Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

BIOCHEMICAL

Vol. 83, No. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 984-990

August 141978

EVIDENCEFORTHE REGULATIONOF PYRIDOXAL5 '-PHOSPHATEFORMATIONIN LIVER BY PYRIDO~AMINE(PYRID~xINE) ~'-PH~SPHATEOKIDASE' Alfred

H. Merrill,a

Kihachiro Horiike,

and Donald B. McCormick

Section of Biochemistry, Molecular and Cell Biology, and the Division of Nutritional Sciences Cornell University, Ithaca, New York 14853 Received

June 21,1978

SUMMARY: Pyridoxamine (pyridoxine) 5*-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5'-phosphate. The Ki, 3 IJ& is considerably layer than the Km for either natural substratz (18 and 24 nM for pyridoxamine 5 -phosphate and 25 and 16 nM for pyridoxine 5 -phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The Ki determined using a lO$ rabbit liver homogenate is the sameas that for the pure enzyme; hence, product inhibition in vivo is probably not diminished significantly by other cellular componentz Similar determinations for a lC$ rat liver homogenate also show strong inhibition by pyridoxal 5*phosphate. Since the reported liver content of free or loosely bound pyridoxal 5 -phosphate is g:eater than Ki, the oxidase in liver is probably associated with pyridoxal 5 -phosphate. These results also suggest that product inhtbition of pyridoxsmine-P oxidase may regulate the --in vivo rate of pyridoxal 5 phosphate formation. INTRODUCTION: Pyridoxamine and pyridoxine nase (EC 2.7.1.35)

(1,2),

then converted into pyridoxal

al-P) by pyridoxamine (pyridoxine) Most pyridoxal-P

are phosphorylated by pyridoxal

acid (7,8).

and oxidized

to pyridoxic

that control

of pyridoxal-P

or hydrolyzed

Li -et al.

(3,k).

by phosphatases (1,3,5,6)

Snell and Haskell (9) first

proposed

metabolism could occur by product inhibition

pyridoxsanine-P oxidase and/or removal of pyridoxal-P tion.

5'-phosphate (pyridox-

5'-phosphate oxidase (EC 1.4.3.5)

is bound by proteins

(10) found that pyridoxal-P

by hydrolysis

is more rapidly

by protein

if product inhibition the rate of pyridoxal-P

binding.

of

and oxida-

hydrolyzed when

free than protein bound, and, hence, the tissue content of pyridoxal-P controlled

ki-

may be

The present study was undertaken to determine

of pyridoxsmine-P oxidase could additionally

regulate

formation.

'Supported by NIH Research Grant AM-04585 from the USPHS. 2Predoctoral trainee supported by NIH Research Service Award 5 T32 GM-07273 from the USPHS. 0006-291X/78/0833-0984$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

984

BIOCHEMICAL

Vol. 83, No. 3, 1978

AND BIOPHYSICAL

Major metabolic transformations Diffusion

and/or

i\

of vitamin Bs by liver.

Transport

to/from

A0 : H: K: 0: PO : T:

//

Protein w

Binding

RESEARCH COMMUNICATIONS

Blood

Aldehyde Oxidose Alkaline Phasphatase Pyridoxol Kinase Pyridoxamine-P Oxidase Pyridaxal Dehydrogenase Transaminases

