Vol. 85, No. 1, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

November 14, 1978

Pages

85-91

H U M A N G R O W T H H O R M O N E A C T I V E SITE F O R M E M B R A N E C O O P E R A T I V E E N Z Y M E S

Ricardo

Norberto

Farias 1 f Lilia

Elena

Ufiates

•

Hortensia

Moreno

I n s t i t u t e de Q u l m i c a B i o l d g i c a - F a c u l t a d de B i o q u l m i c a , Qulmica y Farmacia - Universidad Nacional San M i g u e l

de T u c u m ~ n

de T u c u m ~ n - 4000 - T u c u m ~ n - A r g e n t i n a and

Clara

pefiaI and A l e j a n d r o C. P a l a d i n i 1

D e p a r t a m e n t o de Q u i m i c a B i o l 6 g i c a - F a c u l t a d de F a r m a c i a y Bioqulmica - Universidad Nacional

de B u e n o s A i r e s y

C e n t r e p a r a el E s t u d i o de las H o r m o n a s H i p o f i s i a r i a s . Junln

Received

August

956 - 1113 B u e n o s A i r e s - A r g e n t i n a

23,1978

SUMMARY: The a c t i o n of the h u m a n g r o w t h h o r m o n e (hGH) on the H i l l c o e f f i c i e n t for the i n h i b i t i o n by F of rat e r y t h ~ o c y t e m e m b r a n e a c e t y l c h o l i n e s t e r @ ~ e and for the i n h i b i t i o n by N a - of Escherichia coli m e m b r a n e ( C a ~ - ) A T P a s e was studied. The hGH and s e v e r a l s y n t h e t i c f r a g m e n t s f r o m hGH w e r e able to d e c r e a s e the n v a l u e s in b o t h systems. It can be c o n c l u d e d t h a t 33 r e s i d u e s r e g i o n in hGH, c o r r e s p o n d i n g to p o s i t i o n s 88 t h r o u g h 120, c o n t a i n s the " a c t i v e site" for this h o r m o n a l a c t i o n on m e m b r a n e c o o p e r a t i v e enzymes.

T h e r e is m u c h

j u s t i f i c a t i o n for p e r m a n e n t r e s e a r c h on the

p o s s i b l e e x i s t e n c e of a c o m m o n a c t i v e core(s) hormones.

This

subjects,

suffering

stems

structurally quite hormones

f r o m the u n f o r t u n a t e from hipopituitarism,

s i m i l a r and m o r e

f r o m o t h e r species.

of h u m a n g r o w t h h o r m o n e protein field

in p i t u i t a r y g r o w t h

fact t h a t h u m a n de not r e s p o n d to the

readily available growth

The c o n c e p t that the a c t i v e p r i n c i p l e

(hGH) m a y be a small

f r a g m e n t of t h i s

is u n d e r c o n t i n u o u s e x p e r i m e n t a t i o n and d e b a t e

The use of m e m b r a n e - b o u n d c o o p e r a t i v e e n z y m e s record changes

as p r o b e s

to

in the m e m b r a n e due to the a c t i o n of v a r i o u s

h o r m o n e s has b e e n e x t e n s i v e l y i l l u s t r a t e d 1

in the

(I).

(2-7). Also,

as s h o w e d

C a r e e r I n v e s t i g a t o r of the C o n s e j o N a c i o n a l de I n v e s t i g a c i o n e s Cientlficas y T~cnicas

(CONICET)

85

- Argentina.

0006-291X/78/0851-0085501.00/(] Copyright © 1978 by Academic P~ess, Inc. All rights o[ reproduction in any [orm reserved

Vol. 85, No. 1, 1978

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the case of thyroid a highly

specific

hormones,

tool to study

membrane

cooperative

the action

enzymes

of h o r m o n e

are

analogues

(7). In this paper we show that hGH cooperative

behaviour

erythrocyte

acetylcholinesterase

ATPase.

The c o m b i n a t i o n

availability

of several

to p a r t i a l l y

define

action.

Human

chain of 191 amino the present

MATERIALS

synthetic

Hormone

w o r k was

reported

rat

thus d e v e l o p e d

fragments

with

the

for this b i o l o g i c a l

by a single

two d i s u l f i d e

(Ca2+) -

of hGH m a d e p o s s i b l e

responsible

is formed

acids w i t h

the

enzymes:

and Eseherichia coli

of the assays

the sequence

Growth

is able to change

of the m e m b r a n e - b o u n d

bridges

in p r e l i m i n a r y

form

polypeptide (7). Part of (9).

