Requirements for α-ketoglutarate, ferrous ion and ascorbate by collagen proline hydroxylase

Vol. 24, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL

REQUIREMENTS FOR a-RETGGLUTARATE,

RESEARCH COMMUNICATIONS

FERROUS ION AND ASCORBATE

BY COLLAGEN PROLINE HYDROXYLASE John J. Laboratory National

Hutton,

Jr.,

of Clinical Institutes

A.L,

Tappel

*

and Sidney

Udenfriend

Biochemistry, National Heart of Health, Bethesda, Maryland.

Institute, 20014

ReceivedJune 6, 1966 Collagen

proline

embryo microsomal

hydroxylase

fractions

and was subsequently

(Peterkofsky

obtained

in

1965a),

In both

preparations

obtained

as well

as requirements

Stimulation

by ascorbic

and potassium

ions

However,

it

that

cofactors

the

Recently,

it

dialyzed

(Prockop

acid

collagen

absolute

++

was

cofactors.

and Udenfriend,

of both sufficient

reported.

for

partially

proline

hydroxylase for

1965)

laboratories

utilize

requirements

and Juva,

involvement

1965b) was also

above were not

of

Fe

1965)

activity,

purified

Fe++,

from chick ascorbic

and a-ketoglutarate. Proline

Methods: day-old

chick

centrifuged

*

for

reports

to

in chick

(Prockop

heat-stable

and Juva,

has been possible

embryo to demonstrate

form

(Peterkofsky

from the

listed

preparations

soluble

for

demonstrated and Udenfriend,

evidence

acid

was apparent

was first

University

hydroxylase

was prepared

embryos in O-25 M sucrose. at 105,000

x g and the

of California,

Davis,

179

S-105

by homogenizing The homogenate supernatant

8was

fraction

BIOCHEMICAL

Vol. 24, No. 2, 1966

Following

was dialyzed. buffer,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dialysis,

pH 7.5 and 0.3 vol

and after fuged

standing

at 15,000

brought

to

of water

of 1 M, Tris-HCl sulfate

were added

the mixture

was centri-

The supernatant

with

was collected

vol

15 min,

20 min.

60% saturation

precipitate small

5% streptomycin

at O0 for

x g for

0.05 vol

solid

(NH4)2S04.

by centrifugation,

and dialyzed

solution

The resulting

dissolved

to yield

was

a partially

in a purified

enzyme solution. Tritium

labeled

as described chick

by Hutton

hydroxylase

--et al.

to

into

0.5 N acetic

cold

solutions

inhibit

were generally

800,000

dpm/mg.

peptide-bound

hydroxylase the

to

substrate,

was extracted

Resulting

substrate contain-

the radioactivity

was assayed

shown in the in detail

formation

was in

of peptidyl-hydroxyproline by adding

vacuum distillation.

0.1 vol

(Hutton

, is

One equivalent

in a 2 ml vol

legends

of THO (tritiated

peptidyl-3,4-T-proline

3-T-hydroxyproline.

efficiency

and 1 mM a,a'-

2 mg/ml of protein

1% of

activity

ingredients

and depends upon the

stopped

in the

The substrate

diluted

The assay has been described

alent

buffer

hydroxyproline.

Proline containing

6 g of minced

(T = tritium)

and dialyzed,

Less than

was prepared

by incubating

hydroxylation. acid

substrate

Rrebs-Ringer

of 500 +C 3,4-T-L-proline

dipyridyl

the

(1966)

embryo in 8.5 ml modified

presence

ing

proline

1966)

water)

are presented

8%. 180

when

to peptidyl-

formed

per

Reactions

50% TCA and THO was assayed

Results

tables.

et&.,

converted

of THO is synthesized.

to the

equivwere

after

as cpm THO, counting

Vol. 24, No. 2, 1966

BIOCHEMICAL

Crystalline

bovine

(GDH) . was obtained

Results

in Table

hydroxylase

inactive.

Prockop

and Juva

at

when added in

addition

effect

when added to

enzyme preparations

gether

in the

ysate

to

ascorbic

nucleotide tamins

lesser

were

oxaloacetate

(Table

I),

to be between

could

not be replaced

.

by a number of

-5

of Na+ or K+ did

presence

initial

only

and 10e6M.

Km

sub-

the dialdialyzed screening

amino acids, activity,

and to a

In such studies

Ascorbic

ene-diol

compounds but

acid

could

the was Fe* be re-

not by 2-amino-4-

NADPH or NADH.

influence

or absence of a-ketoglutarate, 181

the

viOf

a-ketoglutarate

+ by Cu .

not

had no

of replacing for

is re-

or to-

a-ketoglutarate

were capable

by

stimu-

number of

to be without

hydroxy-6,7-dimethyltetrahydropteridine, tion

but

containing

nucleotides,

to be

reported

were both

to replace

In this

The apparent 10

++

A large

found

intermediates,

found

placed

++

proline

individually

mixtures

pyridine

ions

cycle

extent

dialysate

and Fe

phosphates,

and metal

~rebs

acid

and Fe

ability

a-

dialysate

the dialysate,

absence of dialysate.

when added to incubation

enzyme,

the

acid

their

collagen

of the

latory

for

to convert

as was previously

for

were tested

of GDH added to in-

of 13 Kmoles/min.

addition

Ascorbic

a concentration

embryos were found

quired

stances

At

dialysis,

found, that

(1965a),

activity.

a rate

from chick

We have

Co.