T

MATERIALSANDMETHODS: Liquid Nz-quick frozen livers from young adult, fullfed rabbits were obtained from Pel-Freez Biologicals (Rogers, AR). Rat livers were from male, Sprague-Dawley rats sacrificed immediately prior to use. The oxidase was purified according to Kazarinoff and McCormick (11). Pyridoxine-P was prepared by reduction of pyridoxal-P with sodium borohydride and purified by recrystallization. All other biochemicals were obtained from Sigma. Livers were homogenized in the designated buffer containing 10 PM phenylmethylsulfonylfluoride by ten strokes of a hand-held TenBroeck apparatus. cytoso1 was prepared by centrifuging the homogenate for one hr at 105,000& All operations were conducted in dim light to prevent photodegradation of the flavin and pyridoxyl compounds. Oxidase activities were determined by incubating the enzyme with substrate (5 ml final volume) at 37’ in a gyrotory shaker (New Brunswick Scientific). After fivetoten min intervals, aliquots were removed, added to trichloroacetic acid (5% final concentration), and pyridoxal and pyridoxal-P were separately quantitated according to Wada and Snell (4). Except where noted in the text, only pyridoxal-P was formed at early incubation times. Rates are initial velocities from plots of product formed versus time. One unit of oxidase activity represents the formation of one nmol of pyridoxal-P/assay/hr. The type of inhibition was ascertained by Lineweaver-Burk plots, but the values for the Michaelis-Menten parameters were determined by replotting the data according to Cornish-Bowden and Eisenthal (12). RESULTSANDDISCUSSION: The Lineweaver-Burk plots for,pyridoxal-P of pyridoxamine-P oxidase in a 1% homogenate of rabbit ure 1. results,

As for the pure enzyme, the inhibition

is clearly

plus those for the pure enzyme and cytosol,

liver

inhibition

are shown in Fig-

competitive.

are summarized in Table 1.

Although the values for Kmvary somewhat, as has been seen previously probably reflects

differences

for Ki are insensitive

in the properties

of the solvent (lb),

to the presence of other cellular

985

These

(13) and the values

components. Only high

Vol. 83, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

PMP

RESEARCH COMMUNICATIONS

+,PLP

,,q

Figure 1: Lineweaver-Burk plots for oxidation of pyridoxsmine-P (PMP) and pyridoxine-P (PNP) by a 1% homogenate of rabbit liver in the absence (o,*) and presence (n,m) of 5 PM pyridoxal-P (PLP). The lines are reflected onto the positive abscissa to show the intercepts. TABLE 1. Michaelis Constants for Rabbit Liver Pyridoxamine-P Oxidase Under Various Conditions Substrate Pyridoxamm+P Pyridox;F;$ Km1 5tl1

Conditions

(IJN Pure enzyme in 0.2 M K phosphate (pH 8) Pure enzyme in-O.2 M K phosphate (pH 7) Pure enzyme in 0.5 M Tris-HCl (pH 8) Cytosol (6 mg) in 0.2 M K phosphate (pH 8)

4

25

3

24

3

16

3

11

19

24

-

15

3

17

4

Homogenate (237 mg) in 0.2 M K phosphate (PH 8)

11

3

17

3

*Calculated

inhibition.

concentrations

of Tris,

base with pyridoxal-P tivity

18

for competitive

an amino group-containing (4), increases the Ki.

buffer

that forms a Schiff

Neither HaOa nor NHa inhibit

at comparable concentrations. The formation of pyridoxal-P

from pyridoxine-P

986

by a 1% homogenate of

ac-

Vol. 83, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

Minutes

RESEARCH COMMUNICATIONS

of Reaction

Figure 2: Time-course of formation of PLP and pyridoxal (PL) from 100 $J¶ PNPby a 1% homogenate of rabbit liver in 50 mMHEPES(pH 7) in the absence (o,O) and presence (a,m) of 10 uM PLP. For ease of comparison, the content of PLP at zero-time was subtracted from subsequent determinations (&M). The formation of PL from 10 PM PLP (-) is also included. TABLR2. Michaelis Constants for Rat Liver 3c Pyridoxsmine-P Oxidase Under Various Conditions

Conditions

Substrate Pvridoxamine-P Pvridoxine-P "Km 'max k 'msx (uM) (Units)

Cytosol (55 mg) in 0.2 M K phosphate (pH 8) cytoso1 plus 5 pM pyridoxal-P Cytosol (55 mg) in 0.2 M K phosphate (pH 7) cytoso1 plus 5 pM pyridoxal-P Homogenate(133 mg) in 0.2 M K phosphate (PH 8) Homogenateplus 5 uM pyridoxal-P

8

(PM) (Units)

u5

3

195

20

150

10

245

__

__

8

260

--

--

18

345

8

100

4

165

39

140

22

220

*Since somehydrolysis of pyridoxal-P occurred, pyridoxal plus pyridoxal-P was used to calculate the velocity.

