Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 13, 1979

Pages

1024-1029

OF VITAMIN K-DEPENDENT CARBOXYLATION

STIMULATION

BY PYRIDOXAL-5'-PHOSPHATE* Adam Dubinl,

Erick

Oklahoma

Medical

Oklahoma

73104;

City, Received

Research

Robert

Delaney,

Foundation,

and University

Oklahoma April

T. Suen,

Andrew

825 N.E.

of Oklahoma

Chiu,

13th

Health

Johnson2

and B. Connor

Street, Sciences

Oklahoma Center,

City, Oklahoma

73190

21, 1979 SUMMARY

This vitamin

paper

K-dependent

of endogenous

to the

while the

evidence

the approximately

of the

substrate,

of the

brought

pentapeptide about

pyridoxal-5'-phosphate

pentapeptide.

pyridoxal,

that

carboxylation

protein

due to binding than

presents

inhibits

increase

PheLeuGluGluLeu,

in

but not

by pyridoxal-5'-phosphate, to microsomal

Pyridoxine

pyridoxamine,

two-fold

this

is

enzyme(s),

peptide

rather

carboxylation,

and pyridoxamine-5'-phosphate

have no effect

on

reaction. INTRODUCTION Amino

require

transaminases,

decarboxylases,

pyridoxal-5'-phosphate

formation of the

acid

of Schiff's

as a coenzyme,

base with

enzyme protein.

It

aqueous

solution

between

several

peptides

(2),

is also

and bovine

system,

carboxylation

of pentapeptide, in part

E-amino known that

requests

Schiff's

of Molecular

01979 by Academic Press, Inc. of reproduction in anyform reserved.

bases

(3).

of Health Biology,

be addressed.

1024

enzymes

through

of a specific

of endogenous

Institutes

should

is bound

Grant

amines

(4).

in an (1),

K-dependent

to stimulate

HL17619.

Jagiellonian

residue

are formed

In the vitamin

substrate

the

lysine

acids,

has been found

but not

by National

other

and amino

serum albumin

0006-291X/79/111024-06$01.00/0 Copyright All rights

group

pyridoxal-5'-phosphate

l0n leave from the Institute University, Krakow, Poland. 2To whom reprint

the

which

pyridoxal-5'-phosphate

carboxylation

*Supported

and several

This

the

Vol. 88, No. 3, 1979

stimulation

BIOCHEMICAL

can be due to either

reaction

the enzyme(s)

or with

evidence

a pyridoxal-5'-phosphate

lation

that

of peptide

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

pentapeptide.

In this enzyme

pyridoxal-5'-phosphate communication,

complex

accounts

with we provide

for

this

stimu-

carboxylation. MATERIALS AND METHODS

Materials: Pyridoxine HCl (vitamin B6), pyridoxal HCl, pyridoxal-5'phosphate, pyridoxamine diHC1, pyridoxamine-5'-phosphateeHC1, Sephadex G-lO120, and Sephadex G-25-150 were obtained from Sigma Chemical Co., St. Louis, MO. Triton X-100 and sodium dithionite were purchased from Eastman Kodak Co., Rochester, N.Y. Vitamin Kl as a Tween susps nsion was obtained from Merck, Sharp and Dohme, Rahway, N.J. and sodium [ C] carbonate (specific activity, 59.9 mCi/nol) from Amersham-Searle. Warfarin was obtained from Endo Laboratories, Inc., Garden City, N.Y. The pentapeptide (PheLeuGluGluLeu) was synthesized using technique as described by Stewart and Young (5), cleaved with by countercurrent distribution by Mr. Craig Ferris. All sources.

other

compounds

were

high-purity

preparations

obtained

the Merrifield HF, and purified from commercial

Methods: Vitamin K deficiency was produced in 10 to 14 days by maintaining Sprague-Dawley male rats on a vitamin K-deficient diet (6) in coprophagypreventing cages (7). Hypoprothrombinemic rats were prepared by treatment with warfarin (5 mg/Kg injected intraperitoneally) 18 hrs before the experiments (8). Vitamin K hydroquinone dithionite (9). The Triton X-100 K-deficient rat liver of Mack -et al. (12).

was prepared

by reduction

of vitamin

soluble system from normal, warfarin-treated, microsomes, was prepared by a modification