dehydrogenase

was sufficient

Following

preparations

totally

acid

quantity

II

to L-glutamate,

and Discussionr

RESEARCH COMMUNICATIONS

L-glutamic

-4 10 M, the

of

described

ketoglutarate

liver

from Sigma Chemical

of a-ketoglutarate cubations

AND-BIOPHYSICAL

reaction

either

Addiin the

BIOCHEMICAL

Vol. 24, No. 2, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Requirements for Formation of Tritiated Water from Peptidyl-3,4-TProline using Purified Chick Embryo Collagen Proline Hydroxylase Proline

Complete

Tritiated Formed

Hvdroxvlase System Additions

Omissions None a-Ketoglutarate Ferrous ion Ascorbic acid Enzyme a-Ketoglutarate a-Ketoglutarate a-Ketoglutarate a-Ketoglutarate a-Ketoglutarate

Water (cpm)

720 0 29 0 0 220 0 117 0 0

None None None None None Dialysate Pyruvate Oxaloacetate Fumarate L-Glutamate

The complete system (2 ml) contained purified hydroxylase fraction (3 mg protein) and, in Fmolest ascorbic acid, 2.5; ferrous ion, 0.15; a-ketoglutarate, 0.02; Tris HCl, pH 7.5, 200; hydroxylase substrate, 800,000 dpm. The amount of dialysate added was equivalent to that obtained from 1.0 ml of s-105. In certain tubes a-ketoglutarate was replaced with 0.02 kmole of the indicated compound. Incubation was for 30 min at 30°. To identify

the

activator

these were preincubated

activity

glutarate certain

of the preparations. was also

that

ium from the proline

the less

tritiated

equilibrium than

destroyed

a-ketoglutarate

II,

such treatment

not

in

of

182

trit-

3-T-hydroxy-

in amounts equivalent

of a-ketoglutarate

The destruction

added a-keto-

some way labilize

containing

a-ketoglutarate

destroyed

Tomake

the resulting

In incubations

10m7MI additional

dehydrogenase,

by such preincubation.

residues,

concentration

to form L-glutamate,

glutamic

and shown to be formed

water,

enzyme preparations,

The activity

did

3,4-T-proline

was isolated

to the

crystalline

As shown in Table

NADPH and NH4Cl. all

with

in undialyzed

the

GDH system,

should

have been

having

by GDH of all

been aminated activity

in

Vol. 24, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II Effect of Glutamic Dehydrogenase Hydroxylase Activity in Crude Additions Durinq

to Buffered Preincubation

(GDH) on Collagen Proline Chick Embryo Extracts

Enzyme

Tritiated Formed

None GDH system minus GDH enzyme GDH system complete a-Ketoglutarate a-Ketoglutarate + complete GDH system

Water (cpm)

815 752 11 1250 18

Undialyzed chick S-105 enzyme fraction (3 mg protein) was preincubated in a 1.0 ml vol containing 100 pmoles Tris HCl, pH 7.5, at 30*, 10 min. The GDH system, where added, contained 1.0 kmole NADPHj 50 ~moles NH4Cl; GDH enzyme as described in the text. a-Ketoglutarate, where added, 0.10 kmole, At the end of the preincubation period, 1.0 ml of solution, containing 100 kmoles Tris KC11 800,000 dpm hydroxylase substrate1 2.5 bmoles ascorbic acid and 0.15 @mole ferrous ion, was added to each tube. Final incubation was for 30 min at 30°.

undialyzed glutarate good

enzyme

preparations

to replace

the

evidence

collagen

that

proline

natural

activity

ical

for

compound.

in Tables

this

I and II

by collagen

is

hydroxylase.

--in vitro

of

added

activator

an sbsolute

is

also

compatible

Experiments that

hydroxylase

skin

for

required

similar

of rat

fairly

requirement

with

a-ketoglutarate

a-keto-

are

The low concentration

indicate

proline

ability

dialyzable

a-ketoglutarate

stimulate role

and the

to

a physiologto

is

those also

and guinea

shown required

pig

gran-

uloma. It

would

appear

collagen

proline

electron

donating

agent,

Preliminary

that

the

hydroxylase agent

hydroxylation utilizes

and iron

studies

indicate 183

reaction

ascorbate

as the that

electron

catalyzed

as the

specific

transferring

a-ketoglutarate

dis-

by

Vol. 24, No. 2, 1966

appears the

BIOCHEMICAL

during

the

absence of Fe

occur.

A likely

is

it

that

is

concentration ability

for

hydroxylase, in collagen

but

that

and ascorbate

explanation an allosteric

for

of a-ketoglutarate

protein

biosynthesis

of the

enzyme,

The tissue of

the biochemical

the avail-

capacity

Thus as an activator this

in

requirement

a good indicator

synchronize

occurs

does not

and of the ATP generating

could with

also

the a-ketoglutarate

is

synthesis.

a-ketoglutarate

disappearance

where hydroxylation

activator

of many amino acids

required

protein

++

reaction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of proline critical

prerequisites

step of

synthesis,

Acknowledgement: We wish to thank Dr. L,A. Fahien for the use of glutamic dehydrogenase in these experiments.

suggesting

REFERENCES Hutton, J.8 Tappel, A,, and Udenfriend, S.# Anal. Biochem,, in press. Peterkofsky, B., and Udenfriend, S., Proc. Natl. Acad, Sci., 53, 335 (1965), Prockop, D., and Juva, K., Proc, Natl. Acad. Sci., 53, 661 (1965a), 150th Meeting Amer. Chem, Sot,, Prockop, D., and Juva, K,, Abst. 1965b, p. 11C.

184