- Email: [email protected]
Mannose-6-P and mannose-1-P in rat brain, kidney and liver
Vol.
89,
No.
1, 1979
July
12, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 279-285
MANNOSE-6-P AND MANNOSE-1-P Nur Asikin" Oklahoma Oklahoma State Received
IN RAT BRAIN, & Roger
KIDNEY AND LIVER
E. Koeppe
Department of Biochemistry Agricultural Experiment University, Stillwater,
Station Oklahoma
74074
#June 4,1979
Summary The approximate concentrations of mannose-6-phosphate and mannose-l-phosphate in female rat brain, kidney and liver are (Man-6-P), and 13, 12, 15 respectively 51, 29 and 99 nmole/g nmol/g Wan-l-P). Intraperitoneal injection of mannose (20 mmol/kg body weight, 15, 30 or 60 min before sacrifice) raises the liver Man-6-P to 0.4 to 4.3 pmol/g and Man-l-P to 100 to 186 nmol/g. Mannose
injection
of
hypoglycemia
to
normal
has
resulting
the by
results
interpreted
to
glucose
tissues
However,
in
1972,
Sloviter
mannose
can
replace
glucose
rat
brain.
Man-6-P
These jin
isolation erythrocytes
it
converted
lished mammals
in
to
is
is
the
(1)
brains
and of
(2).
(3) substrate
also
determined
the
that
mammals Man-6-P,
Man-l-P,
has
brain.
for
that
perfused
concentration
of
been
its
found
in
(5). formed
a synthetic
not
the
reported
has
evidence
microorganisms,
These
and described
Man-1,6-P2
and various
Man-l-P
(3)
made
converted
by
an energy
(4).
is
utilization
brain
restore
rabbits
mannose
colleagues
rat
symptoms to
injection
as
presumed
limited.
relieve
that to
and
GDP-Man via
certain
evidence prior
tissue
humans
of insulin
and normal
this
of
Although is
authors
perfused from
or as
to
hepatectomy
activity
hepatectomy
by other
shown
from
electrical
hypoglycemic were
been
for
been
from
Fru-6-P,
pathway this
reported
estab-
sequence in
in
animal
"Present address: Dept. University of Indonesia, Journal Article ment Stat.ion.
No.
of Biochemistry, School of Medicine, Salemba 6, Jakarta, Indonesia. J-3656 of the Oklahoma Agricultural Experi-
0006-291X/79/130279-07$01.00/0 279
Vol.
89,
No.
tissues, and
PMI+
mammalian
catalyze
the
physiological we report rat
BIOCHEMICAL
1, 1979
tissues,
of
origin
PMM has
not
interconversion role
the
animal
in
this
concentrations with
AND
and without
BIOPHYSICAL
has been
not
been
detected.
of
Man-6-P
reaction of
RESEARCH
mannose
extensively
studied
Although and
remains
Man-6-P
COMMUNICATIONS
and
PGM can
Man-l-P in
(6),
doubt.
Man-l-P
its Herein
in
several
loading.
Methods Materials - Glucose, fructose and perchloric acid were from J. T. Baker; ATP, NADP, hexose-phosphates, mannose and triethanolamine base from Sigma; PM1 from Boehringer, the other enzymes from either Boehringer or Sigma; charcoal (Norit A) from Matheson. All solutions used in fluorometric assays were filtered through a 0.45 p Millipore filter. Preparation of Tissue Extracts - Female Holtzmann rats were used throughout; the sucklings' sex was not determined. Animals were in the laboratory with access to food and water and acclimated to handling for at least two days before being killed; usually at lo-12 A.M. Under mild ether anesthesia, skin above the forehead was removed and the abdominal cavity exposed. The rat was then immersed in liquid nitrogen and desired tissues chiseled from the frozen carcass. When brain was not required, tissues were excised from the anesthetized animal and pressed with a precooled pestle in a mortar filled with liquid nitrogen; a flattened tissue was obtained within 5 set after excision. Mannose was injected intraperitoneally under ether anesthesia, at a dose of 20 mmol/kg body weight given as a 25% solution. The animal was allowed to recover and after an appropriate interval was reanesthetized and killed. After removal of fat and excess blood tissues were put in a steel mortar surrounded verized, transferred to a glass homogenizing ice and weighed.
