Isolation and properties of the FSH and LH-releasing hormone

Vol.43,No.2,1971

BIOCHEMICAL

AND

BIOPHYSICALRESEARCH

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ISOLATION AN0 PROPERTIES OF THE FSH ANI LH-RELEASING HORMONE*

Schally, A. Arimura, Y. Gaba, R.H.G. Nair, H. Matsuo, T.W. Redding, L. Oebeljuk

A.V.

V.A.

Hospital and Tulane University

School of Medicine,

New Orleans,

La.

and W.F. White

Scientific Division, Abbott Laboratories,

Received

March

Chicago, Illinois.

North

15, 1971

Suamarv. LH-releasing hormone (LH-RH) was obtained in apparently a homogeneous state froa extracts of pig hypothalami. The LH-RH preparation isolated has FSH-releasing hormone (FSH-RH) activity, which appears to be intrinsic to LH-RH. The amino acid composition of LH-RH/FSH-RH as determined on acid hydrolysates is: His 1, Arg 1, Ser 1, Glu 1, Pro 1, Gly 2, Leu 1 and Tyr 1. The hormone isolated stimulates the release of FSH and LH in vivo and --in vitro in doses of a few nanograms. This polypeptide appears to represent the hypothalamic hK=uhich controls the secretion of both LH and FSH from the pituitary. Hypothalamus

excercises

hormone (FSH) from the

over the secretion

anterior pituitary gland (1).

of

partial purification

of porcine

of the LH-RH and FSH-RH (4). polypeptide

This report

LH-RH (3). describes

which has both LH-RH and FSH-RH

luteinizing

This control

LH-releasing hormone (LH-RH) and FSH-releasing

designated cribed

control

neurohumoral substance (s)

is mediated by

hormone (FSH-RH)

(2).

We have

Recently, evidence was presented the

and follicle-stimulating

hormone (LH)

previously des-

for the peptide nature

isolation from porcine hypothalamic

activity, its amino acid composition

extracts

of a

chemical and biological

and

properties. The

isolation of LH-RH was acconplished

essentially in 12 steps.

Lyophilized

fragments

hypothalami (Oscar Mayer Co., Madison, Wis.), weighing 2500 grams uere first pulverized, extracted

with 2 N acetic

acid (3,

5).

The

glacial acetic acid (3, 5) and lyophilized. filtration (Fig.

on Sephadex G-25 column (15.5 1, ref.

5) using

1M

X 180 cm) in batches of 45

Plasma LH

levels were determined

in part by USPHS grant

Supported

in effluents

with estrogen

bioassay (a), or by radioimmunoassay

vitro by measuring the stimulation

*

and then with

to 70 g as described previously

The LH-RH activity

elevation of plasma LH in ovariectomized rats pretreated

in

pig

Glacial acetic acid extracts (665 g) were subjected to gel

following

was detected

defatted

lyophilized extract (yield 1075 g) was re-extracted

acetic acid as eluant.

by

of 165,000

AR-07467,

of FSH

and progesterone

for rat LH (7).

release from rat pituitaries

AM-09094 and Population

393

was measured by

Council,

New York,

FSH-RH

(8).

(2-4).

activity

FSHreleased

New York.

Vol.

43,

into

No.

2, 1971

BIOCHEMICAL

incubation medium uas determined

FSH (10). readings d-MSH

The separation at 278 ml. and lysine

pattern

Fractions

concentrated of

by

was followed 913-1082

the release

in 54lO-6OOpg amounts.

RESEARCH

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containing LH-RH and FSH-RH activity emerged between

5).

Lyophilization

of combined active

in vivo in doses of 125-500,ug.

The LH-RH and FSH-RH

extraction with 2400 ml phenol (3, 5).