987

Vol. 83, No. 3, 1978

rabbit

liver

latter

inhibits

was examined using HEPES instead

pyridoxal-P product tion

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

alkaline

inhibited

was slow.

slightly

the initial

and reduced

pyridoxal-P

ing distortion

oxidase

of the curve

tion

inhibition

of Ki;

hibitor

however,

of the rat Inhibition

pyridoxyl

metabolism. liver

ine,

involved

was not involved level

of its

rate

because

formation

a true

of

the inhibi-

did not appear as

5.6 nM/hr.

acid,was

by product,

phosphatases

initial

times.

velocity

as is indicat-

of rat

liver

when considerable

This prevents

an exact

show that pyridoxal-P

spa hybridization

oxidase

estima-

is a powerful

relevant

if

in-

and that

that

this

components

binding

such binding

the oxidase

by rat

liver

Although

they conclude

50 @l pyridoxal-P

did not alter

(pH 7).

was decreased

their

or

inhibited (lo),

plus pyridoxthat

data actually

by 4076, as expected

inhi-

by a whole-

is strongly

cytosol

pre-

"trapped"

was shown by the data of Li --et al.

of pyridoxal-P

adding

out (10,16),

and nonspecific

here establish

and other

has been observed

cellular

by transaminases)

inhibition

by pyridoxal-P

at carbon-b*

as has also been pointed

reported

the oxidase

the equilibrium showed that

for product

inhi-

oxidase.

or not inhibition

of pyridoxal-P

removal

both Km and Vmsx, the latter

formed from pyridoxine,

of an Os-coupled Whether

is also inhibited

pyridoxsmine-P

and phosphate

of pyridoxal-P

the rate bition

liver

the synthesis

ATP, MgCls,

which

by the more-active

homogenate does not take place Similar

2, 10 nM

enzyme.

(e.g.

The results

by pyridoxal-P. which

either

However,

by specific

if

the

of 10 nM.pyridoxal-P

liver

increased

would not be physiologically

pyridoxal-P

of hydrolysis

to pyridoxic

at even early

of rabbit

(4,11,15).

bition

liver

as expected

oxidation

the data clearly

compounds with

viously

rate

in determining occurs

by k$,

of 10 nM pyridoxal,

from rat

Pyridoxal-P

or due to difficulties product

the apparent

and, hence, may reflect

shown in Table 2.

since

As can be seen in Figure

velocity

of disappearance

Pyridoxsmine-P

buffer,

of 2 mM ATP and 0.5 ml4 MgCls increased

Addition

The rate

by 33%

(6).

phosphatase

of phosphate

formation

of pyridoxamine-P from pyridoxamine-P

988

oxidase

regulates

and pyridoxine-P

the --in vivo will

depend

BIOCHEMICAL

Vol. 83, No. 3, 1978

on the relative

and absolute concentrations

studies -in vivo and -in vitro relatively

insensitive

compoundsprimarily centrations

AND BIOPHYSICAL

to mice, approximately

kinase is

and maintains cellular

in the phosphorylated forms. low; even after

Both

of substrates and products.

have found that pyridoxal

to product inhibition

are relatively

ug) of pyridoxine

(2,l7-20)

RESEARCH COMMUNICATIONS

pyridoxyl

Nonetheless, substrate con-

administration

of high levels

(165

3.3 ng (16 nmol) of pyridoxine-P

appears in the liver.

Pyridoxal-P

concentrations,

mally be significant.

In addition

to short-term accumulation of labeled pyri-

aoxal-P after

in viva administration --

on the other hand, may nor-

of radioactive

pyridoxine

high basal levels,

20 to 70 nmol/g wet weight (10,17,20,21),

found in rat liver.

