Fractionation of oyster cilia inhibitor from cystic fibrosis heterozygote serum

Vol.

46,

No.

BIOCHEMICAL

5, 1972

AND

FRACTIONATION FROM

BIOPHYSICAL

OF OYSTER

CYSTIC

FISROSIS

I. R. Schmoyer,

CILIA

J. F. Fischer,

COMMUNICATIONS

INHIBITOR

HETEROZYGOTE

Department State University Fredonia, New

Received February

RESEARCH

SERUM

and S. P. Brooks

of Biology of New York York 14063

7, 1972 SUMMARY

A serum factor found in cystic fibrosis heterozygotes responsible for ciliary asynchrony on gills of the oyster Crassostrea virginica was prepared by ion exchange chromatography and isoelectric focusing. The cationic factor, characterized by an isoelectric point of 8.54, migrated as a single symmetrical band in acrylamide gel electrophoresis. A purified serum factor has considerable potential for detecting heterozygotes and determining the metabolic basis of the disease.

Ciliary upon

asynchrony

exposure

fibrosis

paper

to sera from

(C.F.).

existence

Speck

(I),

of a serum

reports

to heterozygote

serum.

i) investigations

factors

secretions

in many

substance

would

found

exocrine

a causative

fibrosis

This

the possible

may

or may

nature

should

as playing

a role

fibrosis

individuals

number

role in cystic

of exocrine quality.

fibrosis

Press. Inc.

Whether

be unique greatly

of this factor

been suggested

a large

This serum

not

test for heterozygotes.

but also in their

@ 1972, by Academic

parents.

convenient

of cystic

for the

in heterozygote

of this substance causative

(1,2,3) cystic

evidence

and their

which

substance

(4}.

glands

facili-

in the

in the abnormality

of

A humoral

not only

in the

or not this factor

has yet to be determined.

1923 Copyright

victims

tissues

manifesting

have presented

component

preparation

glands

secretions

of individuals

(2) and others

tissue.

of ciliated

of a more

have to affect

of their to play

have

parents

of this

A purified

concerning

in a number

to cystic

on ciliated

and ii) the development Serum

quantity

unique

on the fractionation effect

disease

heterozygotic Bowman

factor

has a distinctive

tate

has been produced

And

is indeed

BIOCHEMICAL

Vol. 46, No. 5, 1972

the existence awaits

of the factor

AND BIOPHYSICAL

in parental

serum

with

RESEARCH COMMUNICATIONS

no obvious

pathological

effects

still

explanation. The existence

developing

a

At present,

large

method

standard

clinical

changes

occuring

clinicafly

factor

to distinguish

scale detection

due to the nature

Prior

of a unique

when

tests

They ciliary

for

depend

when

on the observer’s

ability

serum

was pooled

gill method

individuals

and examined

utilizing

from

several

individuals

with

no history

of cystic

have

no ciliary

effects.

Their

procedure

was also used to assay

heterozygotes.

with active

and coworkers

fibrosis

subtle

from

for

children

ciliary

(2).

was examined

is

to most

to distinguish

from

asynchrony

This

compared

obtained

five

in

population.

is difficult.

to serum

of Bowman

instrumental

the normal

for the disease

are exposed

fibrosis

the oyster

from

prove

are elaborate

heterozygote

cystic

may

individuals

which

systems

to fractionation,

diagnosed

these

of heterozygotes

of the existing analyses.

in heterozygotes

Serum

and shown

all subsequent

to

steps

in the fractionation. The

initial

of Bowman

and coworkers

continuously standard

with buffer

Following with

step in the fractionation

five

one volume

dialysis

was dialyzed examined

0.005

of standard from against

to equilibrium for its effect

to a carboxymethyl

equilibrated

with

of standard

buffer.

buffers:

0.04

standard

M, 0.15

eluted

fractions

against

physiological

both

physiological

(CM) buffer

The

cellulose

at 3O C. The

elution

remaining

was washed

concentrated

and found

The to 3 ml. fraction

to be active

column

(1 .O x 25 cm.)

previously

column

was developed

with

was performed

using

and examined

on oyster

asynchrony

was found

1924

when

2.7 ml. of the preparation

M, and 0.5 M Tris-phosphate

Ciliary

cellulose

C.

and concentrated

of this

saline

at 0’

with

and recentrifuged.

pooled

One-tenth

gilt tissue.

was concentrated saline.

buffer.

was mixed

washed

30 minutes

minutes

were

(9 ml.)

cellulose

g., the DEAE

centrifugations

against

M, 0.25

at pH 7.2 for

for thirty

of the method

serum

(DEAE)

at 12,000 mixed

standard

Stepwise

of heterozygous

M Tris-HCl

buffer,

on oyster

was applied

was a modification

of diethylaminoethyl

of centrifugation

fractions

by vacuum

One volume

volumes

containing

10 minutes

supernatant

(5).

sequence

30 ml.

30 ml. of each of the following at pH 7.5. gill tissue only

upon

Each of the

following application

dialysis of

Vol.

46,Noc5,1972

the 0.04

BIOCHEMICAL

M elutant.

