Glycerol metabolism in higher plants: Glycerol kinase

Vol. March

143,

No. 3, 1987

30,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1987

Pages

GLYCEROL METABOLISM

IN HIGHER PLANTS:

D. Sadava Joint Received

Science

Department,

January

26,

977-983

GLYCEROL KINASE

and K. Moore

The Claremont

Colleges,

Claremont,

CA 91711

1987

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or LJTP could be used as the phosphoryl group donor. The K for glycerol was 55 uM and K values for the nucleoside triphosphateg were 145-620 uM. The V fo? the reaction was 40-78 pmol product per min. Kinetic d a t a indica!!gxthat the enzyme has a sequential
When lipid-storing the

hydrolysis

are

converted

the

into

(l-3).

formation

pathway.

In bacteria

in a wide

is

a glycolytic

studies

beans

fate

such

then

The latter for

of glycerol.

in

leads

to

(4,5), the

glycolytic

metabolism

in initiated

catalyzed

by glycerol

converted

catalyze

[ 14 C]glycerol

in

to molecules

to glycerol-3-phosphate,

by the kinase

to dihydroxyacetone

intermediate. kinase

Later,

investigated.

variety

acids.

and some [14CJC02

can be metabolized and mammals (7),

spherosomes

as a substrate

metabolic

castor

is

in

and fatty

serves

[ 14C] sucrose

bean and peanut were

lipases

of the

germinating

of glycerol (8,9).

then

known

Glycerol-3-phosphate

germinating activity

(6)

of glycerol

The presence labelling

from

glycerol

(EC 2.7.1.30). phosphate,

Less

which

of considerable that

conversion

germinate, to glycerol

succinate.

of tissues

indicating

seeds

of triacylglycerols

gluconeogenesis Incubation

plant

of plants,

the

seeds In the

in

plants

enzyme (10,ll). present

was first

inferred

from

was detected

by direct

assay

However,

properties

study,

and characterized

few we have

its

detected

properties

in

of the the

enzyme

in cucumber

radicles. 0006-291X/87

977

All

Cqvright 0 1987 rights of reproduction

$1.50

by Academic Press, Inc. in any form reserved.

BIOCHEMICAL

Vol. 143, No. 3, 1987

AND BIOPHYSICAL

MATERIALS

RESEARCH COMMUNICATIONS

AND METHODS -~

seeds were soaked in water for Cucumber (Cucumis sativa var. Marketeer) 2 h and then germinated on moist filter paper at 20°C. Other seeds soaked in water for 12 h and germinated in moist vermiSulite. Specialty chemicals were purchased from Sigma (St. Louis, MO) and [Z- HIglycerol, specific activity of 500 mCi/mmol, was purchased from Amersham (Arlington Heights, IL). Unless otherwise stated, all operations were carried out at 4'C. Tissue was chopped with a razor blade and homogenized in a teflon/glass homogenizer in 3 volumes of 0.4M sucrose, 1mM EDTA, 1OmM KCl, 1 mM MgC12: 0.15M Tris-HCl, pH 7.6. The homogenate was centrifuged at 1OOOg for 10 min to remove cell walls and debris. The resulting supernatant was then centrifuged at 21,OOOg for 30 min. The supernatant was made 40% in ammonium sulfate over 2 h. The suspension was centrifuged at 32,OOOg for 45 min and the pellet discarded. The supernatant was brought to 60% ammonium sulfate saturation. This suspension was then centrifuged at 32,000g for 45 min. The resulting pellet was dissolved in homogenizing buffer and dialyzed against this buffer overnight. For further purification, this material was applied to an affinity column of 5'-AMP-Sepharose (12). After washing, the column was eluted with a gradient of O-1M KCl. Glycerol kinase activity eluted around 0.5M KCl. This activity did not differ in the properties examined from the ammonium sulfate precipitated activity: the latter was rountinely used for analyses. Glycerol kinase was assayed by a modification of a radiochemical procedure (13). The reaction mixture contained, in O.lmL volume: tissue extgact, O.lM Tris-HCl, pH 9.0, 1mM EDTA, 4mM ATP, 4mM MgS04 and 1OOuM [2- HIglycerol. Reactions at 37'C were intitated by the addition of substrate and terminated by chilling and the addition of O.lmL of 2M nonradioactive glycerol. The anionic reaction product was retained on DE 81 filter discs (Whatman) and quantitated by liquid scintillation spectrometry. For experiments in which glycerol kinase was assayed as a function of pH, a series of buffers was used (14), all at O.lM: MES (4-morpholineethane-sulfonic acid, pK 6.15), TES (2-(2-hydroxy-1,2-bis(hydroxymethyl)-ethylanlinoethanesulfonicaacid, pK 7.50), TRIS (tris-hydroxymethylaminomethane, pKa 8.30) and CHES (2-(gyclohexylaminoethanesulfonic acid, pK 9.50). Following elution by incubation in 0.5M KCl, two methodsawere used to identify the reaction product. In the first, The R of the anionic product on silica gel thin-layer chromatography in ethyl acte Eate:acetic acid:water: ammonia (6:6:1:1) was compared to those of standard solutions (5 mg/ml) of glycerol-3-phosphate (Rf=0.68), 3-phosphoglyceric acid (Rf=0.30), dihydroxyacetone phosphate (R =0.58), and fructose-1,6-biphosphate (R = was incubated at 37'C with pirified 0.11). In the second method, Ehe product glycerol-3-phosphate dehydrogenase (50 units) in the presence of 0.2mM NAD and O.lM Tris-HCl, pH 9.0 in a volume of O.lmL. After 4 h, 50 UL of the mixture was run on thin layer chromatography as above. Protein was assayed by dye binding (15) and checked with the Folin reagent (16). Catalase, pyruvate kinase, and succinate dehydrogenase were assayed spectrophotometrically (17).

