Myelin basic protein: A substance that releases immunoreactive growth hormone in vitro

Vol. 70, No. 2,1976

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MYF‘LIN BASIC PROTEIN: A SUBSTANCE THAT RELEASES IMMUNORF.ACTIVE GROWTH HORMONEIN VITRO Jose A. Villarreal, Wylie Vale, Marvin Brown, Madalyn Butcher, Paul Braxeau, Catherine Rivier, and Roger Burgus The Salk Institute La Jolla, California 92037 Received

March

22,1976

A protein has been isolated from ovine hypothalamus on the basis of its ability to stimulate release of growth hormone by -in vitro cultures of dispersed pituitary cells. This protein has been identified as being myelin basic protein. With no similar biological activity in vivo, myelin basic protein is thus to be recognized as a potentially interfering substance in any search for the physiological growth hormone releasing factor using -in vitro assay systems. Several

types of hypothalamic

immunoreactive vitro

explain

cell

cultures

(3-6).

growth hormone releasing

the observed biological or identified.

have isolated

from ovine hypothalamic

ful

of the release

has no similar basic protein to report tial

in vivo. --

physiologically

(GRF) has been postulated So far,

attempts extract

at characterizing that

in some detail

or contemplated

GRF we is a power-

growth hormone in vitro,

The substance was identified structural

to

such a GRF has not

a substance

of immunoreactive

(MBP), a well-known

in current

factor

to release

and dogs (3) and -in

A hypothalamic,

In recent

these observations

artifact

vitro

activity

(l-2)

activities.

been isolated

stimulant

have been reported

growth hormone --in vivo in rats

in pituitary

meaningful,

extracts

but

as myelin

component of myelin.

We wish

as they show MBP to be a poten-

programs to isolate

GRF using -in

bioassays. MATERIALSAND METHODS

The residue from a previous extraction (7) of 490,000 ovine hypothalami with 90% C2HSOH-CHClB-HOAc(900:100:5) was homogenized with 2N HOAc at 25'C in a l-gallon Waring blender for 30 min. Homogenates were filtered in a Buchner funnel through Whatman No. 1 paper and filtrates, clarified further by centrifugation, were lyophilized. A 15 x 40 cm column of Whatman CM32 was used for ion-exchange chromatography; elution was carried out with a linear gradient of water and lM N&OAc at pH 7. Gel filtration was perCopyright All rights

0 IY76 by Academic Press, Inc. of reproduction in any form reserved.

551

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Vol. 70, No. 2, 1976

Purification

TABLE I.

AND BIOPHYSICAL

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Sequence Fragments

Stage 1.

Lyophilized

2.

hypothalami

Weight

4.9 x lo5

28 kg

90% C2H50H-CHC13-HOAc extraction

4.9 x lo5

25 kg

3.

2N HOAc extraction

4.9 x lo5

2 kg

4.

Ion-exchange

5.

Gel filtration,

chromatography

(CMC)

G-25

4.2 x lo5

275 g

4.1 x lo5

182 g

formed on a 10 x 200 cm column of Sephadex 625 in 0.5M HOAc. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was done according to previously published methods (8). Digestion with trypsin was done in 0.2M triethylamine-bicarbonate buffer, pH 8.2, at 37' for 16 hr with a 20:1 ratio of protein to enzyme. Two dimensional maps were done as previously described (9). Chromatograms were visualized by fluorescence under DV light after dipping in 0.003% fluorescamine in acetone-pyridine (5O:l). Amino acid analyses and dansylation were done as previously described (7). Maleylation was done according to published methods (10). Digestion with pepsin was done in O.lM acetic acid-axnaonium acetate buffer, pH 3, at 25'C for 30 min with a 5OO:l ratio of protein to enzyme. Samples of characterized bovine and porcine MBP and of the tryptophan containing peptide of MBP, residues 113-121, were generous gifts of Dr. Fred Westall (Salk Institute). Release of growth hormone --in vitro was followed with cultures of enzymatically dispersed rat anterior pituitary cells as used routinely in this laboratory (11). Male Sprague Dawley rats (1OOg) housed in temperature and humidity controlled quarters with 14 hours of light and 12 hours of dark were used in the --in vivo bioassay. All animals were fed Purina rat chow and tap water ad libitum. To ensure uniform basal levels of growth hormone, rats were etherized twice at periods ten minutes apart. Administration of test substances was via the external jugular vein. Blood samples were obtained by decapitation five minutes after each injection. Plasma growth hormone levels were determined utilizing a double antibody method with the following reagents: NIAMDD rat GH standard (GH-R&l); NIAMDDmonkey anti-rat GH (GB-Serum-3); highly purified rat GH for iodination obtained from Dr. Standley Ellis, Ames Research Center, Moffett Field, California. RESULTSAND DISCUSSION The sequence of isolation hypothalamus,

