. D'Arcy, A. and Jost, E. (1978) Cytobiologie

430