K with

sodium

and vitamin of the method

Carboxylation activity was determined as follows. One hundred ~1 of incubation mixture were used per assay. Each assay tube contained 60 ~1 of soluble system (1 mg protein), 20 ~1 pentapeptide sfiution (7.5 mM in 0.25 M potassium phosphate buffer, pH 7.4), 10 ~1 sodium [ C] carbonate (2.5 uCi), and 5 ~1 0.05 M potassium phosphate buffer, pH 7.4, or 5 ~1 vitamin B derivative solution (50 mM in this buffer). The reaction was initiate 8 by the addition of 5 ~1 of vitamin K hydroquinone (25 pg). The samples were incubated in a constantly shaking water bath at 25" for 30 min. The reaction was stopped by adding 1 ml ice-cold solution of 10% (w/v) trichloroacetic acid. After the addition of 100 ~1 of bovine serum albumin solution (10 mg/ml), the protein precipitate was left for 30 min at room temperature and removed by low speed centrifugation. OnT4ml of the supernatant was gassed for 30 min with air to remove unreacted [ Cl-C02, then counted in 5 ml of Scintisol (Isolab Inc., Ohio) in a Packard 3950 liquid scintillation counter. The trichloroacetic acid pellet was dissolved twice in 2% sodium carbonate solution and reprecipitated with 10% trichloroacetic acid and counted in the same way. Modification of enzyme(s) or pentapeptide by reacting with pyridoxal-5'phosphate was carried out as follows. The Triton X-100 soluble system, from vitamin K-deficient rat liver microsomes (10 mg protein) or pentapeptide

1025

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

solution (10 mg), was incubated 24 hrs at 0" with pyridoxal-5'-phosphate (final concentration 2.5 mM), and applied to a column (1 x 30 cm) of Sephadex G-25-150 equilibrated with 0.05 M potassium phosphate buffer, pH 7.4, containing 2% (v/,,) Triton X-100 or Sephadex G-10-120 equilibrated with 0.05 M potassium buffer, pH 7.4. Void volume fractions containing enzyme(s) or pentapeptide bound to pyridoxal-5'-phosphate were yellow in color and were used to estimate activity. Protein was estimated by the method of Esen (11) with bovine albumin as the standard. Pentapeptide and pyridoxal-5'-phosphate-bound pentapeptide were determined by amino acid analysis.

serum

RESULTS AND DISCUSSION Dose Response Figure

-of pyridoxal-5'-phosphate

1 shows that

PheLeuGluGluLeu, deficient

the

stimulation

of carboxylation

by pyridoxal-5'-phosphate,

and when warfarin-treated

of soluble

system.

is much less active.

The soluble

system

The stimulation

at 2.5 mM pyridoxal-5'-phosphate.

vitamin

K-deficient

microsomes,

liver

also

-of Carboxylation: of the pentapeptide

occurs rat

systems

lation

Stimulation

both

when vitamin

microsomes

derived reaches

from

are used as a source

normal

a plateau

The soluble

K-

rat

for

all

liver three

microsomes microsomal

system,

derived

from

shows the most endogenous

protein

carboxy-

activity.

0.5

1.0

1.5

FINALCONCENTRATION Figure 1. Stimulation by pyridoxal-5'-phosphate. H, microsomes. microsomes. 04, microsomes.

2.0

2.5

3.0

OF PLP(mM)

of vitamin K-dependent pentapeptide carboxylation M, soluble system from normal rat liver soluble system from warfarin-treated rat liver soluble system from vitamin K-deficient rat liver

1026

Pentapeptide

75

Pyridoxamine-5'-phosphate

87

108

102

94

94

Endogenous Substrate

Rats*

is

is

is

2100%

3100%

4100%

equivalent

equivalent

equivalent

to

to

to

2,300

1,000

500

cpm for

cpm for

cpm for

pentapeptide,

pentapeptide,

pentapeptide,

I

2,000

cpm for

endogenous

endogenous

endogenous cpm for

cpm for

1,000

170

106

102

101

95

88

Endogenous Substrate

Rats3

substrate.

substrate.

substrate.

102

87

252

91

38

Pentapeptide

Vitamin

Rats4

105

110

103

99

91

Endogenous Substrate

K-deficient

with no added vitamin B6 subtracting the minus The conditions substrate.

System'

PENTAPEPTIDE

of solubilized microsomes of three experiments after and 30 cpm for endogenous

81

80

224

64

39

Pentapeptide

Warfarin-treated

% of

Carboxylation

Control

CARBOXYLATION

K-DEPENDENT

SUBSTRATE

ON VITAMIN

IControl system is defined as the incubation mixture All values expressed are the averages derivatives. vitamin K values which were 100 cpm for pentapeptide of incubation are described in Materials and Methods.

86

160

Pyridoxamine

Pyridoxal-5'-phosphate

71

mM)

Pyridoxal

(2.5

Normal

37

Added

B6 DERIVATIVES

AND ENDOGENOUS

OF VITAMIN

Pyridoxine

Compounds

EFFECT

TABLE

Vol. 88, No. 3, 1979

Effect ---

of Vitamin

The effect lation

is

very

little

BIOCHEMICAL

vitamin

in Table

by adding

three

I.

carboxylation

is

pyridoxine.

by added

from

lation

activity

the

1.6-

of the

carboxylation

but not to influence pyridoxine

may indicate

phosphate

and prevents

Interaction shows that

the

amount

may be altered

that the

stimulation

related

compounds.

pyridoxal,

it

is changed Stimulation

binds

binding

is demonstrated

pyridoxamine, Inhibition

related

and pyridox(61 to 63%)

suggest

to the original

that

part

pentapeptide

carboxylation. to the

carboxy-

some integral

to facilitate

protein

carboxy-

by pyridoxal-5'-phosphate

being

binding,

The inhibition

same site

of

by

as pyridoxal-5'-

of pentapeptide.

