Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
February 28, 1978
Pages
750-758
RADIOENZYMIC ASSAY OF GLYCEROL USING ION-EXCHANGE COLUMN CHROMATOGRAPHY Luis A. Barrera and Ren-jye Ho* of Biochemistry, University of Miami School Miami, Florida
Department
Received
of Medicine
December 5,1977
SUMMARY: Modifications of the glycerol kinase radioenzymic assay for glycerol are This method can be readily employed to measure glycerol kinase activity described. Ion-exchange column chromatography (QAE-Sephadex A-25) in tissue extracts as well. and completely separates the productlkc-glycerol 3-phosphate from lkC!-glycerol, allows all glycerol 3-phosphate formed in an assay to be counted in a single Increasing the Mg2+ concentration significantly increases activity counting vial. These modifications of glycerol kinase and thus the sensitivity of the assay. sensitive and more rapid procedure. provide a simple, reliable, INTRODUCTION: was first with
A spectrophotometric
described
glycerol
3-phosphate
of this
method
Garland
and Randle
Newsholme glycerol
and Suyter
(1)
using
It
measures
DEAE-cellulose
water.
to measure
is
in the sample,
a complete
and a similar
glycerolkinase
method for
glycerol
quantitatively
lkC-G3P
method
was then paper uses
complex
adaptation
was introduced (G3P)
diluted
disks
glycerol.
activity
in crude
tissue
from
column It
by by
by
presence
of
glycerol
and glycerol
ion-exchange
and free
formed
by the
separated
of lkC-G3P
glycerolkinase
reaction
has been described
3-phosphate
ion-exchange
The present separation
assay
14C-glycerol
The radioactivity
onto
off with
(2)
of serum glycerol
A fluoro-spectrophotometric
The radiochemical
(4).
glycerol
by absorption
employed
the determination
dehydrogenase.
(3).
kinase.
to achieve
for
has been reported
et.al.
non-labelled
washed
by Wieland
method
was
chromatography can be easily
extracts.
METHODS AND MATERIALS: Standard Assay Condition for Glycerol: The assay system ContainedTris-HCl buffer, 100 mM,pH 7.6; ATP, 3.2 mM; MgS04, 8.3 mM; EDTA 1 mM; 14C-u- glycerol 4XlO4 cpm and unknown or standard glycerol (l-40 nmole) by adding 0.25 ng of glycerolin a total volume of 120 1. The reactionwasinitiated kinase. After incubation at 300C for 10 minutes, the reaction wasterminated by adding 100 ul of ethanol containing 100 nmoles of G3P and mixing vigorously with * To whom all correspondence should Juvenile Foundation, 76Rl96.
be addressed.
This
work
was partly
supported
Abbreviations: G3P, glycerol 3 phosphate; DEAE cellulose, Diethylamino-Ethyl EDTA, Ethylenediamine Tetraacetic Acid; QAE-sephadex, Diethyl (2-hydropropyl) sephadex; ATP, adenosine 5-Triphosphate. 0006-291x/78/0804-0750$01.00/Q Copyright 0 I978 by Academic Press, Inc. AN rights of reproduction in any form reserved.
750
by
cellulose; Aminoethy
Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a vortex mixer for 5 seconds. Then 800 ~1 of distilled water was added. The content was transferred to QAE sephadex column (0.7 x 2.5 cm) which was previously equilibrated with Tris-HCl buffer. After the loading volume was run through, 30 ml of distilled water was applied to elute the ramaining 14C-glycerol. Then 4 ml of 0.1 N HCl was applied and the eluent containing G3P was collected directly into a glass counting vial and the radioactivity was estimated in a Packard Tricarb liquid scintillation spectrometer (model 3390) in the presence of 15 ml of scintillation fluid "tT 21" (5). The amount of glycerol was calculated from a standard calibration curve which was obtained from the same run. In selected cases an internal standard was added for recovery calculation. Over 95% of added standard was recovered. Glycerolkinase of tissue extracts can be measured by a method that is similar to the standard assay for glycerol. Identification of reaction product: The reaction mixture was applied to a DEAE-cellulose thin layer plate (0.1 mm) and air dried for 20 min. at room temperature and developed in a solvent system consisting of ammonium acetate lM:0.2M EDTA: methanol, 70: 0.5: 20 (v/v/v) for 90 min (6). The solvent frontwas cm. Following air drying for 20 min., the cellulose was scraped off sequentially from the origin to the solvent front, each area is 2 cm2. The cellulose was transferred into a counting vial and extracted with 1 ml of methanol for 20 min. at room temperature before adding 10 ml of scintillation fluid for measuring of radioactivity. Material: lb-C-u-glycerol (lot number 907-2110,131c/nmole) and 14-C-u-glycerol phosphate (lot number 907-227,117 mc/nmole) were obtained from New England Nuclear Corp. Boston,Mass. The initial radiochemical purity was greater than 98.3%. It was purified on Q.AEsephadex column before use. Glycerolkinase, (Candida Mycoderma, Lot number 7234327) was purchased from Boehringer, 1 mg/ml suspended in 3.3 M ammonium sulphate, DEAE cellulose TLC plate, polygram eel 300, precoated plastic sheets 20X20 cm2 0.1 mm cellulose m n 300, was obtained from Brinkman Instrument, Inc. Westbury, N.Y. 11590. RESULTS: Recovery the
elution
first this
of
pattern
GP and QAE-sephadex of glycerol
and G3P.
water.