(without thawing), by dry ice, pultube cooled in dry
Protein-free tissue extracts were prepared according to Williamson and Corkey (7). When Man-l-P was to be determined, neutralization was done with 5M KOH; the use of buffer is a disadvantage here because of the requirement for alkaline and acid hydrolysis. Since some extracts of liver and kidney were yellowish-brown and highly fluorescent, they were decolorized with charcoal (8). Assay of Metabolites - The resence of the NADPH formed (7)
increase in absorbance or was measured. The validity
+Enzyme abbreviations: HK, hexokinase; PGI, phosphoglucose PMI, phosphomannose isomerase; PMM, phosphomannoisomerase; mutase; PGM, phosphoglucomutase; G6PDH, glucose-6-phosphate dehydrogenase.
280
fluoof
Vol.
89,
No.
‘I, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
assay was checked with internal mined by addition of metaboljtes reaction:; were as follows:
RESEARCH
COMMUNICATIONS
standards and recoveries deterto some tissue samples. The
Glc-1-P
Man-6-P i Man
PM1 ~Fru-6-P
H‘K>
+ Mg++
I
PGI -Glc-6-P ATP
F&l
i Glc
All assays were done at 30° mine buffer, pH 7.6, containing MF$~~, 3 mM; ATP, 500 FM; NADP, (yeast), 1.5 U; PGI (yeast), 1 G6PDH (yeast), 1 IT. Some batches of was added before PM1 give an overestimate tor of PGM, was added
PGM G6PDH -7NADP
6-P-G1cA NADPH
In . 2 ml of 0.1 N triethanolathe following as appropriate: 400 PM; PM1 (yeast), 1.5 U; HK U; PGM (rabbit muscle), 1 U;
PM1 contain PGM activitv, therefore PGM to remove any Glc-1-P present which might of Man-6-P. In this case EDTA, an activaat a final concentration of 0.1 mM.
Glucose was measured by the addition of HK after measurement of Glc-6-P. Addition of PGI gave fructose and Fru-6-P and addition of PMI, mannose and Man-6-P. Free fructose and mannose were then obtained by subtracting the Fru-6-P and Man-6-P readjngs in the absence of HK, from those in its presence, respectively. Determination of Man-l-P - Without PMM, mannose-1-P cannot be assayed enzymatically using the system described above. Therefore three other methods were tried; they gave similar results. Two of them involved enzymatic determination of mannose rel.eased from Man-l-P by enzymatic or acid hydrolysis, after removal of hexoses and their 6-phosphates by 1) enzymatic conversion to 6-P-gluconate or 2) reduction to the corresponding polyols with NaBH4. In ithe method of choice, advantage was taken of the differences in stability towards and acid alkali of hexose-lphosphates and reducing sugars (free hexoses and their 6-phosphates. Hexose-l-phosphates are stable in alkali while acid hydrolysis releases an equivalent amount of phosphate and the respective free sugar. Reducing sugars on the other hand are relatively stable in dilute acid whereas treatment with alkali results in degradation products, among others, phosphate, saccharinic acids and lactate. The reducing sugars were first destroyed by boiling in a water bath for 30 min in 0.05 N KOH. Acid was added, HCl or N, and hydrolysis was achieved by heating in H2S04 > to 0.1 boiling vwater for 20 min. The acid hydrolysate was decolorized with charcoal before neutralization and assay. after
Neither free hexoses nor treatment with alkali.
hexose-6-phosphates were After acid hydrolysis,
281
detected Glc-6-P
Vol.