After

areas

The same material

This

material in 5-8 g batches was subjected

4

6

12

16

20

24 lute

to

26

yielded 380 g had FSH-RH

activity in this material was desalted recovery

of the phenol

ion exchange chromatography

32

36

40

44

and

soluble materials, 83 g

extract were obtained which stimulated the release of LH and FSH in doses of 30-15Opg

pectively.

for rat

the Folin-Lowry reaction (11) or by optical density

by

from Sephadex

of LH

BIOPHYSICAL

Steelman-Pohley assay (9) and by a radioimmunoassay

by the

vasopressin (See Fig. 1, ref.

material which stimulated activity --in vitro

AND

and IOOrg

res-

on carboxymethyl

46

number

Figure 1: buffer,

Free flou electrophoresis (FFE) of 277 rag of porcine LH-RH from MC in 0.37 I pyridine acetate pH 6.3. Conditions: 1900 volts, 160 ma, 6' C, 9 hours. Peptide analyses (11) were carried out

on 2511 aliquots.

,

----

Folin c&w Pmaraatirity

p LVP K.076 ma -6ocl 9

$E0.2-

-400

F: 3 O.le

-200

I

L

p E 8 a %

40

80

I201602cO240260320560400440460 Cell number

Figure 2: Countercurrent distribution (CCO) of 94.8 mg of LH-RH from FFE in a systemof 0.1% acetic acid: 1-butanol:pyridine - 11:5:3 (v/v). Louer phase was 3 ml and upper phase 5 ml. The number of transfers 500. Peptide (11) analyses were carried out on 50~1 aliquots lower phase.

394

was

Vol. 43, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

400-

---

Plasrm LH

-40

-

Dry weight/tube

-30

$

-20

2 3

-10

20

40

60 80 loo Fraction number

Ix)

140

3: Partition chromatography I of 8.9 mg of LH-RH from CC0 on a column of Sephadex G-25, 0.9 X 158 CR. The solvent systen consisted of upper phase of Vbntanol:acetic acid:water:benzene=4:1:5:0.33. Fraction size 5 ml; hold-up volume 24.5 ~1. Flow rate 3 ml per hour; T - 68 2 20 F. Figure

cellulose (CNC) columns, as described

(3, 5). Tests on aliquots shoved that LH-RH and FSH-RH

previously

activity was eluted by application of a gradient

conductivity of 6-8 mNHOS (see Fig. 2, ref.

No. 150-190 with colunns

were combined and

3)

doses of

lyophilized.

O.lpg

and 0.5

separator.

When fractions

277 mg of material uas obtained,

purified

obtained Of

of separation

LH-RH (K-2)

by countercurrent from traces

which had detectable

is seen in Figure

distribution

of lysine

LH-RH

(CCO)

vasopressin.

1.

The results

electw

of assays indicated

This material was subjected

3. Lyophilization of fractions FSH-RH

activity had increased.

plates

in

was continued

to partition

No. 45-71 yielded

(94.8

mg)

uas now sub-

in Figure 2. This procedure of the

active area, 14.2 ng were

chromatography

3.6 rg

area (12).

Houever,

395

physiological

with 8.9 mg, equivalent

(12)

clinical

to about 100,OGO

on Sephadex G-25, as shown in Figure

material and assays showed that its LIT-RH and

It showed only one spot on thin layer

to the LH-RH active

This naterial

were used for various

the systemof 1-butanol:acetic acid:water - 4:1:5 (12).

dine corresponded

the recovery

g.

that

activity in doses of 2-25 ng and FSH-RH activity in lo-30 ng amounts.

veterinary (14) studies. The purification

hypothalami.

Y

as indicated

After

this aaterial 5.3 mg, equivalent to 65,000 hypothalami,

(13) and

active in releasing LH and FSH in

flow electrophoresis (FFE) in Brinkman continuous

by free

stimulated LH release at levels of M ng and FSH release in doses of 0.3

separated

3

pg respectively.