Practically
5 ml of distilled
G3P was eluted
fraction.
(5 separate
runs)
of loaded
Determination
of glycerol
determination
of the
repeated
extraction
extractant
also
was 100.4+
2% (from
95.6~
(from
lipid
terminates
(Figure
1, bottom).
the
runs
of human serum).
We routinely
Glycerolkinase
converted
approximately
at 3OoC.
The plot
The recovery
experiments
curve:
of 14Co/l4C!s
with
usedl-40 (candida
10% of the
water
added
Krebs
nmole
(cpm in G3P without
751
column
shows
in the
was recovered
in
98.6% 1.3%
5 ml of HCl. In certain
was made with The
chloroform.
of glycerol Ringer
experiments
cells
washed
in the
bicarbonate
of glycerol
mycoderma)was
14C-glycerol
1 (top)
and more than
medium of fat
medium with
reaction.
glycerol
extraction:
of incubation
Figure from the
1 top)
in the first
7 separate
6 separate
standard
0.1 N HCl (Fig.
incubation
eluted
100% of loaded
chloroform
content
chromatography:
Glycerolwas
GP was recovered following
of the
Glycerol
with
column
phase
buffer)
per tube
usually
aqueous
0.25
and
for
calibration
ng per
tube
to G3P in 10 minutes added
glycerol/cpm
in G3P with
and
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GP
0
5
10
+-----distilled
15
xl
25
t$O--++elution
volume,
non-radioactive
M
0.1 N HCI -3
glycerol,
ml
“moles
Figure 1: top. Separation of I%-u-glycerol 3-phosphate from lkC!-u-glycerol on QAE-SephadzA25 column. Preparation of column and elution profile are described in Methods and Materials section. glycerol; --glycerol phosphate. bottom. Calibration curve of radioenzymic assay of glycerol. Glycerol kinase (Candida Qycoderma) 0.25 rig/tube was used.
added
glycerol)
Mg2+ and other
versus factors
nmoles effecting
have been shown to effect extraction
should
G3P formation buffer.
(Fig.
of non-labelled
this
the
slope
glycerol of glycerol
radioenzymic
be specifically
assay.
mentioned.
Krebs
2 top).
Addition
of Mg2+ alone
A Mg2+ concentration
curve
on G3P formation
752
addedwas
linear.
standard
curve:
Several
factors
Mg2+ concentration
and chloroform
Ringer
buffer
bicarbonate
can mimmick is
the
shown in Fig.
effect
stimulated of this
2, (bottom).
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
50
VI u s
40
M
20 10
0 0
20
60
40 non-radioactive
glycerol
80
nmoles ( s )
0.10
_ (-4)
0.08
0.06
3.0
j :: . 5 s 3 2 g c, z
0.0 OL 0
2
4
8
6 w
2+
10
0.04
2.0
0.02
1.0
0.0
0.0
$
12
mM
Figure 2. top. Effect of Krebs Ringer bicarconate buffer and Mg2+ on glycerol assay There was no or very little G3P detected when and glycerol3-phosphate formation. A) Assay in the presence of the assay was carried out in the presence of no M$+. o,GP formed; )--------a,calibration ix-------50 pl of Krebs Ringer bicarbonate buffer curve. B) Assay in the-presence of 50 1~1 Krebs Ringer bicarbonate buffer and a.3 mM i@+ . o--o, GP formed; calibration curve. C) Assay in the presence of 8.3 mM Mg2+ A-----A GP formed; A------A calibration curve. bottom Effect of increasing concentration of Mg2+ on the activity of cpm and nmoles of GP formed, glycerol glycerol kinase(Candida mycoderma) 0 -0, 0.17 n moles/assay; k-4 cpm, A-----4 n mole of GP formediglycerol 100.17 n moles/ assay. Assay volume was 120 ul. Each data point is a mean of 2 experiments.
