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.
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