Stimulation of protocollagen proline hydroxylase activity by nucleoside triphosphates

Stimulation of protocollagen proline hydroxylase activity by nucleoside triphosphates

BIOCHEMICAL Vol. 69, No. 4, 1976 STIMULATION AND BIOPHYSICAL RESEARCH COMMUNICATIONS OF PROTOCOLLAGEN PROLINE HYDROXYLASE ACTIVITY BY NDCLEOSIDE ...

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BIOCHEMICAL

Vol. 69, No. 4, 1976

STIMULATION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OF PROTOCOLLAGEN PROLINE HYDROXYLASE ACTIVITY BY NDCLEOSIDE TRIPHOSPHATES

Ken Takeda,

Setsuko

Department University,

Received

Kawai,

Tsunao

Tetsuka,

and Kunio

Konno

of Biochemistry, School of Medicine, Hatanodai, Tokyo, JAPAN

February

Showa

23,1976 SUMMARY

Activity of purified protocollagen proline hydroxylase was enhanced several fold by addition of nucleoside triphosphates (3 mM) to the assay were almost inactive. medium, but nucleoside mono- and diphosphates Pyrimidine nucleotides were less effective compared with purine nucleotides, among which GTP was the most effective. dATP and ATP analogues such as adenosine 5'-(B,y-imino) triphosphate (AMP-PNP), adenosine 5'-(B,Y-methylene) triphosphate (AMP-PCP), etc. were inactive. ATP or GTP showed no additive effect on enzyme activity stimulated by dithiothreitol or bovine serum albumin. Protocollagen proline hydroxylase [proline, Z-oxoglutarate dioxygenase: EC 1. 14.

11. 21,

one of the new and unusual

genases,

shows

a stoichiometric

with

proline

hydroxylation

(l),

(3,5)

and the

atmospheric

some additional

factors

the

(3,4),

ascorbate

enzyme progressed (7,8,9)

and catalase

in the presence With there on the

little

increase

by several-fold from that

information

oxyaccompanied

a-ketoglutarate oxygen

(6).

(2),

ferrous

As purification

such as serum

to stimulate

experimentally ascorbate

of this except

fibroblasts.

hydroxylase

Abbreviations: AMP-PNP, adenosine 5'-($,y-methylene) methylene) triphosphate; Copyright All rights

found

function

of a-ketoglutarate

requires

regulation

enzyme of cultured proline

presumed

were

to the

collagen

factor

decarboxylation

of mixed

albumin,

enzymatic

ion of the

dithiothreitol

activities,

even

of cofactors.

reference

has been

(lo),

class

some reports Thus,

in cultured if

lactate injured

or lactate

enzymatic

liver

obtained

the activity

early-log

(ll),

phase

ascorbate (13)

may bring

activity

(12),

was added about

a

in vivo, from

of the cells

studies

proto-

was found

or the

isolated

to the medium.

conversion

of the

adenosine 5'-(B,y-imino) triphosphate; AMP-PCP, triphosphate; AMP-CPP, adenosine 5'-(a,BBSA, bovine serum albumin; DTT, dithiothreitol

0 1976 by Academic Press, Inc. of reproduction in. any form reserved.

957

to

It

was

Vol. 69, No. 4, 1976 inactive

BIOCHEMICAL

precursor

to the

be a direct

one since

stimulation

of enzymatic

of protocollagen of the

active

the

enzyme

cells activity.

proline

In this

collagen

proline

(14).

activity,

enhanced

communication

methylene)

5'-(B,Y-imino)

triphosphate

adenosine

5'-(a,B-methylene)

Laboratories.

[4-jH]

Research,

of ATP to the reaction in vitro

of proto-

were

obtained

and adenosine

from Boehringer

(AMP-CPP)

(21 Ci/mole)

5'-(B,yMannheim

was purchased

was a product

Corp.,

from Miles

of Schwarz

Bio

Inc..

Assay

of Enzyme Activity:

was assayed

essentially

Each assay

mixture

HCl buffer, moles;

(AMP-PNP)

triphosphate

proline

the activity

AND METHODS

triphosphate (AMP-PCP)

that

nucleotides.