753
BIOCHEMICAL
Vol. 80, No. 4, 1978
TABLE l.Glycerolkinase Glyrcol, nmoleslassay
activity
Mg2+
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibited
GSP-fonned, EDTA OillM
by EDTA and stimulated
nmoles EDTA 1mM
EDTA effect %
by Mg2+
Mg2+ EDTA OIRM
effect,% EDTA 1mM
0.17
1.6 4.0 12.5
0.057 0.098 0.11
0.025 0.074 0.11
56 24 0
0 72 93
0 196 340
100.17
1.6 4.0 12.5
0.6 0.7 0.91
0.23 0.59 0.91
62 16 0
0 17 52
0 157 296
*
EDTA effect
is
shown as % inhibition,
values
** Mg2+ effect
is
shown as % stimalation,
in the absence
values
in
of EDTA are
the presence
taken
as 0%
of 1.6 mM Mg2+ as 0%
stimulation
The optimum
Mg2+ concentrationwas The inhibitory
(3.2mM). in the
assay.
Table
effect
0.17
and 100.17
increasing which
a high
inhibitory
calibration
curve
of glycerol
in chloroform
Application
of glycerol
modified
method
as well
as glycerol
adapted
to measure
10 ug of protein single
per tube
of chloroform
chloroform
glycerolkinase from adipose
up an assay
essential
for
measuring
ATP At
G3P formation
for
by
condition
mixture
on G3P formation is
that
nmoles/tube.
in the reaction
to measure fat
cell
from tissue
or 100.17
we set
standard
assay
has been applied in rat
condition
in
as routine.
was observed.
accurate
A
determination
samples.
radioenzymic
content
Mg2+ concentration
mM) inhibited
extra&ion
treated
treated
in an assay
EDTA (1.0 findings,
above ATP concentration
by increasing
was 0.17
(8.3)waamaintained
effect for
mM, glycerol
Based on these
Mg2+ concentration
A slightly
This
glycerol
Mg2+ to 12.4mM.
fold
from experiments
or 12.5
nmole
1.5-2.0
of EDTA can be overcome
1 shows results
was 3.2 ml4 and Mg2+ was 1.6 both
approximately
incubation
chick,
of rat
serum
glycerol glycerol medium
mouse and rat was adequate
and glycerol
kinase:
from mouse and human, (Table
2).
It
also
was
(Table
3).
As little
to detect
this
enzyme in a
as
assay.
Identification
of reaction
product:
The reaction
754
product
of a standard
glycerol
assay
BIOCHEMICAL
Vol. 80, No. 4, 1978
TABLE 2
Source
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Glycerol content in serum and fat incubation medium
of glycerol
Glycerol
Normal mouse serum Human serum Incubation medium before incubation no epinephrine for 30 min. epinephrine 0.1 pg/ml for 30 min.
content,
cell
mnoles/ml
238 l 23 (6) 181 * 0.01 (6) 28.6 * 18 (6) 36 * 12 (6) 310 * 11.8 (6)
Results are mean f SEM number of experiments. For fat cells, 30 mg/ml fat cells of rat epidymal fat were incubated in Krebs Ringer bicarbonate buffer in the presence of 0.1 mM caffeine for 30 min.
was shown to be G3P in two chromatographic with
DEAE cellulose
product
layer
of glycerolkinase
G3P in the In both
same systems.
systems
radioactivity
method
counting
vial.
allows
layer
a significant
glycerol
other
than
up to 100 tubes for
the
assays per
over
in our experience
only
after
glycerol
the product require
time
extracts result
incubation
chromatography
original
is
formed
about
four
the
radioenzymic consuming
as tracer
has been
recently
by Scheidner
(6).
Here too
the blank
values
applied
onto
the thin
layer
or paper
was time disk
consuming. is
755
were
also
identified
is
shown in Fig.
with
glycerolkinase.
as 3 (right) No
also
G3P from glyceroi in a single
of a technicians
time.
recovery
of this
method
assay using
a filter
paper
by high
described
were
high
blanks.
by Thorner or processes
The volume
a limitation.
It
of enzyme and increasing
and affected
32P-ATP
-32p-ATP
hours
and high
using
from"
obtained
The reaction
to be radioassayed
amount
assay method
32p-G3P
left).
to separate
glycerol
to separate
(Fig.3,
The results
and G3P.
The convenience
the it
tissue
run by reducing
assay.
shown.
chromatographic
column all
complete
improvement
Since
from
essentially
Forty
of incubation
(4).
The thin
G3P formed
(TLC and column).
are
mouse adipose
The use of QKE-sephadex
in this
can be scale
chromatography
of crude
was detected
DISCUSSION:
time
thin
systems
of incubation Glycerolkinase
is disk
Another (7)
and
required mixture in
BIOCHEMICAL
Vol. 80, No. 4, 1978
TABLE
Source enzyme
of
3
adipose detectable
of several
glycerol
for
kinase
in adipose
analysis
is
should
with
glycerolkinase
pm&?/assay
2800 3075 914
1333 778 3779
of
formed pmole/mg/min
965 4423 1447
mycoderma
its
optimal
is
in this
kinase
if
standard
accurate
measurement.
sensitive
to increase
activity,
origin ramains
also
radioenzymic
assay
of rat
of the
and other the
activity
glycerolkinase
cells.
should
Our results
utilization reviewed
by
activities. in samples
directly in the indicate
in Mg2+ concentration. require
as to
regarding
compounds
be added
sample
in as little
Subjects
are assayed
animals
willallowinvestigators
such research
they
of
of glycerol-
in mammals have been recently
to be studied.