The pattern

to 500 transfers

CRC

No. 210-270 (see Fig.

migrated toward the cathode and was found in tubes ho. 27-34. The material obtained

the activity

jetted

from thirteen

The material (948 mg) was divided into 4 batches, which were re-

were combined and lyophilized,

This material was further phoretic

5). The active fractions

on an analytical column of CK (1 X 60 cm) as in (3).

chronatographed in ref.

to pH 7, 0.1 fl ammoniumacetate and emerged in fractions

chromatography

This spot revealed

the amino acid ratios

on cellulose uith

indicated

coated

chlorine/o-tolithat

the material

Vol.

43,

No.

2, 1971

BIOCHEMICAL

BIOPHYSICAL

-- -- LH-RH oelivity,plntma Dry wright

8 ‘2 1 k 2 x .P y 150c * 4

AND

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LH Iw*I

-80

50- ,-,;-,/-

-..* ,’

,;

0

20

40

60

80

loo

120

140

FractionNumber Figure 4: Partition chmwatography G-25, 0.9 X 150 cm. The solvent Pyridine:Benzene-25:7:30:2:1:10. T = 70 + 2O F.

was still

not homogeneous.

vent systemas illustrated

The material was further in Figure

The final purification aliquots of fractions After

lyophilization

the recovery product

of 3.6 ng of LH-RH from partition chromatography I on a column of Sephadex system consisted of upper phase of I-butanol:ethanol:water:acetic acid: Fraction size 5 al; hold-up volume 24.5 al. flow rate 5 al per hour;

of material

tional methods for pmof of purity

Tyr 1 (Table

of zone electrophoresis

activity migrated strongly was obtained

at each step was essentially complete,

appeared to be homogeneous,

hydrolysis

LH-AH

weight of active fractions

The

step consisted

showed that 83Opg

4.

repurified by partition chmnatography

but due

as show in Figure 5. Assays on

toward

the

cathode under these conditions.

both LH-RH and FSH-RR

the purification

was over 2 million

to the very limited amounts of material The amino acid

were inapplicable.

sol-

uas reduced to 2.3 wg.

(15)

which contained

in another

activity.

fold.

Since

The

available manyconven-

composition, determined after

with 6 N HCl (22 hrs, 110' C) was: His 1, Arg 1, Ser 1, I& 1, Pm 1, Uy 2, Leu 1 and I).

8

---I,4 ;:

-

LH-RH ochvily ploomo LH Id Dry weight

I ! : : ] i :, I i :

$$ Y

0

Y !G t P 140

.p

100

i

P @J .: 20 0

E

+i

-20

0

IO

20304050

Fraction Number

Fiaure 5:

High voltage electmphoresis of ‘2.3 mg LH-RH in 0.18 M pyridine acetate buffer, pH 6.3. Vertical coluan 0.9 X 91.6 cm, with external cooling at 5o C, packed uith cellulose pouder (15). After the electrophoretic separation at 2570 V; 20 MA for 18 hrs, the column was eluted with buffer and 1.3 ml fractions were collected.

396

Vol.

43, No.

BIOCHEMICAL

2, 1971

AND

BIOPHYSICAL

Table

RESEARCH

COMMUNICATIONS

I

AMINO AC10 COMPOSITION OF PORCINE LH-RH/FSH-RH

mumoles/lOrug material*

l

Amino Acid Ratio”

Integral

Nearest

Histidine

2.351

1.10

1

Arginine

2.459

1.15

1

Swine

1.773

0.83

1

Glutamic Acid

1.987

0.93

1

Proline

2.708

1.27

1

Glycine

4.271

2.00

2

Leucine

2.234

1.05

1

Tyrosine

1.563

0.73

1

Total Amino Acid Content - 30.5%. The

view that the polypeptide

gical activity

A and a).

moiety,

which probably

of LH-RH and FSH-RH is supported

and FSH-RH activities

subtilisin,

w Gly - 2.0.

were

(G-200 preparation,

a blocked supplied

N-terminus.

by Dr. R.