89,
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Man-6-P were detected, presumably due to the presence of the respective 1,6-bisphosphates. Free glucose and mannose, detected after acid hvdrolysis (above), originated from the respective l-phosphates: In standard runs known Man-l-P was quantitatively recovered as mannose. The sequence
of
[Hexose Hexose-1-P Hexose-6-P Hexose-(1,6)--P2 UDP-Glc
events
is:
OH-
1
Hexose-1-P Hexose-(1,6)-P2
a
H+ Hexose -Hexose-6-P A
Addition of amino acids to standard runs did not cause interference. GDP-Man, if present, might contribute to the mannose measured after acid hydrolysis. However, no mannose was detected when a solution of GDP-Man was put through the procedure. Similar treatment of UDP-Glc, which differs from GDP-Man at the hydroxyl group on C2 of the hexose, yielded glucose in the acid hydrolysate. That hexose-1-P is present in an alkaline hydrolysate of a liver extract was verified by separating the sugar phosphates from other intermediates by anion exchange chromatography. Enzymatic analysis showed hexose-6-P to be absent. Hydrolysis of the hexose-P peak gave a ratio of glucose + mannose:P of i 0.8. Results The
results
concerning
the
are
are
not
animals
are
in
are (10).
fact
used
concentrations hence
ranges
and
not
assay 60 vary
the
data.
In normal
female
rats
the
23 nmol/g
* The authors are indebted for supplying the freeze
from
normal
reported
by
several
215
g.
those
of
However,
to
100 Veech
several
brain * others tissue size
of
metabolite animal
size;
of
Man-6-P
concentration
kidney
of
The
with
to Drs. Richard blown brains.
282
blown
satisfactory.
average in
freeze
methodology
substantially
combined
about
obtain
to,
our is
to
we have
from
we
from
in
data
Fru-6-P
comparable that
Considerable
and
those
obtained
but
from
did
with
those
indicates
varied
I.
The values
than,
extraction
Table
Glc-6-P
agreement
(9); lower
This
freezing, animal
good
in of
presented.
laboratories slightly
presented
concentration
tissues
other
and Discussion
nmol/g
in
liver.
and Todd King
Vol.
89,
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
Table
RESEARCH
COMMUNICATIONS
1
Concentration (nmol/g) of Mannose, Man-6-P and Man-l-P in Rat Tissue Treatment
Brain __suckling freeze blown normal
f mannose
15 min
30 min 60 min
Man-6-P
Man-l-P
Mannose
41,52 51 * 3 (3)" 21 f 4 (5)
11,14,15
0
85-162
15,28
(4)
58
14
2300-3900 4500 1800
32 29 2 4 (6)
12,lZ
0
14,28
35100,35000
112,195
(3)
Kidney suckling
normal + mannose 15 min 30 min
23,24,44 53,82,107 32
60 min
15100
Liver -suckling norma
48,54 99 -+ 6 (9)
1
+ rnannose 15 min 30 min 60 min
410-2620 790-4310 1010,156O
(5) (4)
15 +- 3 (5)
0
100,134
11000-27500 10600 6900,247OO
186
Blood -control + rnannose 15 min 60
0 5600,120OO 7350
min
-parentheses are the number of observations values or used in calculation of S.E.M.
The
brain
killed brain
Man-6-P in
are
brains*
this
value
hands
the
latter
value
for
Man-6-P
and brains
values
colleagues perfused
need
laboratory
approximately
the
(4)
is
(see
approximately seems rat
When the
Methods)
21 nmol/g.
figure in
comment.
in
However,
brain.
be
the
with
found
considerably
with
either
glucose
283
(Table more
We should
(3)
of
animals
were
Man-6-P
51 nmol/g to
a range
note
more or mannose.
values
for
freeze
blown
I).
In our
nearly
correct
that
Sloviter
Man-6-P
in
rat
The reason
for
Vol.
89,
the
No.
difference
between It
apparent. in
BIOCHEMICAL
1, 1979
brain et
nmol/g)
al.
PM1 reaction 1;
Our
is
the
direction
the
time
the
data presence
concentrations
of
of
lo-15
(Man-l-P
---
(Keq
see
in
+2+ 10,
61,
somewhat
removed
from
However,
because
the
from
equilibrium
Some animals manner
similar wi-th
Treatment little
effect
liver, istration with
mannose
about
1.5
magnitude
the
in animal,
Umol/g
blood changes
resulted of
at
li~ver
of
the
is
Man-l-P. apparent
concentrations
to
it
loading.
concentrations
of
the
fructose
adminLoading
liver. concentration extent
mannose
of
in
brain
administration,
In another,
Man-6-P
60 min
concentration umol /g.
was Although
concentrations
in
did
order
of
not
a
had
of
pmol/g.
in
sacrifice
a lesser
24.7
fructose
mannose
though
in
to
fructose mM,
or
content
about
284
tissues
this
for
shown),
liver
in
the
reaction
limited,
Man-l-P
30 min after
several in
or
mannose
this
prior
exceeded4
and
that
equilibrium
direction
(Methods)
and
administration,
c
for
in
be near
glucose
increase
liver
to
are
Man-l-P
a striking
suggest
Whereas
the
with
not
the
(Keq
rat
brain
fructose
(data
the
be overemphasized.