756
was not
modification,
This
of unknown
an internal
of mammalian
fat
accelerate
the possibility reaction
paper,
the
tissue
In view
measured.
in isolated
should
Using
the present
step.
obtained
glycerolkinase
to assure
Using
low.
a limiting
method
(1,8).
time
It
to be low or absent.
in adipose
can be adequately
out that
glycerolkinase 1 for
but
of glycerol
Therefore,
sample
was used
was studied
of glycerolkinase
be pointed
from Candida
than
cells
Our convenient
of glycerol.
activity
of rat
and regulation
interference
tissue
fat
regulation
of glycerol
method
no longer
tissue
10 mg of isolated
It
tissue
CPm
has been reported
was detectable
volume
(14).
for
L3P
2.10 3.95 0.33
animals
kinase
Activity
Lin
assay animals
assay
90.7 90.7 200
when the photometric
(9-13).
radioenzymic experimental
from adipose tissue of chicken, rat and mouse were isolated by ammonium sulfate (60, 60 and 40X by weight for chicken, rat and mourse respectively). The assay except 20 min for rat glycerolkinase. Results are mean of duplicates.
tissue
Newsholme
glycerol
glycerol in nmole/assay,cpm/pmole
46 6.8 64
Glycerolkinase fractionation was 30 min
unknown
of
Enz. protein pg/=.Wy
Chicken Rat MOUSS
study
Application
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
without assay that
for
isolation each
glycerolkinase
Whether
a Mg2+ to ATP molar
may cause
ratio
adipose greater
Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12
12
A - Standards
A - standards
8 4 0
0 0
40
20
60
80
100 12
12 B - zero time reaction
m ‘0,
B - zero time reactlon
mixture
8
”
”E
E v
4
U
0
0 12
A
xl C - reaction
40
60 R
mixture
80
1W
4
A
20 C - reactlon
40
60
80
100
mixture
:: I I 1 I ’ \ ’ \
8 4
8 0 x
l---l
x
mixture
’ I
\
6
0
Figure 3. Identification of the reaction product of glycerol and glycerol kinase assay by thin layer chromatography. Left: glycerol kinase (40 rig/assay) and no nonlabelled glycerol (7~10~ cpm/tube).Practically 100% of the applied radioactivity was recovered. from the plate. A) glycerol and GP standards B) reaction mixture before incuba.tion, 5 ul applied onto TLC plate C) reaction mixture after 20 min incubation at 300C, 5 ul applied onto TLC plate. Right. Glycerolkinase assay, glycerol kinase from lean normal mouse epididymal fa.t pad (162 &tube) and glycerol ( 3~10~ cpm/tube). A) glycerol and GP standard B) reaction mixture before incubation, 5 ul applied C) reaction mixture after 20 min. incubation at 300C, 5 ul applied. Practically 100% of the applied radioactivity was recovered from the plate.
REFERENCES: 1. Wieland. O., and Suyter, M. (1957) Biochem. Z., 329, 320-331. 2. Dalziel; K..(1962) Biochemical J, 84, 244-254. 3. Garland, P.B. and Randle, P.J. (1962) Nature, 196, 987-988. 4. Newsholme, E. A., and Tayler K., (1968) Biochim. Biophys. Acta. 158, 11-24. 5. Patterson, M.S. and Greene, R.C., (1965) Anal.Chem. 37, 854-857. 6. Schneider, P.B. (1977) J. Lipid Res. 18, 396-399. 7. Thorner, J.W. in Method in Enzymology, Vol 42, pp 149-156, 1975, Academic Press New York, S. P. Colowick and N.O. Kaplan edts.