RESULTS Glycerol cotyledons germinated) maize lost

kinase

was detected

(1 day germinated), of 7 plants:

and oat. activity

activity

rapidly

and hypocotyls

cucumber,

The specific upon

in the

activity storage

broad

bean,

16,OOOg

supernatants

and radicles

or roots

kidney

was 0.1-3.5 at 4'C 978

but

bean,

nmol/min/mg were

stable

of (4 days

mung bean, protein. frozen

pea, Extracts

at -2O'C.

BIOCHEMICAL

Vol. 143, No. 3, 1987

AND BIoPHYSICAL

Fraction Fig.

1.

number

AFFINITY CHROMATOGRAPHYOF GLYCEROL KINASE. The 60% ammonium sulfate fraction dissolved in 2 mL of 5mM MgCl and dialyzed against this buf i!'er applied to a 0.5 x 6.0 cm column

above buffer washed

with

(flow

0.70

cases, (range

phosphate

the Rf of the 0.68-0.73).

Glycerol germinated

2 days.

almost

300-

showed

at

pure,

fold least

of the activity routine

purification

The cucumber l-20mM

dithiothreitol

abolished

the

activity;

of the

purified

was

then in the

the column was mL gradient of and analyzed KC1 (refractive

chromatography

product

gel

filtration

glycerol

The

1 ).

on acrylamide

the

from

with

was

glycerol-3-

from

eluate

on Sephadex 210,000.

ammonium kinase

This

sulfate

was not

divalent

cations 979

of cucumber

(Figure

electrophoresis,

or NaF to the reaction other

radicles

chromatography

(Table

was approximately

characterization,

on thin-layer

incubation

affinity

by gel

was

Rf was 0.57.

Following

As determined

tissue

overnight. The mgterlal packed with 5'-AMP-Sepharose

product

was partially

6 bands

radicle

of 0.2mL/min). After loading, and then eluted with a 14 Fractions (0.5 mL) were collected (squares), protein (circles) and

reaction

its

kinase

cucumber

buffer

Following

dehydrogenase,

from

1mM EDTA, 1OmM KH,P04: pH 6.5

rate

2 mL of

O-1M KC1 in buffer. for glycerol kinase index, upper points).

In all

RESEARCH COMMUNKATIONS

precipitate

the

the

affinity

was an column

molecular

weight

was unstable;

for

was used.

affected

by the

mixture.

Omission

(Ca

there

and so was not

G-100, activity

l),

seeds

addition

of

of Mg+'

++ or Mn++ ) could

not

act

BIOCHEMICAL

Vol. 143, No. 3. 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 PURIFICATION OF GLYCEROL KINASE Fraction

Protein (w)

Enzyme Act. (pmol/min)

Specific Act. (pmol/min/mg)

13.92

202.2

15

Amm. SO4 ppt.

2.40

156.5

65

Affinity

0.02

80.7

4035

21,000g

supernt.

column

Radicle tissue from 2-day germinated cucumber seeds was homogenized, centrifuged, and subjected to ammonium sulfate fractionation as described Materials and Methods. Affinity chromatography was performed as described the legend to Figure 1.

as replacements.

The activity

1OmM: glycerol-3-phosphate, phosphate,

phosphoenol

phosphate,

pyruvate,

Glycerol protein.

kinase

For routine

The pH optimum

Fig.

2.

of the

was not

affected

by the

fructose-1,6-biphosphate, pyruvate,

cyclic

following

added

in in

at O.l-

dihydroxyacetone

AMP, glucose-6-phosphate,

fructose-6-

and glucose. activity assays, enzyme

was linear

up to 10 min and 100 ug of tissue

8 min incubations was around

9.0

and over (Figure

2).