AND METHODS

Rat e r y t h r o c y t e

acetylcholinesterase.

Male S p r a g u e - D a w l e y rats, grown after w e a n i n g on basic diet s u p p l e m e n t e d w i t h 5 % corn oil w e r e used to o b t a i n e r y t h r o c y t e membrane-bound acetylcholinesterase. Details c o n c e r n i n g e r y t h r o c y t e preparations, assay of the e n z y m a t i c activities and c a l c u l a t i o n of kinetic p a r a m e t e r s for the i n h i b i t i o n by F- have been g i v e n in a previous article (6).

Escherichia

coli

(Ca 2+) ATPase.

The b a c t e r i a l strain used was E.coli K-12 Hfr M 1 (pho-alkaline phosphatase). The b a c t e r i a w e r e grown a e r o b i c a l l y in a g y r a t o r y shaker at 20°C in n u t r i e n t broth m e d i u m (3). Details of p r e p a r a t i o n of the m e m b r a n e s as well as those r e l a t e d to the p r o c e d u r e for the enzymatic assaysand c a l c u l a t i o n of the k i n e t i c p a r a m e t e r s for the i n h i b i t i o n by Na w e r e p r e v i o u s l y d e s c r i b e d (i0).

Human Growth Hormone It was p r e p a r e d a c c o r d i n g to Roos et al step of p u r i f i c a t i o n by gel filtration.

(ii) w i t h

an a d d i t i o n a l

Human Growth Hormone f r a g m e n t s . They w e r e s y n t h e s i z e d by the solid phase method, as i n d i c a t e d b e f o r e (12). All s y n t h e t i c p e p t i d e s had g l y c i n a m i d e as C - t e r m i n a l amino acid and those c o n t a i n i n g a c y s t e i n e r e s i d u e had the free sulfydryl b l o c k e d by an a c e t a m i d o m e t h y l group. The synthetic p e p t i d e s p u r i f i e d by gel f i l t r a t i o n and p a r t i t i o n c h r o m a t o g r a p h y on silica gel w e r e c h a r a c t e r i z e d as single c o m p o n e n t s by SDS gel e l e c t r o p h o r e s i s , thin layer c h r o m a t o g r a p h y , amino acid c o m p o s i t i o n and amino terminal residue d e t e c t i o n . Human G r o w t h H o r m o n e and s y n t h e t i c fragments w e r e d i s s o l v e d in water w i t h the a d d i t i o n of small v o l u m e s of N a O H 0.1 M. Thereafter, the stock solutions w e r e p r e p a r e d in 310 m o s M S o d i u m ~ h o s p h a t e (pH 8.0) or 20 m M HCI Tris b u f f e r (pH 9.0) and w e r e stored at -20°C until used. C.Pe~a, J . M . S t e w a r t preparation.

and A . C . P a l a d i n i

86

(1978)

manuscript

in

VoI. 85, No. 1, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.o

olt

06

,-,i 0 4

I

I

I

I

1 .o

a

I

I 40

2 .o

F-mM ±g.l.-

Effect

Na ~ mM

of the h G H on t h e H i l l

coefficient

for

the

inhibition

(A) a n d

the

E.coli

(B).

was

inhibition

used

fragments (0.18

~

Hill

i: E f f e c t

b y Na + of

(Ca2+)-ATPase

a n d in the p r e s e n c e plots

for the

of h G H

of s a m e d a t a .

The

(00)

of h G H

and h o r m o n e

tests.

fragments

o n the H i l l

coefficient.

for

In the

10-8M.

same membrane

control

Inset

preparation

n values x

added

~g/ml)

Acetylcholinesterase (rat e r y t h r o c y t e )

None -

,!

acetylcholinesterase

shows

hGH

I 80

by F- of r a t e r y t h r o c y t e

absence

TABLE

I

1.55 ~ 0.05 a

(Ca 2+)

ATPase

(E.coli) 2.20

-+ 0 . 0 5 a

0.80 ~ 0.10 b

128

±9 - 128

0.90

34 - 128

1 00 ~ 0.i0 b

~ 0.15 b

44 - 128

1 05 ~ 0 . 0 5 b

64 - 128

0 93 ~ 0.08 b

88 - 128

0 90 ~ 0 . 1 0 b

1.40

+ 0.i0 b

98 - 128

1 55 ~ 0.i0 a

2.20

+- 0 . 1 5 a

108

- 128

1 53 ~ 0 . 0 7 a

2.20

+ 0.I0 a

81 - 120

0.90 ~ 0.15 b

1.40

-+ 0 . 0 2 b

Mean

of n v a l u e s o b t a i n e d w i t h 3-5 d i f f e r e n t e n z y m a t i c + p r e p a r a t i o n s - SEM. V a l u e s f o l l o w e d by d i f f e r e n t l e t t e r s significantly

different

(p

40.001).