Man-6-P
brain
one
loading and
or
concentration
mannose
brain
glucose
increase
In
after
fructose
described
Man-6-P
I).
Man-6-P
that
caused
and
(Table the
or
did
Man-l-P
injected
on
kidney
not
were
to
and
data
should
by
brain
report
three
I).
in
Man-l-P
rat
al.
we
seems
equilibrium
reported
in
et
in
(Table
kidney
concentrations
equilibrium
limited,
M-6-P)
immediately
Man-6-P.
Man-l-P
nmol/g
PMM reaction
near
Sloviter of
not
nmol/g
that
is
more
is
COMMUNICATIONS
Fru-6-P
indicate
of
are
ours
12-15
Fru-6-P)
results
in
and
the
data
----
RESEARCH
we found
to
the
Although
shift
that
whereas
reaction
first
(3).
BIOPHYSICAL
values
close
(Man-6-P
see 11)
this
their
may be noted
(19
Sloviter
AND
cause
Fru-6-P
(data
liver, of not
Vol.
89,
No.
shown). of
BIOCHEMICAL
1, 1979
We conclude
mannose,
rapidly
rat
forms
tions trations
liver,
of
of
step
of in
ing to liver been
note
are
several
(Ki
be due which unable
the
in
= 0.46
extent very
liver
high
it
for
brain,
concentrathe
following
Ki
Fru-6-P
rat
Since
the
mM; 121,
concentrations
concenmannose
Man-6-P
as
may be inhibiting
this were
concentrations
an
not
shown).
the
loading to
lesser to
COMMUNICATIONS
large
removal.
fold
although
not
a
to
accumulates
observe
is
mannose
which
to
we
(data
Of
and
RESEARCH
exposed
Man-6-P
glycolysis,
elevated
when
rapid
PGI
a
BIOPHYSICAL
less
administration inhibitor
that
Man-6-P
because
AND
large
increase
(Table
I).
presence
interconverts to detect
in
At
of
PMM in
present
an as yet
Man-6-P rat
Man-l-P this
in
liver
must
undescribed
and Man-l-P.
followbe assumed PMM in
To date
rat
we have
tissues.
References 1. 2. 3. 4. 5. 6. 7.
a. 9. 10. 11. 12.
Mann, F. C. (1925) Ergebn. der Physiol. 24, 379-384. Maddock, S., Hawkins, J. E., Jr., and Holmes, E. (1939) Am. J. Physiol. 125, 551-565. 11972) J. Gosh, A. K., Mukherji, B., and Sloviter, H. A. Neurochem. 19, 1279-1285. Saraswathi, S., Gosh, A. K., and Sloviter, H. A. (1973) Fed. Proc. 32, 525. Bartlett, G. R. (1968) Biochim. Biophys. Acta 156, 231239. Lowry, (1969) J. Biol. 0. H. annd Passonneau, .J. V. Chem. 244, 910-916. Williamson, J. R., and Corkey, B. E. (1969) Methods Enzymol. m, 434-513. Hurlbert, R. B. (19571 Methods Enzymol. III, 793. Williamson, D. H., and Brosnan, J. T. (19m Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) vol 4, pp. 2266-2302, Academic Press, New York. Ruderman, N. B., Ross, P. S., Berger, M., and Goodman, M. N. (1974) Biochem. J. 138, l-10. (1968) J. Biol. Chem. Gracy, R. W., and Noltmann, E. A. 243, 5410-5419. Sal'as, M., Vinuela, E., and Sols, A. (1965) J. Biol. Chem. 3, 561-568.