(leucine

Doolittle)

acids, is essential for bioloenzymes (4).

with endopeptidases

aminopeptidase,

and failure

Inactivation

of 9 amino

with proteolytic

simultaneously abolished by incubation

inactivation by Edman procedure

Oansyl method indicate

consists

by experiments

thermolysin) but not by exopeptidases Lack of

9 residues

to detect

by pyrrolidone

suggests that

(chymotrypsin,

aninopeptidase any

Both LH-RH papain,

M, carboxypeptidase

N-terminal amino acid by

carboxylyl

peptidase

(16)

the N-tetminus may be occuppied by pyroglu-

Table II EFFECT OF LH-RH ON PLASMA LH LEVELS IN OVARIECTOMIZEO RATS PRETREATEOWITH ESTROGENAND PROGESTERONE

Sample

dose rig/rat

Plasma LH (RIA)

ng/ml*L

Saline

----

7.6

+ 0.6

mm-m

LH-RH

0.1

9.2

+ 0.6

NS

LH-RH

0.25

11.0

+ 0.6

0.01

LH-RH

0.625

13.8

+

2.0

0.05

LH-RH

1.56

26.0

+

2.2

0.001

LH-RH

3.89

60.3

+

7.2

0.001

* In terms of NIH-LH-S-14.

397

S.E.

P Value

Vol. 43, No. 2, 1971

tamic acid (17). similar

BIOCHEMICAL

massspectral fragmentation

The

peaks at 83, 84, 111, and 112 mass units,

Inactivation with N-bromosuccininide

As suggested

as described

illustrated

progesterone.

increased

Table III

LH and FSH

in vitro

The highest

castrated

rabbits

the

acid (4) suggests

LH-RH, exhibit

that

tyrosine

appears to be intrinsic

Partition

to LH-RH.

FSH-RH activity

from LH-RH.

The material

(9) and

with estrogen

in incubation

in FSH and LH activity 10 and 45 min after

LH-RH also increased

intracarotid

both LH-RH and FSH-RH

activity and it represents'the

of both The stimulation

of FSH as well

that the nonapeptide hypothalamic

Table IV shows

of LH-RH/FSH-RH

that in humans administration as LH (13).

indicates lack of species specificity

reported

medium.

injection

It is of interest

the levels

mamnals. The results

here indicate

radioimmunoassays(7, 10) of media.

and

log dose of LH-RH/FSH-RH over the range 1.5 - 13.5 ng/rl.

with testosterone.

This

Results

administration of doses as small as 0.25 - 0.625 ng

to the

is a 4 fold increase

also respond to LH-RH (12, 14);

and/or

(4).

levels in ovariectonized rats pretreated

related

N-terminus.

release of both LH and FSH --in vivo as well as -d in vitro

levels were increased

porcine

and trifluoroacetylated

doses as small as 0.5 ng LH-RH/ml augmented the release

male rats pretreated

highly purified

activities

by bioassays

was linearly

that plasma LH and FSH

of

shows that

dose resulted

due to the presence of a pyroglutamyl

systems did not separate

plasma LH

as ascertained

of FSH and LH release

probably sulfanilic

II indicate that intravenous

significantly

LH-RH

solvent

above stimulated

in Table

of free

(4, 18) the FSH-RH activity

in 10 different

chromatography isolated

previously

patterns

and diazotized

histidine are necessary for both biological

into

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

isolated

Monkeys,

sheep and

to LH-RH/FSH-RH in

from porcine

hormone which controls

hypothalami

has

the secretion

both LH and FSH from the pituitary.

Table

III

STIMULATION BY LH-RH/FSH-RH OF FSH AND LH RELEASE -m IN VITRO FROH PITUITARIES OF HALE RATS FSH CONTENT OF MEDIUM

Steelman - Pohlev Assav Sample

Dose rig/ml*

Ovarian

P Value

Wt mg + S.E.

FSH**pg/ml

LH CONTENT RIA LH ng*/ml

Control

---

71.4

+

4.2

----

21

238

LH-AH

0.5

94.9

L 4.9

.Ol

28

362

LH-AH

1.5

101.6

+

6.4

.005

32

460

LH-RH

4.5

143.2

+

20.0

.Ol

53

628

LH-RH

13.5

169.6

+

10.0

.OOl

II

978

l

Total

volume 10 ml.