(LKB-Produkter

This fraction AB)

Isoelectric column

(LKB).

of protein

in preparation

focusing

for

was carried

sucrose

as a stabilizing

midway

was applied

followed

by 12 hours

pH was noted

from

absorbance

at 280 mu.

only

the fractions

asynchrony

activity.

one-fourth

the time

these fractions which

(3-7

contained contained

was prepared

The dialyzed

sample

of Lowry

24 hours.

Fifty

against

produced

seen for heterozygous

serum

only

l-2 mg./ml.

as compared

60-70

mg./ml.

increased

to 600 V were

was estimated

found ciliary

min.)

of protein

was of 300 V

physiological

1, Peak II) were

fractions

(6).

2 ml. fractions content

(17-20

from

5.6 mg.

potential

was then

and the protein

8 to 9 (Figure

containing

An initial

at 900 V.

dialysis

series ampholyte

and coworkers

The voltage

vacuum

focused

ampholyte

for a pH range

of focusing

Isoelectrically

carrier

focusing.

of the gradient.

Following

1% light

gradient

for each sample

in the pH range

against

COMMUNICATIONS

8100

matrix.

C for

RESEARCH

in a 110 ml. capacity,

by the method

at 3’

The

dialyzed

out

the preparation

to the column

for 24 hours collected.

through

BIOPHYSICAL

isoelectric

stepwise

in 1 ml., as determined

applied

serum

was then

A discontinuous,

3 to 10 using

AND

saline

to have ciliary asynchrony

min.).

in

Moreover,

to active

heterozygous

of protein.

II IO 9 e 7: 6 s 4 3

Tube

Numbrr

Figure 1. Isoelectric focusing at pH 3-10 of the CM cellulose and broken lines represent absorbance and pH, respectively.

Acrylamide

gel electrophoresis

and all of the active Davis

(8).

Two

fractions

hundred

using

micrograms

at pH 8.9 was performed a modification of heterozygous

1925

active

Solid

on heterozygous

of the procedures serum

fraction.

of Clark

and lesser amounts

serum (7) and of

Vol.46,No.5,

1972

subsequent

fractions

to migrate tion

for

resulted

active

BIOCHEMICAL

were

applied

25 minutes

geneous

profile

focusing

on a shallow

active

peaks

units,

migrated

a single

of heterozygous

(9).

elution

in the number

displayed

symmetrical serum

peaks,

to identical

RESEARCH

with

bands. This

2).

However,

resulted

The

in acrylamide

isoelectrically

is contrasted we found

in isoionic

and allowed

Each step in the fractiona-

in the resolution

differences

positions

dye.

band.

(Figure

COMMUNICATIONS

gels, respectively,

of the tracker of observed

7-9 pH gradient

These

BIOPHYSICAL

to 7 and 5% acrylamide

following

in a decrease

fraction

AND

with

the hetero-

that

isoelectric

of at least

points

focused

two

of less then

separate 0.3 pH

gels.

: I-I b

a

Figure 2. Absorption profiles at 540 mu of amido shwarz stained acrylamide electrophoresis gels of a) heterozygous serum (5% gel) and b) active fraction pH 3-10 isoelectric focusing column (7% gel).

The resolution focusing after

is interesting

elution

of these

in light

of homozygous

of 60,000

of a number

This work

of Speck’s

weight (1).

New

York

University

of New

York

(GR

of active

finding.

He discovered

a Sephadex

of at least

200,000

Therefore,

of larger

a number

from

molecular

was supported

of Western

into

serum

had a molecular

on the order subunit

of the factor

(024-7137A).

G-200

with two

isoelectric active

gel filtration

and the smaller

the possibility

exists

that

fractions column.

a molecular the factor

One weight

may

be a

complexes.

by research

36FRE71)

peaks

disc from the

grants

and The

from

Research

J. F. Fischer

1926

the United Foundation

was supported

Health

Foundations

of The State in part

by a United

Vol.

46, No.

5, 1972

Health

Foundation

his valuable for their

BIOCHEMICAL

Fellowship

assistance technical

in obtaining

AND

BIOPHYSICAL

(PFT-64-Fre-71). blood

The authors

specimens

and Diane

RESEARCH

thank Pennica

COMMUNICATIONS

Dr. J. Forbes and

Karen

for 0:Keefe

assistance.

References 1. 2. 3. 4.

5. 6. 7. 8. 9.

A. Speck, H.M.C. Heick, H. Cress, W.S. Logan, Pediat. Res. 3 173 (1967). B.H. Bowman, L.H. Lockhart, M.L. McCombs, sew, 325 (1969). G.T.N. Besley. A.D. Patrick, A.P. Norman,& Med. Genet. 4 278 (1969). A. Speck in Cystic Fibrosis and Related HumaGdzal Diseases: Symposium Highlights, S. Jakowska, Conference Ed., (Gordon and Breach, New York, 1970), p. 70. B.H. Bowman, M.L. McCombs, L.H. Lockhart, Science 167,871 (1970). O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 -m---1 265 11951). J.T. Clarke 8&Ann N-‘-‘-‘-‘-* Y Acad SC 121 428 (1964). B.J. Davis, --8 ibid. 121 428 (1964). I.R. Schmoyer, S.P. Brooks, J.F. Fischer, in preparation.

1927