285
89,
No.
1, 1979
July
12, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 279-285
MANNOSE-6-P AND MANNOSE-1-P Nur Asikin" Oklahoma Oklahoma State Received
IN RAT BRAIN, & Roger
KIDNEY AND LIVER
E. Koeppe
Department of Biochemistry Agricultural Experiment University, Stillwater,
Station Oklahoma
74074
#June 4,1979
Summary The approximate concentrations of mannose-6-phosphate and mannose-l-phosphate in female rat brain, kidney and liver are (Man-6-P), and 13, 12, 15 respectively 51, 29 and 99 nmole/g nmol/g Wan-l-P). Intraperitoneal injection of mannose (20 mmol/kg body weight, 15, 30 or 60 min before sacrifice) raises the liver Man-6-P to 0.4 to 4.3 pmol/g and Man-l-P to 100 to 186 nmol/g. Mannose
injection
of
hypoglycemia
to
normal
has
resulting
the by
results
interpreted
to
glucose
tissues
However,
in
1972,
Sloviter
mannose
can
replace
glucose
rat
brain.
Man-6-P
These jin
isolation erythrocytes
it
converted
lished mammals
in
to
is
is
the
(1)
brains
and of
(2).
(3) substrate
also
determined
the
that
mammals Man-6-P,
Man-l-P,
has
brain.
for
that
perfused
concentration
of
been
its
found
in
(5). formed
a synthetic
not
the
reported
has
evidence
microorganisms,
These
and described
Man-1,6-P2
and various
Man-l-P
(3)
made
converted
by
an energy
(4).
is
utilization
brain
restore
rabbits
mannose
colleagues
rat
symptoms to
injection
as
presumed
limited.
relieve
that to
and
GDP-Man via
certain
evidence prior
tissue
humans
of insulin
and normal
this
of
Although is
authors
perfused from
or as
to
hepatectomy
activity
hepatectomy
by other
shown
from
electrical
hypoglycemic were
been
for
been
from
Fru-6-P,
pathway this
reported
estab-
sequence in
in
animal
"Present address: Dept. University of Indonesia, Journal Article ment Stat.ion.
No.
of Biochemistry, School of Medicine, Salemba 6, Jakarta, Indonesia. J-3656 of the Oklahoma Agricultural Experi-
0006-291X/79/130279-07$01.00/0 279
Vol.
89,
No.
tissues, and
PMI+
mammalian
catalyze
the
physiological we report rat
BIOCHEMICAL
1, 1979
tissues,
of
origin
PMM has
not
interconversion role
the
animal
in
this
concentrations with
AND
and without
BIOPHYSICAL
has been
not
been
detected.
of
Man-6-P
reaction of
RESEARCH
mannose
extensively
studied
Although and
remains
Man-6-P
COMMUNICATIONS
and
PGM can
Man-l-P in
(6),
doubt.
Man-l-P
its Herein
in
several
loading.
Methods Materials - Glucose, fructose and perchloric acid were from J. T. Baker; ATP, NADP, hexose-phosphates, mannose and triethanolamine base from Sigma; PM1 from Boehringer, the other enzymes from either Boehringer or Sigma; charcoal (Norit A) from Matheson. All solutions used in fluorometric assays were filtered through a 0.45 p Millipore filter. Preparation of Tissue Extracts - Female Holtzmann rats were used throughout; the sucklings' sex was not determined. Animals were in the laboratory with access to food and water and acclimated to handling for at least two days before being killed; usually at lo-12 A.M. Under mild ether anesthesia, skin above the forehead was removed and the abdominal cavity exposed. The rat was then immersed in liquid nitrogen and desired tissues chiseled from the frozen carcass. When brain was not required, tissues were excised from the anesthetized animal and pressed with a precooled pestle in a mortar filled with liquid nitrogen; a flattened tissue was obtained within 5 set after excision. Mannose was injected intraperitoneally under ether anesthesia, at a dose of 20 mmol/kg body weight given as a 25% solution. The animal was allowed to recover and after an appropriate interval was reanesthetized and killed. After removal of fat and excess blood tissues were put in a steel mortar surrounded verized, transferred to a glass homogenizing ice and weighed.
