The stimulation of partially purified bovine caudate tyrosine hydroxylase by phosphatidyl-L-serine

Vol. 59, No. 4, 1974

THE

BIOCHEMICAL

STIMULATION

AND BIOPHYSICAL

OF PARTIALLY HYDROXYLASE T.

PURIFIED BOVINE CAUDATE BY PHOSPHATIDYL-L-SERINE

Lloyd

and

National Institute Laboratory Building Bethesda,

Received

July

2,

RESEARCH COMMUNICATIONS

S.

TYROSINE

Kaufman

of Mental Health Neurochemistry 36, Room 3D-30 Maryland 20014 of

1974

SUMMARY. A new procedure has been developed for the purification of tyrosine hydroxylase from bovine caudate nucleus. This procedure yields a soluble and stable enzyme which has been purified 50-fold from an acetone powder extract. The enzyme is stimulated greater than 3-fold by phosphatidyl-L-serine and other polyanions which lower the Km of the enzyme for the pterin cofactor. Substrate inhibition and an ability to readily hydroxylate phenylalanine in the presence of tetrahydrobiopterin have also been observed with this preparation. Although

INTRODUCTION. tissue

has been documented

has not have

been

it

proteases

for

bovine

to

caudate

Our interest

in the

hydroxylase

We have

that

lated observed

by certain with

phenylalanine

tyrosine

Copyright All rights

appears

hydroxylase

a soluble

of

the

tyrosine

(6) than

to

@ 1974 by Awdemic Press, Inc. of reproduction in uny form reserved.

to that that

with

here

the

were

a relatively tyrosine

simple hydroxylase

with

fold

hydroxylase

caudate of rat

liver

effect,

The mechanism

observed

with

than

that

thus

of this on crude

on phenylalanine

(4,5).

be stimu-

can also

examined

heparin

tyrosine

by lysolecithin

is much less

phospholipids

lysolecithin

1262

or

which

activity

20-50

stimulation

greatest

analagous

that

Of the

particles

polyanions.

about

the

tissue studies

with

in preparations

and stable

observation

although

this

of detergents

of phospholipids

caudate

system

Earlier

action

We report

hydroxylase.

produces to be more

resulted

be stimulated

activity

from

associated

by the

by certain

our previous

enzyme

is largely

interaction

phospholipids,

phosphatidyl-L-serine lation

stimulation

the

nervous

characterized.

only

of

in central

(l),

irreversibly.

could

the

brain

however,

possible

from

phenylalanine found

from

purification

and its

stemmed

a decade

solubilized

aggregate

a 50-fold

hydroxylase

enzyme

Such procedures,

or tended

procedure

the

activity

or adequately

be partially

(2,3).

unstable

nearly

purified

that

could

hydroxylase

for

extensively

demonstrated

and that

from

tyrosine

far, stimubrain

hydroxlase

BIOCHEMICAL

Vol. 59, No. 4, 1974

MATERIALS AND MFTHODS. L-tyrosine

(14c)

Nuclear

e L-(3,5-3H

chemical

Center

L-Phenylalanine

unifomy tyrosine),

Aldrich

were

30 Ci/mmole,

and purified

according

Co.

in this

laboratory

(8,9).

Boehringer

~annhe~

from

and Nephew Research,

Crystalline Corp.

prepaxed

each

aay by sonication

from

the

commercial

L-tyrosine

hydroxylase

activity

of the

0.95-2.20

nmoles

shakex. acid.

2.0

assay

compounds

from

tyrosine

Harlow,

nmoles

Amersham

Radio-

(7).

The

purchased

according

was purchased NSD-lO$,

Essex,

EngIana-

and from All

was performed

from

Supelco; other

(6-MPH,,)

to published from

the

wes a kind

gift

Phosphatidyl-L-serine fresh

compounds

as previously

50 umoles

solutions were

obtained

0.50

umoles

to be assayed

potassium

liver

and water

was terminated

to be linear

used

was for

by the

10 min

addition

with

to

time

of

20.0

nmoles

specified),

0.50

ml. 1

The specific

in the

kinetic

studies

at 37'

of

reductase

otherwise

0.50

up to

(10). (pH 6,1),

phosphate

dihydropteridine

6-MPH& (unless

preparations

Incubation

described

(3 X 105 cpm),

TPNH and sheep

hydroxylase

was found

New Rngland

et aI.

Milstien

3,5-3H-L-tyrosine

DOPA/mg/min.

the

were

inhibitor,

contained:

fraction

the

was

in a metabolic

ml 10% trichloroacetie

20 min and with

protein

mg/ml.