87

were

Vol. 85, No. 1, 1978

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

r~

~..~ ..~ O

0

•,-[ ,.~

..~ 0

L~ O

•

O O

I I I

+1

O >~ ,.~ 0 ,....-I ~.~ 4-)

I I I

I I I

I I I

o '..o

I.~ O

o

o

+1

+1

o o

>

o ~o

o o

+I

0

o

~o

•O

"v

I oo o

4-)

o

O

O ~

~ O

~ O

~ O

~ O

O

O

O

O

O

O

O

O

+1

+1

+1

+1

+1

+1

+1

O

O

O

O

O

O

4-)

O

•M 4-)

-r-I "O

N

-~

4-~

O '~ LN ",~

oo

I oo o~

0)

I o

.;-I

CO I O ~-I

0.4

CO

0o I O

I O ~

OO

Ln

N CO ~

OO

OO ~

O~

I OO

•~

4~ 4J

0 ,-I I ~o

0

I oo o

oo o ,-I

..~ -I-

(11 0

w

t~

~

1.4 g.4

~

w

o

o

O

Rq

+ o~

0 0 ,---I

0

>i

v I

I

I

o

~

>

0

o 0

cq

oo

0

I

I

Co

Co

O O -IK

88

Vol. 85, No. 1, 1978

TABLE

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

3: S p e c i f i c i t y

of the b l o c k i n g

action

of p e p t i d e s

hGH 98-

128 and hGH 108-128.

Hormone

n values

of hGH synthetic

fragment

added

(M)

None

2.20 + 0.05 a

Insulin

(i x 10 -9 )

Insulin

(i x 10-9)+

L-T 3

(i x 10 -9 )

L-T 3

(i x 10-9)+

1.40 108-128

(8 x 10 -7 )

-+ 0.05 b

1.40 + 0.05 b 1.00 -+ 0.i0 b

hCS

( 1 x 10 -8 )

hCS

(i x 10-8)+

hCS

( 1 x 10-8)+

Mean

(E.coli}

(Ca 2+) A T P a s e

8 x 10 -7)

98-128

1.10

-+ 0.10 b

1.30 + 0.05 b 108-128

8 x 10 -7)

1.10

-+ 0.15 b

98-128

8 x 10 -7)

1.20

+ 0.I0 b

of n values o b t a i n e d with 3-5 d i f f e r e n t e n z y m a t i c + - SEM. Values f o l l o w e d by d i f f e r e n t letters

preparations

significantly RESULTS

different

(p { 0.001).

AND DISCUSSION

Fig.

1 shows

the action

from both m a m m a l i a n inhibitors

activity

hGH were

also

erythrocyte

of hGH on m e m b r a n e

and b a c t e r i a l

(F- or Na +)

specific

enzymes.

able to decrease

and E.co£i systems of hGH from amino

shorter

like

ones

as intact position

hGH.

(Table

F r o m those region the

(Table acid

98-128

in hGH,

"active

GH fragments,

of the

88-128,

chain,

the

were

comprising as

as active

advanced

the a c t i v i t y

Advancing

from

in the

128 as well

acid was

enzymes

fragments

The P e p t i d e s

amino

past

dissapeared

the C - t e r m i n a l

120 did not affect

the

81-120).

it can be c o n c l u d e d to p o s i t i o n s

for this p a r t i c u l a r

like hGH

coefficient

i).

and 108-128).

corresponding

site"

synthetic

1 through

128 to p o s i t i o n

i, p e p t d i e results

In the absence

64-128,

the N - t e r m i n a l

from p o s i t i o n

activity

the Hill

34-128,

cooperative

of hGH did not affect Several

88 in the hGH p o l y p e p t i d e

i, p e p t i d e s

residue

19-128,

When

systems.

the p r e s e n c e

of both

the sequence

(Table

were

95-136

the 3 3 - r e s i d u e s

88 t h r o u g h

action.