Vol. 80, No. 4,1978
8. 9. 10. 11. . 12. 13. 14.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Margolis S., and Vaughan, M. (1962) J. Biol. Chem. 237, 44-48. Robinson J. and Newsholme, E.A. (1967) Biochem. J. 104-2c-4c. Persico, P.A., Gerchio, M. and Jeffay, H. (1975) Am. J. Physiol. 228,1868-18 Thenen, S.W. ,and Mayer, J. (1975) Horm. Metab. Res. 8, 80-81. Gries, F.A. and Herberg, L. (1971) Diabetologia, 7, 316-22. Welton, R.F. and Martin, R. J. Scholz, R.W. Baumgardt, B.R., (1973) J. Nutr. 103, 890-898. Ann Rev. Biochm. 46, 765-95, 1977. Lin, E.C.C.,
758
'4.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
February 28, 1978
Pages
750-758
RADIOENZYMIC ASSAY OF GLYCEROL USING ION-EXCHANGE COLUMN CHROMATOGRAPHY Luis A. Barrera and Ren-jye Ho* of Biochemistry, University of Miami School Miami, Florida
Department
Received
of Medicine
December 5,1977
SUMMARY: Modifications of the glycerol kinase radioenzymic assay for glycerol are This method can be readily employed to measure glycerol kinase activity described. Ion-exchange column chromatography (QAE-Sephadex A-25) in tissue extracts as well. and completely separates the productlkc-glycerol 3-phosphate from lkC!-glycerol, allows all glycerol 3-phosphate formed in an assay to be counted in a single Increasing the Mg2+ concentration significantly increases activity counting vial. These modifications of glycerol kinase and thus the sensitivity of the assay. sensitive and more rapid procedure. provide a simple, reliable, INTRODUCTION: was first with
A spectrophotometric
described
glycerol
3-phosphate
of this
method
Garland
and Randle
Newsholme glycerol
and Suyter
(1)
using
It
measures
DEAE-cellulose
water.
to measure
is
in the sample,
a complete
and a similar
glycerolkinase
method for
glycerol
quantitatively
lkC-G3P
method
was then paper uses
complex
adaptation
was introduced (G3P)
diluted
disks
glycerol.
activity
in crude
tissue
from
column It
by by
by
presence
of
glycerol
and glycerol
ion-exchange
and free
formed
by the
separated
of lkC-G3P
glycerolkinase
reaction
has been described
3-phosphate
ion-exchange
The present separation
assay
14C-glycerol
The radioactivity
onto
off with
(2)
of serum glycerol
A fluoro-spectrophotometric
The radiochemical
(4).
glycerol
by absorption
employed
the determination
dehydrogenase.
(3).
kinase.
to achieve
for
has been reported
et.al.
non-labelled
washed
by Wieland
method
was
chromatography can be easily
extracts.
METHODS AND MATERIALS: Standard Assay Condition for Glycerol: The assay system ContainedTris-HCl buffer, 100 mM,pH 7.6; ATP, 3.2 mM; MgS04, 8.3 mM; EDTA 1 mM; 14C-u- glycerol 4XlO4 cpm and unknown or standard glycerol (l-40 nmole) by adding 0.25 ng of glycerolin a total volume of 120 1. The reactionwasinitiated kinase. After incubation at 300C for 10 minutes, the reaction wasterminated by adding 100 ul of ethanol containing 100 nmoles of G3P and mixing vigorously with * To whom all correspondence should Juvenile Foundation, 76Rl96.
be addressed.
This
work
was partly
supported
Abbreviations: G3P, glycerol 3 phosphate; DEAE cellulose, Diethylamino-Ethyl EDTA, Ethylenediamine Tetraacetic Acid; QAE-sephadex, Diethyl (2-hydropropyl) sephadex; ATP, adenosine 5-Triphosphate. 0006-291x/78/0804-0750$01.00/Q Copyright 0 I978 by Academic Press, Inc. AN rights of reproduction in any form reserved.
750
by
cellulose; Aminoethy
Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a vortex mixer for 5 seconds. Then 800 ~1 of distilled water was added. The content was transferred to QAE sephadex column (0.7 x 2.5 cm) which was previously equilibrated with Tris-HCl buffer. After the loading volume was run through, 30 ml of distilled water was applied to elute the ramaining 14C-glycerol. Then 4 ml of 0.1 N HCl was applied and the eluent containing G3P was collected directly into a glass counting vial and the radioactivity was estimated in a Packard Tricarb liquid scintillation spectrometer (model 3390) in the presence of 15 ml of scintillation fluid "tT 21" (5). The amount of glycerol was calculated from a standard calibration curve which was obtained from the same run. In selected cases an internal standard was added for recovery calculation. Over 95% of added standard was recovered. Glycerolkinase of tissue extracts can be measured by a method that is similar to the standard assay for glycerol. Identification of reaction product: The reaction mixture was applied to a DEAE-cellulose thin layer plate (0.1 mm) and air dried for 20 min. at room temperature and developed in a solvent system consisting of ammonium acetate lM:0.2M EDTA: methanol, 70: 0.5: 20 (v/v/v) for 90 min (6). The solvent frontwas cm. Following air drying for 20 min., the cellulose was scraped off sequentially from the origin to the solvent front, each area is 2 cm2. The cellulose was transferred into a counting vial and extracted with 1 ml of methanol for 20 min. at room temperature before adding 10 ml of scintillation fluid for measuring of radioactivity. Material: lb-C-u-glycerol (lot number 907-2110,131c/nmole) and 14-C-u-glycerol phosphate (lot number 907-227,117 mc/nmole) were obtained from New England Nuclear Corp. Boston,Mass. The initial radiochemical purity was greater than 98.3%. It was purified on Q.AEsephadex column before use. Glycerolkinase, (Candida Mycoderma, Lot number 7234327) was purchased from Boehringer, 1 mg/ml suspended in 3.3 M ammonium sulphate, DEAE cellulose TLC plate, polygram eel 300, precoated plastic sheets 20X20 cm2 0.1 mm cellulose m n 300, was obtained from Brinkman Instrument, Inc. Westbury, N.Y. 11590. RESULTS: Recovery the
elution
first this
of
pattern
GP and QAE-sephadex of glycerol
and G3P.
water.