(without thawing), by dry ice, pultube cooled in dry
Protein-free tissue extracts were prepared according to Williamson and Corkey (7). When Man-l-P was to be determined, neutralization was done with 5M KOH; the use of buffer is a disadvantage here because of the requirement for alkaline and acid hydrolysis. Since some extracts of liver and kidney were yellowish-brown and highly fluorescent, they were decolorized with charcoal (8). Assay of Metabolites - The resence of the NADPH formed (7)
increase in absorbance or was measured. The validity
+Enzyme abbreviations: HK, hexokinase; PGI, phosphoglucose PMI, phosphomannose isomerase; PMM, phosphomannoisomerase; mutase; PGM, phosphoglucomutase; G6PDH, glucose-6-phosphate dehydrogenase.
280
fluoof
Vol.
89,
No.
‘I, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
assay was checked with internal mined by addition of metaboljtes reaction:; were as follows:
RESEARCH
COMMUNICATIONS
standards and recoveries deterto some tissue samples. The
Glc-1-P
Man-6-P i Man
PM1 ~Fru-6-P
H‘K>
+ Mg++
I
PGI -Glc-6-P ATP
F&l
i Glc
All assays were done at 30° mine buffer, pH 7.6, containing MF$~~, 3 mM; ATP, 500 FM; NADP, (yeast), 1.5 U; PGI (yeast), 1 G6PDH (yeast), 1 IT. Some batches of was added before PM1 give an overestimate tor of PGM, was added
PGM G6PDH -7NADP
6-P-G1cA NADPH
In . 2 ml of 0.1 N triethanolathe following as appropriate: 400 PM; PM1 (yeast), 1.5 U; HK U; PGM (rabbit muscle), 1 U;
PM1 contain PGM activitv, therefore PGM to remove any Glc-1-P present which might of Man-6-P. In this case EDTA, an activaat a final concentration of 0.1 mM.
Glucose was measured by the addition of HK after measurement of Glc-6-P. Addition of PGI gave fructose and Fru-6-P and addition of PMI, mannose and Man-6-P. Free fructose and mannose were then obtained by subtracting the Fru-6-P and Man-6-P readjngs in the absence of HK, from those in its presence, respectively. Determination of Man-l-P - Without PMM, mannose-1-P cannot be assayed enzymatically using the system described above. Therefore three other methods were tried; they gave similar results. Two of them involved enzymatic determination of mannose rel.eased from Man-l-P by enzymatic or acid hydrolysis, after removal of hexoses and their 6-phosphates by 1) enzymatic conversion to 6-P-gluconate or 2) reduction to the corresponding polyols with NaBH4. In ithe method of choice, advantage was taken of the differences in stability towards and acid alkali of hexose-lphosphates and reducing sugars (free hexoses and their 6-phosphates. Hexose-l-phosphates are stable in alkali while acid hydrolysis releases an equivalent amount of phosphate and the respective free sugar. Reducing sugars on the other hand are relatively stable in dilute acid whereas treatment with alkali results in degradation products, among others, phosphate, saccharinic acids and lactate. The reducing sugars were first destroyed by boiling in a water bath for 30 min in 0.05 N KOH. Acid was added, HCl or N, and hydrolysis was achieved by heating in H2S04 > to 0.1 boiling vwater for 20 min. The acid hydrolysate was decolorized with charcoal before neutralization and assay. after
Neither free hexoses nor treatment with alkali.
hexose-6-phosphates were After acid hydrolysis,
281
detected Glc-6-P
Vol.