When lb C-tyrosine tyrosine

from

and 6-methyltetrahydropterin

Sheldon

in water.

assay

catalase>

The reaction This

up to

0.25

2000 units

tyrosine

obtained

and

available,

containing

(or

(BHb)

Biochemicals

sources

mixture

2-mercaptoethanol in excess),

General

hydroxylase

incubation

nmoles

384 mCi/mmole,

of Ikeda

in suspension

Ltd.,

were

0.05

method

The decarboxylase

both

A standard

to the

catalase

from

The tyrosine

were

labeled,

was obtained

by Dr.

was purchased

best

uniformly

Tetrahydrobiopterin

procedures

Smith

RESEARCH COMMUNICATIONS

(DMPH4) and 3-iodo-L-tyrosine

Chemical

synthesized

(14C)

368 rn~i/~ole,

labeled,

6,74imethyltetrahydropterin, the

AND BIOPHYSICAL

formation after

or 1' C-phenylalanine were

they

measured

had been

were

used

by determination separated

as substrates,

of radioactivity

by thin-layer

1 The results described in this report were obtained regenerating system for the cofactor, although equivalent in each case tith the enzymatic regenerating system.

1263

chromatography

with the results

DOPA and in these of the

super-

chem%caI were obtained

Vol.

59,

natant

No.

4, 1974

fractions

utilizing

BIOCHEMICAL

from

the

solvent

by the

procedure

standard

(11).

AND

reaction

mixtures

system:

n-propanol:NHbOH:H20;

of Lowry

et al. --

(Eastman

with

the

The 3-iodo-L-tyrosine-substituted to

a previous

report

from

Cuatrecasas

(13).

after

which

15 mg of

at 4’

for

the

uncoupled

of

The extract was subjected

phosphate

was washed

tyrosine

M TRIS-chloride,

chromatography

at pH 10, with

elution

M potassium

phosphate, of the

by an ammonium

sulfate

fractionation

aqueous

elsewhere.

extracts

resultant be about

We have

soluble

and stable

40% pure

by disc-gel

RESULTS AND DISCUSSION. of

solubilized

presence

1 shows that

substrate

substrate;

We have

by a 04.3 of the

a lo-20%

tyrosine

previously

bovine but

caudate

LB, and mixed

an aqueous

of

extract

techniques

(14).

4B, the

active

material

fraction

on calcium

hydroxylase

activity

was chromatography M KC1 gradient)

hydroxylase

followed

procedure

purifications recovery

on

was precipitated

purification

of

will

50-fold

over

The

of activity.

hydroxylase

inhibition

reported

adrenal not

tyrosine

inhibition

no substrate

of

has been estimated

to

electrophoresis.

of tetrahydrobiopterin, Purified

the

caudate

procedure

was added

active

step

achieved

with

according

and 1 mM dithiothreitol)

of tyrosine

The detailed

tissue

and particulate

DMPHb (12).

of the

The third

routinely

as the

by standard

Sepharose

portion

(most

albumin

of Sepharose

from

8% sucrose,

substituted

saturation).

of caudate

was purified

enzyme peak

was determined

gel.

pH 8.6,

major

Protein

general

gel

sheets

to be 8.4% by measurement

was prepared

pH 6.8.

(elution

be reported

powder

of the

25 and 40 percent

the

by fractionation

DEAE-cellulose

between

was found

from

on the

followed

the

150 mg CNBr/ml

hydroxylase

0.02

gel,

by 0.025

of coupling

The acetone

to

following

with

(#6064)

was synthesized

per ml of activated

powder. (in

eluted

which

caudate

an acetone

being

The extent

ligand

The bovine

(12)

3-iodo-L-tyrosine

serum

4B gel

COMMUNICATIONS

cellulose

8:l:l).

use of bovine

laboratory

was activated

RESEARCH

precoated

Sepharose

this

The gel

20 hrs.

BIOPHYSICAL

occurs

medulla

in the

occurs

1264

inhibition

tyrosine

presence

hydroxylase above

marked

of the

shows similar 0.05

hydroxylase synthetic

in the cofactor,

behavior.

mM when tyrosine

when phenylalanine

by tyrosine

is is used

Figure used

as

as the

Vol. 59, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

r

050

[M]

SU3STRATE

Figure 1. Effect of Substrate Concentration on Bovine Caudate Tyrosine Hydroxylase Activity in the Presence of 260 nM Tetrahydrobiopterin. Constant specific activity 14C-ty7cosine 2n # 14C-phenylalanine was used in this experiment (41.6 pCt/umole}, NSD 1034 at 10K was included %n the assays to prevent decarboxylation of the DOPA. Reaction products were isolated by thin-layer chromatography as described in M2terials and Nethods. After chromatography, the separated amino acids were visualized by spraying with ninhydrin, the chromatogram channels cut into 0.5 cm strips and the radioactivity determined by liquid scintillation spectrometry. The solvent system routinely yielded Rf values of 0.66, 0.39 and 0.13 for phenylalanine, tyrosine and DOPA, respectively. Tyrosine hydroxylase activity is expressed as nmoILes product formed in 10 ul of a 110 ul incubation mixture which contained 0.35 mg tyrosine hydroxylase preparation.