MATERIALS Adenosine

not

on the regulation

we observed

the stimulation

by various

could

the observable

of studies

by the addition

we report

hydroxylase

the effect

to produce

In the course

hydroxylase

RESEARCH COMMUNICATIONS

However,

had to be intact

enzyme was considerably

mixture.

AND BIOPHYSICAL

pH 7,5,

cpm);

prepared

according

FeS04,

2 u moles;

enzyme preperation

of a,e'-dipyridyl,

followed

of Hutton,

2 p moles;

catalase, (0.1-1.0

minced

hydroxylase

of 1 ml and contained

200 u moles;

by incubating

proline

to the method

had a volume

a-ketoglutarate,

(60,000

Protocollagen

rat

et al

the

following;

sodium

ascorbate

200 pg;

peutidyl

ug of protein).

fetuses

with

by extracting

activity

[4-3H]

(15). Tris10 u

[C-'HI-substrate The substrate

proline

was

in the presence

the labelled

protocollagen

15 min at 37" with

shaking

according

to the same authors. The reaction stopped

by adding

was collected liquid

scintillation

chick

ammonium

out

for

0.2 ml of 50 % trichloroacetic

embryos sulfate

ammonium sulfate

spectrometer of Enzyme: according fractionation extraction,

using

Bray's

(30-65

X),

and passage

958

formed

in a

solution. from decapitated

of Rhoads and Udenfriend DEAE-Sephadex through

and

water

was determined

The enzyme was purified to the method

in air

Tritiated

acid.

Radioactivity

by vacuum distillation.

Preperation old

was carried

12-day(9),

using

chromatography,

two Sephadex

G-200

columns.

BIOCHEMICAL

Vol. 69, No. 4, 1976

AND BIOPHYSICAL Table

Effect activity

of various

nucleotides

3 H-water

hydroxylase

% of control

formed(cpm)

483 -L 16

100

AMP

355 f 11

73

Cyclic-AMP

432 f

89

7

GMP

418 2 10

87

ADP

522 f 32

108

GDP

418 + 10

149

ATP

1503 + 24

311

GTP

2644 f 13

486

ITP

1995 + 22

365

CTP

1160 f 13

212

UTP

1049 * 10

192

The peak

of enzymatic

with

small

are given in Materials used was 3 mM. Values

activity

eluted

from

peak of protein

eluted

in

than

that

was 2,000-fold

natant

proline

None (Control)

The standard incubation conditions Concentration of the nucleotides of four determinations.

activity

I

on the protocollagen

Additives

the

RESEARCH COMMUNICATIONS

greater

Sephadex

the

are

column

and Methods. means i S.E.

was coincident

same position,

and the specific

of the original

105,OOOg

super-

fraction. RESULTS AND DISCUSSION As shown

collagen

in Table

proline

was even seen

1, addition

hydroxylase

activity

at the nucleotide

maximum stimulation

was attained

other

also

than

than ATP,

ATP were and it

enhanced

the

the enhancing

(Figure

Among the nucleoside

in the

enzymatic

This

stimulatory

Nucleoside

stimulation;

activity

effect

triphosphates GTP was more effective

by about

of GTP reached

upto

triphosphates,

pyrimidine

959

of proto-

as low as 0.1 mM, and the

at 3 mM ATP.

effect

an enhancement

by 2-4-fold.

concentration

effective

Occasionally 1).

of ATP resulted

&fold

5-fold. of the control nucleotides,

CTP

Vol. 69, No. 4, 1976

BlOCHEMlCAl

AND BlOPHYSlCAL

NONE

BSA

Figure 1. Effect of BSA and DTT on protocollagen proline hydroxylase. are given in Materials and Methods. nor DTT, Middle; BSA (2 mg/ml) solely, (0.01 mM), were added, respectively. 1-64); m, GTP (3 mM).

and UTP showed

less

and diphosphates effect

were

These

stronger

than

that

AMP-PNP,

was also

the

of adenine

effect

is not

yet

activation

increase

for

the

stimulatory On the other

extent. activities

of both

of guanine

mono-

nucleotides

ATP is

analogues

of ATP (16),

and some of them were

data

may suggest triphosphates

the breakdown

were

ineffective dATP

inhibitory.

the possibility would

all

that

the

be energy-dependent,

of Y-phosphate

is

really

necessary

but in

process.