Of this basal content, -12046is readily

cellulose

chromatography (22), *5$

of pyridoxal-P

liver

information

is not available

is removed by exhaustive dialysis

(24) is sufficiently

and distribution of pyridoxal-P Overall,

liver,

(lo),

may be analogous in these species.

and

Although

the Be content of rabbit

similar to the rat (25) that the pyridoxal-P

content

Hence, from 4 to 14 nmol

is present/g wet weight in a free or loosely bound form. these results

establish that product inhibition

oxidase is not merely a property vivo.

for rabbit

are

removed by DEAE-

55 to 6oq6 is removed (20) by apotryptophanase (Km = 0.7 mM) (23).

similar

fairly

(18,19),

Complete evaluation

metabolism awaits further

of the relative work.

whereas protein binding will pyridoxamine-P oxidase will

of the purified

of pyridoxamine-P

enzyme but also can occur -in

contributions

of each step in Bs

However, the evidence thus far suggests that,

determine the basal level play a kinetic

of pyridoxal-P

role in regulating

(LO),

pyridoxal-P

formation. IXZFERBNCES 1. Hurwitz, J. (1953) J. Biol. Chem. 205: 935-947. 2. McCormick, D.B., Gregory, M.E., andTell, E.E. (1961) J. Biol. cbem. 236: 2076-2084. Pogell, B.M. (1958) J. Biol. Chem. 232: 761-776. ;: Wada, H. and Snell, E.E. (1961) J. sl. Chem.236: 2089-2095. 5. Wada, H., Morisue, T., Nishimura, Y., Morino, Y., Saksmoto, Y., and Ichihara, K. (1959) Proc. Japan Acad. 35: 299-304. 6. Lumeng, L. and Li, T.K. (1975) J. BiolrChem. 250: 8126-8131. Schwartz, R. and Kjeldgaard, N.O. (1951) BiochG J. 48: 333-337. 87: Stanulovic, M., Jeremic, V., Leskovas, V., and ChaykinyS. (1976) Enzyme -21: 357-369. 989

Vol. 83, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

9. Snell, E.E. and Haskell, B.E. (1971) Camp. Biochem. 21: 47-67. 10. Li, T.K., Lumeng, L., and Veitch, R.L. (1974) BiochK Biophys. Res. ccmmun.g: 677-684. ll. Kazarinoff, M.N. and McCormick, D.B. (1975) J. Biol. Chem. 250: 3&36-j&42. 12. Cornish-Bowden, A. and Eisenthal, R. (1974) Biochem. J. ~~21-730. 13. Bell, R.R. and Haskell, B.E. (1971) Arch. Biochem. Biophys. 147: 588-601. 14. Horiike, K., Merrill, A.H., and McCormick, D.B. (1978) Biochzstry, submitted.

15. Korytnyk, W., Hakala, M.T., Potti, P.G.G., Angelino, N., and Chang, S.C. (1976) Biochemistry 15: 5458-5466. 16. Anonymous (1975) Nut;;; Rev. 3: 214-217. 17. Lumeng, L. and Li, T.K. (1978f Fed. Proc. x: 1976. : 533-538. 18. Colcmbini, C.E. and McCoy, E.E. (1970) Biochemistry 19. Johansson, S., Lindstedt, S., and Tiselius, H.G. (197 9 ) J. Biol. Chea. 249: 6040-6049. 20. Bukin, Y.V. (1976) Biokhimiya 41: 81-90. 21. Lloyd, M.E. (1964) Masters Thezs, Cornell University, Ithaca, NY, p, 36.

22. Bosron, W.F., Veitch, R.L., Lumeng, L., and Li, T.K. (1978) J. Biol. Chem. 253: 1488-l&2. Y. and Snell, E.E. (1967) Proc. Natl. Acad. Sci. USA 57: 1692-1699. 23. Kino, 24. Hove, E.L. and Herndon, J.F. (1957) J. Nutr. 61: ~7-136. 25. Beaton, G.H. and McHenry, E.W. (1953) Brit. JyNutr. 1: 357-363.

990