89,
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Man-6-P were detected, presumably due to the presence of the respective 1,6-bisphosphates. Free glucose and mannose, detected after acid hvdrolysis (above), originated from the respective l-phosphates: In standard runs known Man-l-P was quantitatively recovered as mannose. The sequence
of
[Hexose Hexose-1-P Hexose-6-P Hexose-(1,6)--P2 UDP-Glc
events
is:
OH-
1
Hexose-1-P Hexose-(1,6)-P2
a
H+ Hexose -Hexose-6-P A
Addition of amino acids to standard runs did not cause interference. GDP-Man, if present, might contribute to the mannose measured after acid hydrolysis. However, no mannose was detected when a solution of GDP-Man was put through the procedure. Similar treatment of UDP-Glc, which differs from GDP-Man at the hydroxyl group on C2 of the hexose, yielded glucose in the acid hydrolysate. That hexose-1-P is present in an alkaline hydrolysate of a liver extract was verified by separating the sugar phosphates from other intermediates by anion exchange chromatography. Enzymatic analysis showed hexose-6-P to be absent. Hydrolysis of the hexose-P peak gave a ratio of glucose + mannose:P of i 0.8. Results The
results
concerning
the
are
are
not
animals
are
in
are (10).
fact
used
concentrations hence
ranges
and
not
assay 60 vary
the
data.
In normal
female
rats
the
23 nmol/g
* The authors are indebted for supplying the freeze
from
normal
reported
by
several
215
g.
those
of
However,
to
100 Veech
several
brain * others tissue size
of
metabolite animal
size;
of
Man-6-P
concentration
kidney
of
The
with
to Drs. Richard blown brains.
282
blown
satisfactory.
average in
freeze
methodology
substantially
combined
about
obtain
to,
our is
to
we have
from
we
from
in
data
Fru-6-P
comparable that
Considerable
and
those
obtained
but
from
did
with
those
indicates
varied
I.
The values
than,
extraction
Table
Glc-6-P
agreement
(9); lower
This
freezing, animal
good
in of
presented.
laboratories slightly
presented
concentration
tissues
other
and Discussion
nmol/g
in
liver.
and Todd King
Vol.
89,
No.
1, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
Table
RESEARCH
COMMUNICATIONS
1
Concentration (nmol/g) of Mannose, Man-6-P and Man-l-P in Rat Tissue Treatment
Brain __suckling freeze blown normal
f mannose
15 min
30 min 60 min
Man-6-P
Man-l-P
Mannose
41,52 51 * 3 (3)" 21 f 4 (5)
11,14,15
0
85-162
15,28
(4)
58
14
2300-3900 4500 1800
32 29 2 4 (6)
12,lZ
0
14,28
35100,35000
112,195
(3)
Kidney suckling
normal + mannose 15 min 30 min
23,24,44 53,82,107 32
60 min
15100
Liver -suckling norma
48,54 99 -+ 6 (9)
1
+ rnannose 15 min 30 min 60 min
410-2620 790-4310 1010,156O
(5) (4)
15 +- 3 (5)
0
100,134
11000-27500 10600 6900,247OO
186
Blood -control + rnannose 15 min 60
0 5600,120OO 7350
min
-parentheses are the number of observations values or used in calculation of S.E.M.
The
brain
killed brain
Man-6-P in
are
brains*
this
value
hands
the
latter
value
for
Man-6-P
and brains
values
colleagues perfused
need
laboratory
approximately
the
(4)
is
(see
approximately seems rat
When the
Methods)
21 nmol/g.
figure in
comment.
in
However,
brain.
be
the
with
found
considerably
with
either
glucose
283
(Table more
We should
(3)
of
animals
were
Man-6-P
51 nmol/g to
a range
note
more or mannose.
values
for
freeze
blown
I).
In our
nearly
correct
that
Sloviter
Man-6-P
in
rat
The reason
for
Vol.
89,
the
No.
difference
between It
apparent. in
BIOCHEMICAL
1, 1979
brain et
nmol/g)
al.
PM1 reaction 1;
Our
is
the
direction
the
time
the
data presence
concentrations
of
of
lo-15
(Man-l-P
---
(Keq
see
in
+2+ 10,
61,
somewhat
removed
from
However,
because
the
from
equilibrium
Some animals manner
similar wi-th
Treatment little
effect
liver, istration with
mannose
about
1.5
magnitude
the
in animal,
Umol/g
blood changes
resulted of
at
li~ver
of
the
is
Man-l-P. apparent
concentrations
to
it
loading.
concentrations
of
the
fructose
adminLoading
liver. concentration extent
mannose
of
in
brain
administration,
In another,
Man-6-P
60 min
concentration umol /g.
was Although
concentrations
in
did
order
of
not
a
had
of
pmol/g.
in
sacrifice
a lesser
24.7
fructose
mannose
though
in
to
fructose mM,
or
content
about
284
tissues
this
for
shown),
liver
in
the
reaction
limited,
Man-l-P
30 min after
several in
or
mannose
this
prior
exceeded4
and
that
equilibrium
direction
(Methods)
and
administration,
c
for
in
be near
glucose
increase
liver
to
are
Man-l-P
a striking
suggest
Whereas
the
with
not
the
(Keq
rat
brain
fructose
(data
the
be overemphasized.