Lineweaver-Burk tides-stimulated

that

the affinity

activity,

of nucleoside

clear

ADP inhibited

of stimulatory

that

Nucleoside

nucleotides.

nucleotides.

These

ineffective.

stimulatory

enzyme

suggest

enzymatic

purine

of GTP to a lesser

inhibitor

AMP-PCP and AMP-CPP,

in enhancing

the nucleotides-stimulation of The standard incubation conditions Left (shown as NONE); Neither BSA Right; Both BSA (2 mg/ml) and DTT 0 , no nucleotide; m , ATP (3

with

and that

results

BSA + DTl

In addition,

inactive.

GDP was a more potent

and GTP.

this

compared

of ATP competitively

hand,

it

effect

RESEARCH COMMUNICATIONS

plots activities

substrate,

of the exhibited

and that

data

showed a little

the primary

Vmax. 960

that

the

difference action

control

and nucleo-

in the affinity

of nucleotides

was to

of

Vol. 69, No. 4, 1976

As shown the

in Fig.

enhancement

report

less

showing

dithiothreitol

activity more

markedly, (not

shown).

together

with

lo-fold. GTP nor

(9).

it

Also

showed in

ATP caused

mM concentration

by addition

activity In the

presence

(DTT)

mM concentration

0.01

at 0.1 case,

further

mM DTT showed activity

not

than

the

enzymatic it

any

BSA (2 mg)

BSA alone,

i.e.,

by

concentrations, activity.

and the activity

the

respectively.

stimulate

no stimulation,

of the enzymatic

inhibitory,

enhanced did

with

GTP and ATP

and 1.5-fold,

of BSA and DTT at these

enhancement

was rather

l.l-

resulted

in agreement

the nucleotides

more enhancing

medium

of BSA, however,

of only

the presence

RESEARCH COMMUNICATIONS

by 4.6-fold,

stimulations

and in this Although

AND BIOPHYSICAL

of BSA (2 mg) to the assay

enzymatic

et al.

active,

Similarly,

1, addition

of the

of Rhoads

were

0.1

BIOCHEMICAL

neither DTT above

was not

restored

of GTP or ATP.

REFERENCES 1. Rhoads, R.E., and Udenfriend, S. (1968) Proc. Nat. Acad. Sci. U.S.A. 60, 1473-1478 2. Hutton, J.J., Tappel, A.L., and Udenfriend, S. (1966) Biochem. Biophys. Res. Commun. 24, 179-184 3. Pererkofsky, B., and Udenfriend, S. (1965) Proc. Nat. Acad. Sci. U.S.A. 53, 335-342 4. Prockop, D.J., and Juva, K. (1965) Proc. Nat. Acad. Sci. U.S.A. 53, 661668 5. Hutton, J.J., and Udenfriend, S. (1966) Proc. Nat. Acad. Sci. U.S.A. 56, 198-202 6. Fujimoto, D., and Tamiya, N. (1962) Biochem. J. 84, 333-335 7. Rhoads, T.E., Hutton, J.J., and Udenfriend, S. (1967) Arch. Biochem. Biophys. 122, 805-807 8. Popenoe, E.A.,Aronson, R.B., and Van Slyke, D.D. (1969) Arch. Biochem. Biophys. 133, 286-292 9. Rhoads, R.E., and Udenfriend, S. (1970) Arch. Biochem. Biophys. 139, 329-339 10. Kivirikko, K.I., and Prockop, D.J. (1967) J. Biol. Chem. 242, 4007-4012 11. Cornstock, J.P., and Udenfriend, S. (1970) Proc. Nat. Acad. Sci. U.S.A. 66, 552-557 12. Stassen, F.L.H., Cardinale, G.J., and Udenfriend, S. (1973) Proc. Nat. Acad. Sci. U.S.A. 70, 1090-1093 13. McGee, J.O'D., O'Hare, T.P., and Patrick, R.S. (1973) Nature New Biology 243, 121-123 14, McGee, J.O'D., Langness, O., and Udenfriend, S. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 1585-1589 15. Hutton, J.J., Tappel, A.L., and Udenfriend, S. (1966) Anal. Biochem. 16, 384-394 16. Yount, R.G., Babcock, D., Ballantyne, W., and Ojala, D. (1971) Biochemistry 10, 2487-2489

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