and b o v i n e

89

that

Some

GH 96-133

120,

closely

contains related

have b e e n

found

Vol. 85, No. I, 1978

active

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

in g r o w t h

assays

(13-14).

have not b e e n g e n e r a l l y The

inative

specifically (Table

confirmed

peptides

98-128

the hGH a c t i o n

2). These

"active

site"

facts

Up to now though,

findings

(15).

and 108-128

were

able to b l o c k

and that of the active

suggest

responsible

these

the e x i s t e n c e

for its effect

peptide

81-120

in hGH of o n l y one

on m e m b r a n e

cooperative

enzymes. Table

3 shows

do not inhibit

somatomammotrophin, hormones. blocking

that the

the action hCS)

inactive

or u n r e l a t e d

A high molecular action

We think

peptides

of hGH r e l a t e d

(insulin,

specificity

of the inactive

98-128

and 108-128

(human c h o r i o n i c

is thus

L-thriiodothyronine suggested,

in the

peptides.

that the i n t r o d u c t i o n

of this new m e t h o d o l o g i c a l

system

in the hGH field m a y be i m p o r t a n t

active

core

to detect

the "in vivo"

of the hormone.

ACKNOWLEDGMENTS This w o r k was p a r t i a l l y s u p p o r t e d by grants from the CONICET, the S e c r e t a r f a de C i e n c i a y T ~ c n i c a U.N.T. and the Regional P r o g r ~ of S c i e n t i f i c and T e c n o l o g i c a l D e v e l o p m e n t of O.A.S. REFERENCES i.- B e w l e y T.A. and Li C.H. (1975) in A d v a n c e s in E n z y m o l o g y 42, 73-166; A . P e c i l e and E . E . M u l l e r (Eds) (1976) Growth H o r m o n e and r e l a t e d peptides. E x c e r p t a M e d i c a - Amsterdam. 2.- M a s s a E.M., M o r e r o R.D., Biophys. R e s e a r c h Commun. 3.- M o r e n o H. and Farfas 72, 74-80. 4.- M e l l O n E.M., M a s s a FEBS Letters.

Bloj B. and Farfas 66, 115-122.

R.N.

E.M.,

(1976) Morero

5.- de M e n d o z a D., M a s s a E.M., FEBS L e t t e r s 84, 199-203. 6.- de M e n d o z a July).

D. and Farlas

7.- de M e n d o z a D., M o r e n o press July).

R.D.

and FarIas

R.D.

(1978)

H. and Farlas

8.- Li C.H. (1975) in Li C.H. A c a d e m i c Press, N e w York,

(1975)Biochir

B i o c h i m . B i o p h y s . Res.Commun.

Morero

R.N.

R.N.

R.N.

and FarIas

J.Biol.Chem.

R.N.

Proteins

(1977)

(in press

R.N.J.Biol.Chem.

(Ed) H o r m o n a l p. 36.

(1978)

(in

and P e p t i d e s

9.- Farfas R.N., Ufiates L.E., M o r e n o H., Pe£a C. and P a l a d i n i A. C. (1977) A b s t r a c t N°70, XIII N a t i o n a l M e e t i n g of the S o c i e d a A r g e n t i n a de I n v e s t i g a c i o n e s Bioqufmicas, La Falda - A r g e n t i na, N o v e m b e r 2-4. 10.- M o r e n o H., 7701-7706.

Si~eriz

F. and Farlas

90

R.N.

(1974)

J.Biol.Chem.

249

Vol. 85,. No. 1.1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ii.- Roos P., Fevol H.R., A c t a 74, 525-531.

Gemzell

C.A.

(1963)

Biochim.Biophys.

12.- Pe~a C., S t e w a r t J.M., P a l a d i n i A.C., D e l l a c h a J.M. and S a n t o m ~ J.A. (1975) in R . W a l t e r and J . M e i e n h o f e r (Eds). Peptides: Chemistry, S t r u c t u r e and Biology, A n n A r b o r S c i e n c e P u b l i s h e r s Inc, A n n Arbor, 523-528. 13.- B l a k e J. and Li C.H. 123-125.

(1973)

14.- Y a m a s a k i N., K i k u t a n i 9, 1107-1114.

M.and

15.- K o s t y o

J.L.

and W i l h e l m i

Int.J.Peptide Sonenberg

A.E.

9!

(1975)

M.

Protein

Res.

5,

(1970)Biochemistry

Metabolism

25,

105-124.