-of Pyridoxal-5'-phosphate

the

carboxylation

effects.

The data

endogenqus

K-dependent

protein

The stimulation

system.

system

while

have significant

to 2.5-fold,

Carboxylation:

by pyridoxal-5'-phosphate

systems,

do not

varies

pyridoxine

of pentapeptide

K-dependent

on vitamin

The endogenous

amine-5'-phosphate caused

--on Vitamin

B6 compounds

any of the

Qf the CarbOXylatiQn in all

k& Derivatives

of various shown

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

of carboxylation

Enzyme -or Substrate:

of pentapeptide

Table

can be observed

TABLE II EFFECT

OF PYRIDOXAL-5'-PHOSPHATE OR SUBSTRATE

Enzyme

ASSOCIATE

ON PENTAPEPTIDE

CARBOXYLATION'

System

Soluble

System4

Soluble

System

No PLP2

Pentapeptide

plus plus

PLP bound

(1.5 mM)

PLP-bound Soluble Pentapeptide

System

PLP-bound Soluble System PLP-bound Pentapeptide

of

six

separate

'From

250

25

40

240

240

23

25

plus plus

experiments

(different

microsomes),

mM vitamin

K-deficient

PLP3

100

2Pyridoxal-5'-phosphate 32.5

Plus

to

Pentapeptide

'Averages duplicate.

WITH ENZYME(S)

rat

liver

microsomes

1028

(10

mg protein/ml)

each

run

in

II by

Vol. 88, No. 3, 1979

BIOCHEMICAL

adding

pyridoxal-5'-phosphate

the

the

other

protein-bound hand,

activity

with

the

of the carboxylation

enzyme E-amino

is presumably

The relationship carboxylation

This

of the

supplied

in which

pentapeptide between

of the

appears

which

enhances

stimulation interaction

of a solubilized

protein

serves

poor

as a very

and the

with

a lysine

system. to the

to indicate the

was not

pyridoxal-5'-phosphate

pyridoxal-5'-phosphate

pentapeptide

that

pyridoxal-5'-phosphate

carboxylation

same

peptide

not due to pyridoxal-5'-phosphate

interaction

of the

the

On 25% of the

the

demonstrates

be due to an alteration

the reaction

site,

shows only

solution

clearly

The modified

of the

of the

at the carboxylation

is

but must

of a protein

This

in the experiments

system.

The nature

group

as used

to pentapeptide.

pentapeptide

pentapeptide.

carboxylation

pentapeptide,

substrate.

free

pyridoxal-5'-phosphate.

of pentapeptide with

with

of pentapeptide

reacted

fraction

pyridoxal-5'-phosphate-bound

of that

amount

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

vitamin

a change

availability

K-dependent

in conformation

of the

pentapeptide.

ACKNOWLEDGEMENT We wish to thank manuscript.

MS. Patricia

Ownbey for

aid

in the

preparation

of this

REFERENCES 1. 2. i: 5. 6. 7. 8. 76.

11. 12.

Matsuo, Y. (1957) J. Am. Chem. Sot. 79, 2011-2015. Christensen, H.N. (1958) J. Am. Chem. Sot. 80, 99-105. Dempsey, W.B. and Christensen, H.N. (1962) J. Biol. Chem. 234, 1113-1120. Suttie, J.W., Lehrman, S.R., Geweke, L.D., Hageman, J.M., and Rich, D.H. (1979) Biochem. Biophys. Res. Cornmun. 86, 500-507. Stewart, J.W. and Young, J.D. (1969) Ssid Phase Peptide Synthesis. , W.H. Freeman, San Francisco. pp. 103 Mameesh, M.S. and Johnson, B.C. (1959) Proc. Sot. Exptl. Biol. Med. 101, 467-468. Metta, V.C., Nash, L. and Johnson, B.C. (1961) J. Nutr. 74, 473-476. J.W. and Grant, G.A. (1973) Arch. Bizhem. Biophys. Shah, D.V., Suttie, 159, 483-491. Fieser, L.F. (1940) J. Biol. Chem. 133, 391-396. Mack, D.O., Suen, E.T., Girardot, J.M., Miller, J.A., Delaney, R. and Johnson, B.C. (1976) J. Biol. Chem. 251, 3269-3276. Esen, A. (1978) Anal. Biochem. 89, 264-273. Mack, D.O., Wolfensberger, M., mrardot, J.M., Miller, J.A. and Johnson, B.C. (1979) J. Biol. Chem. (In Press).

1029