Practically
5 ml of distilled
G3P was eluted
fraction.
(5 separate
runs)
of loaded
Determination
of glycerol
determination
of the
repeated
extraction
extractant
also
was 100.4+
2% (from
95.6~
(from
lipid
terminates
(Figure
1, bottom).
the
runs
of human serum).
We routinely
Glycerolkinase
converted
approximately
at 3OoC.
The plot
The recovery
experiments
curve:
of 14Co/l4C!s
with
usedl-40 (candida
10% of the
water
added
Krebs
nmole
(cpm in G3P without
751
column
shows
in the
was recovered
in
98.6% 1.3%
5 ml of HCl. In certain
was made with The
chloroform.
of glycerol Ringer
experiments
cells
washed
in the
bicarbonate
of glycerol
mycoderma)was
14C-glycerol
1 (top)
and more than
medium of fat
medium with
reaction.
glycerol
extraction:
of incubation
Figure from the
1 top)
in the first
7 separate
6 separate
standard
0.1 N HCl (Fig.
incubation
eluted
100% of loaded
chloroform
content
chromatography:
Glycerolwas
GP was recovered following
of the
Glycerol
with
column
phase
buffer)
per tube
usually
aqueous
0.25
and
for
calibration
ng per
tube
to G3P in 10 minutes added
glycerol/cpm
in G3P with
and
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GP
0
5
10
+-----distilled
15
xl
25
t$O--++elution
volume,
non-radioactive
M
0.1 N HCI -3
glycerol,
ml
“moles
Figure 1: top. Separation of I%-u-glycerol 3-phosphate from lkC!-u-glycerol on QAE-SephadzA25 column. Preparation of column and elution profile are described in Methods and Materials section. glycerol; --glycerol phosphate. bottom. Calibration curve of radioenzymic assay of glycerol. Glycerol kinase (Candida Qycoderma) 0.25 rig/tube was used.
added
glycerol)
Mg2+ and other
versus factors
nmoles effecting
have been shown to effect extraction
should
G3P formation buffer.
(Fig.
of non-labelled
this
the
slope
glycerol of glycerol
radioenzymic
be specifically
assay.
mentioned.
Krebs
2 top).
Addition
of Mg2+ alone
A Mg2+ concentration
curve
on G3P formation
752
addedwas
linear.
standard
curve:
Several
factors
Mg2+ concentration
and chloroform
Ringer
buffer
bicarbonate
can mimmick is
the
shown in Fig.
effect
stimulated of this
2, (bottom).
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
50
VI u s
40
M
20 10
0 0
20
60
40 non-radioactive
glycerol
80
nmoles ( s )
0.10
_ (-4)
0.08
0.06
3.0
j :: . 5 s 3 2 g c, z
0.0 OL 0
2
4
8
6 w
2+
10
0.04
2.0
0.02
1.0
0.0
0.0
$
12
mM
Figure 2. top. Effect of Krebs Ringer bicarconate buffer and Mg2+ on glycerol assay There was no or very little G3P detected when and glycerol3-phosphate formation. A) Assay in the presence of the assay was carried out in the presence of no M$+. o,GP formed; )--------a,calibration ix-------50 pl of Krebs Ringer bicarbonate buffer curve. B) Assay in the-presence of 50 1~1 Krebs Ringer bicarbonate buffer and a.3 mM i@+ . o--o, GP formed; calibration curve. C) Assay in the presence of 8.3 mM Mg2+ A-----A GP formed; A------A calibration curve. bottom Effect of increasing concentration of Mg2+ on the activity of cpm and nmoles of GP formed, glycerol glycerol kinase(Candida mycoderma) 0 -0, 0.17 n moles/assay; k-4 cpm, A-----4 n mole of GP formediglycerol 100.17 n moles/ assay. Assay volume was 120 ul. Each data point is a mean of 2 experiments.