*

Expressed

as NIH-FSH-S-4.

398

+ As NIH-LH-S-W.

of

BIOCHEMICAL

Vol. 43, No. 2, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table EFFECT OF LH-RH/FSH-RH

IV

ON PLASMA LH AND FSH LEVELS

IN CASTRATED MALE RATS TREATED WITH TESTOSTERONE

PLASMA FSH ACTIVITY Steelnan - Pohley Assay Sample

Dose

rig/rat

Ovarian

+

mg

FSH*

95%limits

LH-RH

1

mm--

mm-m

12

l

4.5

LH-RH

5

----

m-w-

42

+

4.0

LH-RH

25

66

L

5.9

As NIH-FSH-S-8,

45 ain

after

injection.

IO ( 6.5

with

----

134.y

6

pg/nl

Saline

+

89.4

wt.

PLASMA LH ACTIVITY RIA LH rig/ml+

+ 10.9

- 15.3)

19 (12.1 w

P - 0.05

vs

0

- 28.9)

saline.

+ As NIH-LH-S-14,

10 min after

injection.

REFERENCES 1. 2.

Harris,

3. 4.

Schally, Schally,

i:

Parlow,

Schally,

Schally,

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

G.W. in

Neural

Control

of the

Pituitary

Gland,

E. Arnold,

London,

1955.

A.V., Arimura, A., Bowers, C.Y., Kastin, A.J., Sawano, S., and Redding, T.W.: Recent Proqr. Hormone Res, 24: 497, 1968, Academic Press. A.V., Bowers, C.Y3hite, W.F. and Cohen, A.I.: EndocrinoloqyQ 77, 1967. A.V., Baba, Y., Arimura, A., Redding, T.W. and White, W.F.: Biocham. Bioohys. Res. Comn. Q 50, 1971. A.V., Redding, T.W., Bowers, C.Y. and Barrett, J.F.: J. Biol. Chem. 244: 4077, 1969. in Albert, A. (ad.), Human Pituitary Gonadotropins, Charles C. Thomas, Springfield, A.F.:

Ill., 1961, p. 300. Niswender, G.D., Midgley, A.R., Jr., Monroe, S.E. and Reichert, L.E., Jr.: Proc. Sot. Exp. Biol. Med. 728: 807, 1968. Schallg.V., Mittler, J.C., and White, W.F.: Endocrinoloqy& 903, 1970. Steelman, S.L. and Pohley, F.M.: Endocrinoloay 53: 604, 1953. Przar of The Endocrine Society, 51st Meeting, New York, Parlow, A.F., Oaane, T.A. and Schally, A.V.: 1969, p. 83. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: J. Viol, Chem. l& 265, 1951. Schally, A.V., Arimura, A., Kastin, A.J., Reeves, J.J., Bowers, C.Y., and Baba, Y.: in Nammalian Reproduction H. Gibian and E.J. Plotz, eds., pg. 45, 1970, Springer-Verlag, Berlin. Kastin, A.J., Schally, A.V., &al, C., Ilidgley, A.R., Jr., Bowers, C.Y., Gonet-Perez, F.: & J. Obstet. Gynec. 108: 177,'1970. Reeves, J.J., Arimura, A.,and Schally, A.V.: J. Animal Science 31: 933, 1970. Porath, J.: A&iv for Kemi 111 259, 1957. Doolittle, R.F. and Armentrout, R.W.: Biochemistry2 516, 1968. Amoss, M., Burgus, R., Ward, D.N., Fellers, R.E., and Guillemin, R.: Program of The Endocrine Society, 52nd Meeting, St. Louis, 1970, p. 61. !4hite, W.F.: in Mamaalian Reproduction H. Gibian and E.J. Plotz, eds., pg. 84, 1970, SpringerVerlag, Berlin.

399