substrate the

up to

possibly

tyrosine, system,

it since

phospholipids out

mM phenylalanine.

physiological is

a potential

inhibition

The dramatic

ried

0.40

not

yet

established

of the

substrate

Inhibition

observed

regulatory

mechanism

for

adrenergic

nervous

occurs

stimulation

purified

we have

significance

has been previously with

Although

caudate

just of rat

above

tissue

liver

phenylalanine

reported tyrosine

the

(4,5). hydroxylase,

1265

levels

of tyrosine

(15).

hydroxylase

by several

When a similar only

survey

phosphatidyl-L-serine

with

was carwas

Vol.

59,

No.

4, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 2. Effect of Phosphatidyl-L-Serine on Bovine Caudate Tyrosine Hydroxylase Activity. The assay was performed in the presence of 50 uM 6-MPH4. The control value of 100 percent was the formation of 0.52 nmoles DOPA/lO min in the absence of any phosphatidyl-L-serine. The tritium-release assay was used as described in Materials and Methods with 0.50 mg tyrosine hydroxylase preparation/ml.

found

to

in Fig.

stimulate 2 where

it

can be seen that

phospholipid.

The nature

concentration

of enzyme,

of this

by about

b-fold.

conditions.

Km's of Km for

(Fig.

tyrosine A recent

hydroxylase

in the

has been

on Vrna

were

to the

series

of reports

by heparin

have

of either on the

described

activity

a slight

1266

of

presence

out

or absence the

of this

Km of the under

in reducing phospholipid

Km of tyrosine.

of

crude

pH dependence

of pterin these

6,'7-DMF'H4 or ~-MPH,+,

with effect

as a

rat

their

Km

on the The apparent

6,7-DMPH4 or 6-MPH4 is 0.076 stimulation

0.4 mM

as a function

has been observed

on the

at

and of pH.

a similar

had no effect

studied

decreases

effect

is presented

achieved

hydroxylase

carried

In contrast

presence

is

phospholipid

to have

it

stimulation

in the

was observed

tetrahydropterins,

stimulation

3) of tyrosine

effect

studies

b-fold

tyrosine

the

No appreciable

b-fold.

maximal

concentration

When identical

by 3 to

3 to

stimulation

shows that

phosphatidyl-L-serine values

plot

of tetrahydrobiopterin

phosphatidyl-L-serine

the

tetrahydropterin,

The Lineweaver-Burk function

A typical

significantly.

brain

of this

mM. tyrosine stimulation

(16,

Vol. 59, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PS 20

1

7

1 I

0

! 2

V[Bti4],

, 3

, 4

1 5

1 6

mM

Figure 3. Determination of the K,,, of Bovine Caudate Tyrosine Hydroxylase for Tetrahydrobiopterin 1~ the Presence and Absence of Phosphatidyl-L-Serine. The velocity is expressed as concentration of 3,5-3H-tyros2.ne was 50 $.iM. Reaction nmoles DOPA formed in 10 min. The tritium-release assay used as described in Material.s and Methods with 0.50 mg tyrosine hydroxylase preparation/ml.

171.

The activity

of purified

presence

of either

Fig,

4.

Although

rat

brain

with

the

60 pg/m.l heparin

tyrosine bovine

potassium

tt

naturally

to

the

was essential occurring

the

5.9 to

to pterin

to

shtft

6.1,

this

buffer.

as a function is

pH optimum

effect

was not

of 6.0

for

of pH ln the shown

from

6.0-6.1

observed

in either

to

j.n

soluble

100 mM

Phosphatidyl-L-serine,

however,

6.6-6.8

in either

buffer. of rat

brain

presence

of the

synthetic

investigate

this

stimulation

cofactor.

the

had a pH optimum

enzyme pH optimum

stimulation

in the

hydroxylase

or 0.4 mM phosphaticlyl-L-serine

50 mM TRIS-acetate

or TRIS-acetate

heparin only

from

enzyme which

shift

phosphate

been reported that

or

tyr&ine

has been reported

caudate

has been observed

Since

heparin

hydroxylase

phosphate

potassium

caudate

tyrosine

hydroxylase

cofactor, in the

When tetrahydrobiopterin

1267

has previously

DMPH4, we thought presence was used

of a as the

Vol. 59, No. 4, 1974

BlOCHEMiCAl

AND B~OPHYSICAL RESEARCH COMMUNICATIONS

20I al6l4-

0402I

54

!