Man-6-P
brain
one
loading and
or
concentration
mannose
brain
glucose
increase
In
after
fructose
described
Man-6-P
I).
Man-6-P
that
caused
and
(Table the
or
did
Man-l-P
injected
on
kidney
not
were
to
and
data
should
by
brain
report
three
I).
in
Man-l-P
rat
al.
we
seems
equilibrium
reported
in
et
in
(Table
kidney
concentrations
equilibrium
limited,
M-6-P)
immediately
Man-6-P.
Man-l-P
nmol/g
PMM reaction
near
Sloviter of
not
nmol/g
that
is
more
is
COMMUNICATIONS
Fru-6-P
indicate
of
are
ours
12-15
Fru-6-P)
results
in
and
the
data
----
RESEARCH
we found
to
the
Although
shift
that
whereas
reaction
first
(3).
BIOPHYSICAL
values
close
(Man-6-P
see 11)
this
their
may be noted
(19
Sloviter
AND
cause
Fru-6-P
(data
liver, of not
Vol.
89,
No.
shown). of
BIOCHEMICAL
1, 1979
We conclude
mannose,
rapidly
rat
forms
tions trations
liver,
of
of
step
of in
ing to liver been
note
are
several
(Ki
be due which unable
the
in
= 0.46
extent very
liver
high
it
for
brain,
concentrathe
following
Ki
Fru-6-P
rat
Since
the
mM; 121,
concentrations
concenmannose
Man-6-P
as
may be inhibiting
this were
concentrations
an
not
shown).
the
loading to
lesser to
COMMUNICATIONS
large
removal.
fold
although
not
a
to
accumulates
observe
is
mannose
which
to
we
(data
Of
and
RESEARCH
exposed
Man-6-P
glycolysis,
elevated
when
rapid
PGI
a
BIOPHYSICAL
less
administration inhibitor
that
Man-6-P
because
AND
large
increase
(Table
I).
presence
interconverts to detect
in
At
of
PMM in
present
an as yet
Man-6-P rat
Man-l-P this
in
liver
must
undescribed
and Man-l-P.
followbe assumed PMM in
To date
rat
we have
tissues.
References 1. 2. 3. 4. 5. 6. 7.
a. 9. 10. 11. 12.
Mann, F. C. (1925) Ergebn. der Physiol. 24, 379-384. Maddock, S., Hawkins, J. E., Jr., and Holmes, E. (1939) Am. J. Physiol. 125, 551-565. 11972) J. Gosh, A. K., Mukherji, B., and Sloviter, H. A. Neurochem. 19, 1279-1285. Saraswathi, S., Gosh, A. K., and Sloviter, H. A. (1973) Fed. Proc. 32, 525. Bartlett, G. R. (1968) Biochim. Biophys. Acta 156, 231239. Lowry, (1969) J. Biol. 0. H. annd Passonneau, .J. V. Chem. 244, 910-916. Williamson, J. R., and Corkey, B. E. (1969) Methods Enzymol. m, 434-513. Hurlbert, R. B. (19571 Methods Enzymol. III, 793. Williamson, D. H., and Brosnan, J. T. (19m Methods of Enzymatic Analysis (H. U. Bergmeyer, ed.) vol 4, pp. 2266-2302, Academic Press, New York. Ruderman, N. B., Ross, P. S., Berger, M., and Goodman, M. N. (1974) Biochem. J. 138, l-10. (1968) J. Biol. Chem. Gracy, R. W., and Noltmann, E. A. 243, 5410-5419. Sal'as, M., Vinuela, E., and Sols, A. (1965) J. Biol. Chem. 3, 561-568.
285