of the active

material

shown in Table I, was accomplished

of growth hormone release --in vitro. (Table I) to be nearly

from ovine

by following

We judged the material

pure myelin basic protein

552

the zone from stage 5

on the basis of several

Vol. 70, No. 2, 1976

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AND BIOPHYSICAL

Amino Acid Ratios PepOvine ti e Bovine MBP MBPl Pl !I

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TABLE II.

Pepti e P29

Bovine MBP Resid es Residue l-88 Y 89-169 9

133 10

13.14 9.6

12.8 9.2

6.1 4.4

7.0 2.4

5 8

8 2

18 11

16.2 11.7

17.1 11.3

7.7 5.2

8.5 4.3

10 7

8 4

7 18

7.0 15.9

6.8 15.2

3.4 7.6

2.8 8.8

4 8

Glu Pro

10 12 25 14 3 2

10.5 12.8 25.0

5.1 6.2

GUY Ala Val Met

10.6 13.2 25.0

4.0 6.0 14.8

6 6 10

3 10 4

14.2 2.0 1.7

14.2 1.8 1.8

5.5 0.6 0.9

9 2 1

Ileu Leu5

3 10

2.4 10.0

2.3 10.0

Tyr Phe

4 8

4.0 8.2

4.0 8.0

1.2 5.0 2.1

1.4 5.0 2.0

3.8

3.9

1 5 2 4

Trp

1

LYS His Arg

Asp Thr Ser

1. 2. 3. 4. 5.

11.9 6.8 0.8 0.8

6 15 5 1 1 2 5 2 4

0

1

Material from stage 5 (Table I). Obtained from pepsin digest of ovine MBP. Peptide Pl contained minor impurities as judged by polyacrylamide gel electrophoresis; peptide P2 appeared nearly pure. Ref. 9-10. Experimental values. Ratios normalized to leucine.

criteria.

SDS-polyacrylamide

band corresponding (Table II) In addition,

material

The two dimensional

essentially

of an acetyl

peptide

to those of bovine MBP (9,12). NG,NG-

which are known to occur was detectable

by dansylation

blocked N-terminus

maps of the tryptic

and of bovine MBP were very similar

553

one

The amino acid ratios

to the unusual amino acids,

No free N-terminus

the occurrence

revealed

of 20,000.

and NG-monomethyl-L-arginine,

were present.

stage 5 material

weight

were very similar

peaks corresponding

in agreement with MBP (9).

to a molecular

for this

dimethyl-L-arginine in MRP (9),

gel electrophoresis

digests

in bovine of

but not identical.

Vol. 70, No. 2, 1976

BIOCHEMICAL

TABLE III.

Effect

AND BIOPHYSICAL

of MBP on Growth

Hormone

Dose Wml)

Treatment

MBP

MBP

20

330 2

26

c.05

60

527 A 12

c.01

1730

60

1052

1.

P value for difference determined by Dunnett's

Digestion the

with

active

with

acid

in

of bovine The data

similar cell

ized

(0.1

above

(13),

factor

minimum

times

and somatostatin E2 (PGE2)

mone levels,

an observation

results

in the 1-88

were

ovine,

that

doses

50

c.01

on CMC of

case

II),

of Pl and

and 89-169,

obtained

hormone

-10

respec-

secretion

M) at which factor

and somatostatin, --in vivo

(Table

greater

than

minimally

In the

previously

when either

554

pituitary

are

active

the

well

active

in the

MBP have

from

(TRF),

are IV)

in a significant

we have

and porcine

proteins

releasing

(14).

resulted

bovine,

these

(10

thyrotropin

activity

prostaglandin

negative

+

c.01

Pl and P2 (Table

similar

growth early

(LRF),

500 to 10,000

LRF (13)

fragments,

II)

that

stimulating

the

MBP lacked

umol)

shows

factors,

releasing

thermore,

III

for

hypothalamic

hormone

<.Ol

28

chromatography

of P2 to residues

We recognized

well

ion-exchange

(Table

case

in Table

cultures.