1

58

62

!

,

66 70 PH

1

1

74

78

Figure 4. The Effect of pH on Stimulation of Bovine Caudate Tyrosine Hydroxylase by Phosphatidyl-L-Serine or Heparin. All incubations were done in the presence of 100 uM 6-MPH4, 50 UM 3,5-3H-tyrosine, and 100 mM potassium phosphate buffer at the indicated pH values. The concentration of phosphatidyl-L-serine used was 0.40 mM; 60 ng heparin/nii was used for the assays containing the latter compound. Reaction velocity is expressed as nmoles DOPA formed/l0 min. The tritiumrelease assay was used as described in Materials and Methods with 0.64 mg tyrosine hydroxylase preparation/ml.

cofactor

in

studies

pH dependence in the

identical

was observed,

pH optimum

preliminary caudate L-serine, increases

from

studies, tyrosine

V

max

The value

i.e.,

we have

polyanion

one described

no shift

also

observed

only

presence

presence a 3-4

by poly-L-glutamic not

in Fig.

in the

pH 6.1 to 6.6 in the

hydroxylase

this

to the

decreases

the

fold

4, virtually

the

of heparin

same

and a shift

of phosphatidyl-L-serine. stimulation

acid.

In contrast

Km for

the

In

of bovine to phosphatidyl-

cofactor,

but

also

2 *

of

0.22

mM for

the

Km of brain

TH for

BHk in the

absence

of

2Although Kuczenski and Mandell (6) have concluded that the heparin activation of brain tyrosine hydroxylase is a highly specific effect, detailed studies of the activation by other anions, including the effect of polyglutamate described above, strongly indicate that activation is non-specific, being related primarily to the charge density of the anion (I. Katz and S. Kaufman, unpublished results).

1268

Vol.

59,

No.

4, 1974

BIOCHEMICAL

phosphatidyl-L-serine

is about

of BHb (18)

uniform

(assuming

question

about

how tyrosine

cofactor

levels

apparently

that

phosphatidyl-L-serine

this

apparent

likely

candidates

important

role

tissue

so far

in the

below

Although

or other regulation

higher

than

distribution).

hydroxylase

as naturally

BIOPHYSICAL

200 times

decreases

discrepancy.

phosphatidyl-L-serine

AND

RESEARCH

the

reported

This

in this

the

Km value.

the

BHk Km four-fold

heparin

occurring endogenous

tissue

cerebral

of central

adrenergic

levels raises

with

may partially

of brain

a

pterin

The finding

or poly-L-glutamic

modifiers

tissue

observation

can function cofactor

COMMUNICATIONS

acid tyrosine

phospholipids

explain are not hydroxylase,

may play

an

activity.

REFERENCES 1. 2. Z: 2: 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Nagatsu, T., Levitt, M. and Udenfriend, S., J. Biol. Chem. a, 2910rzg1-r , 1964. Poillon, W.N., Biochem. Biophys. Res. Commun. g, 64-70, 1971. Nagatsu, T., Sudo, Y. and Nagatsu, I., J. Neurochem. l.8, 217+218g, 1971. Fisher, D.B. and Kaufman, S., J. Biol. Chem. 247, 2250-2253, 1972. Fisher, D.B. and Kaufman, S., J. Biol. Chem. m, 4345-4353, 1973. Kuczenski, R.T. and Mandell, A.J., J. Neurochem. I& 131-137, 1972. Ikeda, M., Fahien, M. and Udenfriend, S., J. Biol. Chem. 2& 4452-4456, 1966. Storm, C.B., Shiman, R. and Kaufman, S., J. Org. Chem. 3&, 3925-3927, 1973. Andrews, K.J.M., Barber, W.E. and Tong, B.P., J. Chem. Sot.(c), 928+X0, 1969. Lloyd, T., Mori, T. and Kaufman, S., Biochemistry g, 2330-2336, 1971. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem. 193, 265-275, 1951. Oman, R., Akino, M. and Kaufman, S., J. Biol. Chem. 2& 1330-1340, 1971. Cuatrecasas, P., J. Biol. Chem. 2& ?,OFg-3065, 1970. Tabor, H., Methods in Enzymology, vol. 1, Academic Press, New York pp. 609-610, 1955. Kandera, J., Levi, G. and Lajtha, A., Arch. Biochem. Biophys. &, 24g-z6o, 1968. Kuczenski, R., J. Biol. Chem. N, 5074-5080, 1973. J. Biol. Chem. x, 3114-3122, 1972. Kuczenski, R. and Mandell, A.J., (W. Pfleiderer and E.C. Taylor, eds.), Rembold, H., in Pteridine Chemistr The MacMillan Co., New York, pp. 4 5-484, 1964.

1269