753
BIOCHEMICAL
Vol. 80, No. 4, 1978
TABLE l.Glycerolkinase Glyrcol, nmoleslassay
activity
Mg2+
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibited
GSP-fonned, EDTA OillM
by EDTA and stimulated
nmoles EDTA 1mM
EDTA effect %
by Mg2+
Mg2+ EDTA OIRM
effect,% EDTA 1mM
0.17
1.6 4.0 12.5
0.057 0.098 0.11
0.025 0.074 0.11
56 24 0
0 72 93
0 196 340
100.17
1.6 4.0 12.5
0.6 0.7 0.91
0.23 0.59 0.91
62 16 0
0 17 52
0 157 296
*
EDTA effect
is
shown as % inhibition,
values
** Mg2+ effect
is
shown as % stimalation,
in the absence
values
in
of EDTA are
the presence
taken
as 0%
of 1.6 mM Mg2+ as 0%
stimulation
The optimum
Mg2+ concentrationwas The inhibitory
(3.2mM). in the
assay.
Table
effect
0.17
and 100.17
increasing which
a high
inhibitory
calibration
curve
of glycerol
in chloroform
Application
of glycerol
modified
method
as well
as glycerol
adapted
to measure
10 ug of protein single
per tube
of chloroform
chloroform
glycerolkinase from adipose
up an assay
essential
for
measuring
ATP At
G3P formation
for
by
condition
mixture
on G3P formation is
that
nmoles/tube.
in the reaction
to measure fat
cell
from tissue
or 100.17
we set
standard
assay
has been applied in rat
condition
in
as routine.
was observed.
accurate
A
determination
samples.
radioenzymic
content
Mg2+ concentration
mM) inhibited
extra&ion
treated
treated
in an assay
EDTA (1.0 findings,
above ATP concentration
by increasing
was 0.17
(8.3)waamaintained
effect for
mM, glycerol
Based on these
Mg2+ concentration
A slightly
This
glycerol
Mg2+ to 12.4mM.
fold
from experiments
or 12.5
nmole
1.5-2.0
of EDTA can be overcome
1 shows results
was 3.2 ml4 and Mg2+ was 1.6 both
approximately
incubation
chick,
of rat
serum
glycerol glycerol medium
mouse and rat was adequate
and glycerol
kinase:
from mouse and human, (Table
2).
It
also
was
(Table
3).
As little
to detect
this
enzyme in a
as
assay.
Identification
of reaction
product:
The reaction
754
product
of a standard
glycerol
assay
BIOCHEMICAL
Vol. 80, No. 4, 1978
TABLE 2
Source
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Glycerol content in serum and fat incubation medium
of glycerol
Glycerol
Normal mouse serum Human serum Incubation medium before incubation no epinephrine for 30 min. epinephrine 0.1 pg/ml for 30 min.
content,
cell
mnoles/ml
238 l 23 (6) 181 * 0.01 (6) 28.6 * 18 (6) 36 * 12 (6) 310 * 11.8 (6)
Results are mean f SEM number of experiments. For fat cells, 30 mg/ml fat cells of rat epidymal fat were incubated in Krebs Ringer bicarbonate buffer in the presence of 0.1 mM caffeine for 30 min.
was shown to be G3P in two chromatographic with
DEAE cellulose
product
layer
of glycerolkinase
G3P in the In both
same systems.
systems
radioactivity
method
counting
vial.
allows
layer
a significant
glycerol
other
than
up to 100 tubes for
the
assays per
over
in our experience
only
after
glycerol
the product require
time
extracts result
incubation
chromatography
original
is
formed
about
four
the
radioenzymic consuming
as tracer
has been
recently
by Scheidner
(6).
Here too
the blank
values
applied
onto
the thin
layer
or paper
was time disk
consuming. is
755
were
also
identified
is
shown in Fig.
with
glycerolkinase.
as 3 (right) No
also
G3P from glyceroi in a single
of a technicians
time.
recovery
of this
method
assay using
a filter
paper
by high
described
were
high
blanks.
by Thorner or processes
The volume
a limitation.
It
of enzyme and increasing
and affected
32P-ATP
-32p-ATP
hours
and high
using
from"
obtained
The reaction
to be radioassayed
amount
assay method
32p-G3P
left).
to separate
glycerol
to separate
(Fig.3,
The results
and G3P.
The convenience
the it
tissue
run by reducing
assay.
shown.
chromatographic
column all
complete
improvement
Since
from
essentially
Forty
of incubation
(4).
The thin
G3P formed
(TLC and column).
are
mouse adipose
The use of QKE-sephadex
in this
can be scale
chromatography
of crude
was detected
DISCUSSION:
time
thin
systems
of incubation Glycerolkinase
is disk
Another (7)
and
required mixture in
BIOCHEMICAL
Vol. 80, No. 4, 1978
TABLE
Source enzyme
of
3
adipose detectable
of several
glycerol
for
kinase
in adipose
analysis
is
should
with
glycerolkinase
pm&?/assay
2800 3075 914
1333 778 3779
of
formed pmole/mg/min
965 4423 1447
mycoderma
its
optimal
is
in this
kinase
if
standard
accurate
measurement.
sensitive
to increase
activity,
origin ramains
also
radioenzymic
assay
of rat
of the
and other the
activity
glycerolkinase
cells.
should
Our results
utilization reviewed
by
activities. in samples
directly in the indicate
in Mg2+ concentration. require
as to
regarding
compounds
be added
sample
in as little
Subjects
are assayed
animals
willallowinvestigators
such research
they
of
of glycerol-
in mammals have been recently
to be studied.