+ 282

MBP (9,12).

capacities

(10m6M)

the

1500

two peptide

compositions

identical

tively,

yielded

<.Ol

between control and other treatments test on log transformed values.

and subsequent

material

amino

nearly

pepsin

31

390 t

200

c.01

f 447

445 f

60

MBP

P1 22

200 Porcine

in vitro.

176 f

200 Bovine

Release

rGH (ngfdish)

Control Ovine

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rat, active

at doses character-

luteinizing (13,14).

Fur-

even

at doses

doses

of TRF

same experiments

in

elevation

of growth

reported our highly

the

(15).

Similarly

purified

material

rat, hor-

or

BIOCHEMICAL

Vol. 70, No. 2, 1976

TABLE IV.

Effect

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of MBP on Release of Growth Hormone in vivo. GH ngfml plasma

Treatment

P1

Control Ovine MBP (1.6 mg)

41.8 5 11.0 35.3 + 4.0

Bovine MBP (1.6 mg) PGEp (10 ug)

30.6 t 2.6 246.6 f 110.6

1.

P Value for difference by Dunnett's test.

characterized

MBP of bovine origin

Grumbach and collaborators

responsible

for its

fied material Maleylation,

exists

activity

in several i.e.,

ways.

and other

San Francisco).

that a small fragment we generated

The trypsin

digest

peptides

followed

by digestion

with

groups at pH 2.4,

was inactive

Fragments Pl and P2 (Table II),

peptide

were also inactive of MBP, residues

peptides, the entire

especially

sibly

responsible

maleylated

--in vitro,

113-121.

of the large peptic

basic nature for its

gave a mixture

fragments

in vitro,

plus arginine)

ones.

Two other highly

thymus histone

and poly-L-lysine,

showed toxicity

555

by pepsin

containing of these

of MBP is pos-

in view of the inactivity

to acidic

as MBP. In addition,

that

in vitro. --

MBP, in which the basic side chains of the lysine

at the same level

and

of peptides generated

by

Pl and P2, suggests that

for activity

(ca. 18% lysine -

activity

residues

trypsin

as was the tryptophan

been converted

tested

--in vitro.

The complete lack of activity

sequence of MBP is necessary

The highly

from our puri-

was inactive

removal of the blocking

digestion,

of MBP may be

of the c-amino groups of the lysine

anhydride,

--in vitro.

treatments

in the dog by Dr. M.

of California,

in vitro, --

blocking

with maleic

were tested

(University

Because the possibility

reaction

between control

c.01

residues

basic substances, effects our material

in vitro --

of have

calf when

appeared to

Vol. 70, No. 2,1976

release

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other pituitary

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hormones (TSH, LH, FSH) in vitro,

time it was realized

that MBP interfered

with

but at the same

the radioimmunoassays

for

these hormones. Of the previously activities,

all

of extracting is toxic levels

reported

have been extracted

in our hands.

(8-300 up) nearly (Table III).

one, called

(4:1:5),

The other

with

exhibiting

in the system n-butanol-

in the same system, its described

by Currie

to MBP on the basis

chromatography

(16).

ently

on Sephadex 625

eluting

as one band centered

aliquots

of this

material

from MBP. The "GRF" reported

by Krulich

material (cutoff

being retarded of 1000).

Using an antiserum Lennon (Salk Institute),

against

dilution

Our

Rechromatography gave a single

of the "GRF" reported

of band

by Sawano

we are unable to distinguish et al. --

It is also soluble retarded

and passing through

in 90% ethanol.

Cur

by both UM 2 and UM 10

in 90% ethanol. bovine MBP, kindly

Dr. M. Dubois (IN&

556

it

(5) does appear to be of

on Sephadex 615

is excluded on Sephadex 615, of lO,OOO), and insoluble

(3) is appar-

the bulk of the active

higher

detailed,

UM 2 (cutoff

on ion exchange

at -ca. 1.8 Vo.

at a lo-fold

of load.

"B-CRRR", does not

by Sawano --et al.

on Sephadex G25,

(3) was not sufficiently

Diaflo

(4)

material

as a function

(41,

behavior

Because the chromatography

size,

et al. --

acid-water

and is adsorbed to CM-Sephadex.

material

small molecular

Our purified

et al. --

The "GRF" reported

behaved anomalously

et al. --

dose of MBP

by Currie

acetic

Rf varying

of its

material

at -ca. 1.1 Vo.

at

(4) excluded on Bio Gel P2, has properties

appear similar

retarded

which

(3-5) were tested

reported

a wide range of Rf values.

The second activity

(3140 pg/ml)

those of MBP. "A-CRRR" appears to be spread (4)

chromatography

behaves similarly

activities

which are capable

equal to or above the minimum active

"A-CEWI", reportedly

on partition

solvents

at a dose level

Of the two activities

which are consistent

growth hormone releasing

with acidic

MBP. One (6) was tested

in vitro --

in vitro --

(3-6) --in vitro

furnished

Nouzilly,

by Dr. V.

France) was un-

BIOCHEMICAL

Vol. 70, No. 2,1976

AND BIOPHYSICAL

able to observe by isnmurohistofluorescence material clearly fibers.

in the perikarya observing

the expected outer

acid extracts isolated

(18) and later

sheath.

In fact,

by another

similarity

basic protein

which must be recognized and bioassay8

In consequence,

closely

the presence of myelinated the location

the presence of MBP in

a protein

activity)

neurons while

of MBP in myelinated

(17) but also reaffirm

regard to biological

recognized

the basis of its Myelin

tissue

is not surprising.

(without

axonal location

not only demonstrate

in hypothalamic

of MBP in the myelin

any evidence of MBP-like

or the axoplasms of hypothalamic

These observations

nerve fibers

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was previously

from porcine

group (19) to be porcine

hypothalami MBP on

to bovine MBP. thus appears to be a powerful

artifact,

by others using methods of chemical purification

related

to those reported

here.

ACKNOWLEDGMENTS We would like to thank L. Chan, L. Clark, C. Maith, A. Nussey, L. Osaki, K. Setbacken, R. Schroeder, and R. Wolbers for excellent technical assistance. We are grateful to our various collaborators mentioned in the text for their generous contributions. We thank Dr. Roger Guillemin for his continuing interest and encouragement during the course of this research. This research was supported by NIH (Grant Nos. AM 16707, AM 18811, and RD 09690). REFERENCES 1. 2. 3.

4. 5. 6. 7. 8. 9. 10.

Szabo, M. and Frohman, L. A. (1975) Endocrinology 96, 955-961. Malacara, J. M., Valverde-R., C., Reichlin, S., and Bollinger, J. (1972) Endocrinology 91, 1189-1198. Sawano, S., Matsumo, T., Yamazaki, M., Baba, Y., and Takahashi, K. (1974) Psychoneuroendocrinology, Workshop Conf. Intern. Sot. Psychoneuroendocrinology (Miekin, ea.), pp. 232-242, Base1 Karger (publ.). Currie, B. L., Johansson, K. N.-G., Grelbrokk, T., and Polkers, K. (1974) Biochem. Biophys. Res. Commun. 60, 605-609. Krulich, L., Illner, P., Fawcett, C. P., Quijada, M., and McCann, S. M. (1972) Growth and Growth Hormone (Pecile, A. and Muller, E. E., eds.) pp. 306-316, Excerpta Medica 263, Amsterdam. Wilber, J. F., Nagel, T., and White, W. F. (1971) Endocrinology 89, 1419-1424. Burgus, R., Ling, N., Butcher, M., and Guillemin, R. (1973) Proc. Nat. Acad. Sci., USA 70, 684-688. Weber, K. and Osbom, M. (1969) J. Biol. Chem. 244, 4406-4412. Eylar, E. Ii., Brostoff, S., Hashim, G., Caccam, J., and Burnett, P. (1971) J. Biol. Chem. 246, 5770-5784. Butler, P. J. G., Harris, J. I., Hartley, B. S., and Leberman, R. (1969) Biochem. J. l&, 679-689.

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15. 16. 17. 18. 19.

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