756
was not
modification,
This
of unknown
an internal
of mammalian
fat
accelerate
the possibility reaction
paper,
the
tissue
In view
measured.
in isolated
should
Using
the present
step.
obtained
glycerolkinase
to assure
Using
low.
a limiting
method
(1,8).
time
It
to be low or absent.
in adipose
can be adequately
out that
glycerolkinase 1 for
but
of glycerol
Therefore,
sample
was used
was studied
of glycerolkinase
be pointed
from Candida
than
cells
Our convenient
of glycerol.
activity
of rat
and regulation
interference
tissue
fat
regulation
of glycerol
method
no longer
tissue
10 mg of isolated
It
tissue
CPm
has been reported
was detectable
volume
(14).
for
L3P
2.10 3.95 0.33
animals
kinase
Activity
Lin
assay animals
assay
90.7 90.7 200
when the photometric
(9-13).
radioenzymic experimental
from adipose tissue of chicken, rat and mouse were isolated by ammonium sulfate (60, 60 and 40X by weight for chicken, rat and mourse respectively). The assay except 20 min for rat glycerolkinase. Results are mean of duplicates.
tissue
Newsholme
glycerol
glycerol in nmole/assay,cpm/pmole
46 6.8 64
Glycerolkinase fractionation was 30 min
unknown
of
Enz. protein pg/=.Wy
Chicken Rat MOUSS
study
Application
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
without assay that
for
isolation each
glycerolkinase
Whether
a Mg2+ to ATP molar
may cause
ratio
adipose greater
Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12
12
A - Standards
A - standards
8 4 0
0 0
40
20
60
80
100 12
12 B - zero time reaction
m ‘0,
B - zero time reactlon
mixture
8
”
”E
E v
4
U
0
0 12
A
xl C - reaction
40
60 R
mixture
80
1W
4
A
20 C - reactlon
40
60
80
100
mixture
:: I I 1 I ’ \ ’ \
8 4
8 0 x
l---l
x
mixture
’ I
\
6
0
Figure 3. Identification of the reaction product of glycerol and glycerol kinase assay by thin layer chromatography. Left: glycerol kinase (40 rig/assay) and no nonlabelled glycerol (7~10~ cpm/tube).Practically 100% of the applied radioactivity was recovered. from the plate. A) glycerol and GP standards B) reaction mixture before incuba.tion, 5 ul applied onto TLC plate C) reaction mixture after 20 min incubation at 300C, 5 ul applied onto TLC plate. Right. Glycerolkinase assay, glycerol kinase from lean normal mouse epididymal fa.t pad (162 &tube) and glycerol ( 3~10~ cpm/tube). A) glycerol and GP standard B) reaction mixture before incubation, 5 ul applied C) reaction mixture after 20 min. incubation at 300C, 5 ul applied. Practically 100% of the applied radioactivity was recovered from the plate.
REFERENCES: 1. Wieland. O., and Suyter, M. (1957) Biochem. Z., 329, 320-331. 2. Dalziel; K..(1962) Biochemical J, 84, 244-254. 3. Garland, P.B. and Randle, P.J. (1962) Nature, 196, 987-988. 4. Newsholme, E. A., and Tayler K., (1968) Biochim. Biophys. Acta. 158, 11-24. 5. Patterson, M.S. and Greene, R.C., (1965) Anal.Chem. 37, 854-857. 6. Schneider, P.B. (1977) J. Lipid Res. 18, 396-399. 7. Thorner, J.W. in Method in Enzymology, Vol 42, pp 149-156, 1975, Academic Press New York, S. P. Colowick and N.O. Kaplan edts.
Vol. 80, No. 4,1978
8. 9. 10. 11. . 12. 13. 14.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Margolis S., and Vaughan, M. (1962) J. Biol. Chem. 237, 44-48. Robinson J. and Newsholme, E.A. (1967) Biochem. J. 104-2c-4c. Persico, P.A., Gerchio, M. and Jeffay, H. (1975) Am. J. Physiol. 228,1868-18 Thenen, S.W. ,and Mayer, J. (1975) Horm. Metab. Res. 8, 80-81. Gries, F.A. and Herberg, L. (1971) Diabetologia, 7, 316-22. Welton, R.F. and Martin, R. J. Scholz, R.W. Baumgardt, B.R., (1973) J. Nutr. 103, 890-898. Ann Rev. Biochm. 46, 765-95, 1977. Lin, E.C.C.,
758
'4.