100 ug were Results

used.

of

pH PROFILE OF GLYCEROL KINASE ACTIVITY. Enzyme was 132 ug cucumber radicle protein. Buffers used were MES (closed circles), TES (open circles), CHES (closed squares) and TRIS (open squares). Each point represents the mean of 3 determinations. 980

BIOCHEMICAL

Vol. 143, No. 3, 1987

AND BIOPHYSKAL

TABLE SUBCELLULAR

DISTRIBUTION

2

OF GLYCEROL

Homogenate

Protein

(up)

Catalase

(mmol/min)

Pyruvate

kinase

(mmol/min)

Succinate

Dehydrogenase (mmollmin) Glycerol kinase (nmol/min)

fractionation

supernatant

along

pellet,

with

(succinate Glycerol 55uM,

studies

with

which

the

dehydrogenase) kinase

as determined

37.8 (87)

5.6 (13)

0.56

0

0.56 (100)

1.22

1.25

(100)

1.68

0.01

(1)

nonspecific

(Table

3).

Glycerol

kinase

activity

TABLE

Substrate

Glycerol

PARAMETERS

in

kinase,

but

not

(19,ZO)

requirement was analyzed

glycerol

K m, uM

was rather vs.

glycerol

KINASE

V max,

pmollmin

55

(12)

78

(18)

ATP

320

(90)

48

(16)

CTP

515

(165)

42

(19)

GTP

145

(35)

55

(15)

UTP

620

(90)

40

(10)

Cucumber radicle enzyme (120 ug protein) was used. When glycerol concentration was varied, ATP was 2mM. When nucleoside triphosphate concentrations were varied, glycerol was 100uM. Data are means with S.E. in parentheses.

981

in

was

and checked

3 OF GLYCEROL

the

and mitochondria

The Km for

analysis

triphosphate

activtiy

(18)

kinetics.

by computer

The nucleoside

pyruvate

(99)

30 min. The pellet pellet and and the other

the

(catalase)

hyperbolic

3).

KINETIC

at 21,OOOg for the homogenate, radiochemically

marker,

associated.

0 1.58

2) localized

were

(Table

(W)

50.6

microbodies

graphically

Supernatant

4668 (80)

(Table

statistically

(W)

1207 (20)

for

displayed

ACTIVITY

5750

cytoplasmic

markers

KINASE

Pellet

Cucumber radicle homogenate was centrifuged was resuspended in homogenizing buffer, and supernatant were assayed for glycerol kinase enzymes spectrophotometrically.

subcellular

RESEARCH COMMUNICATIONS

the

BIOCHEMICAL

Vol. 143, No. 3, 1987

0.05

AND BIOPHYSICAL

Fig.

3.

at fixed

concentrations

the

suboptimal

(0.05mM

of ATP to investigate intersection

X-axis,

was held

0.20

0.25

(Glycerd, UM)

SUBSTRATE DEPENDENCEOF GLYCEROL KINASE. Enzyme was 136 ug of cucumber radicle protein. Concentrations of ATP were 1mM (closed circles), 0.2mM (open circles) and 0.05mM (squares). Each point represents the mean of 3 determinations.

concentration

resulting

0.15

0.10

l/S

RESEARCH COMMUNICATIONS

point

was also at 10,

the mechanism

(Figure

obtained

and 0.2mM)

3),

to the

when ATP was the

and optimal of the

left

enzyme

of the

substrate

(1mM) (21).

Y-axis

varied

The

and above

and glycerol

30 and 100uM. DISCUSSION

Two criteria glycerol

indicate

kinase

is

chromatography, for

and second,

mammalian

Properties previous sharp

optima

contrast

with

ATP.

(11)

pH optimum

biphasic

In addition

the

tissues

(Figure

values

to differences

product

they

could

act

co-migrated

as the

and be converted of the

of the

enzyme

p lant

on thin-layer

specific

substrate

to dihydroxyacetone (Table

2) is

similar

to

(7). kinase

of 8 and 10. reported

product

on an activity

of 9.0

react ion First,

localization

of glycerol

report

anionic

dehydrogenase

The cytoplasmic

in most

the

glycerol-3-phosphate.

glycerol-3-phosphate

phosphate. that

that

in

this

study

differ

in unfractionated 2) contrasts

The Km for of 5 uM for in purity, 982

with

glycerol

pea leaf the

previously

from

extracts.

a The

reported

of 55uM and of ATP of 320uM

glycerol the

considerably

and nonlinear

present

study

kinetics differs

for

in using

Vol.

143,

No. 3, 1987

a more

BIOCHEMICAL

sensitive

variations

enzyme assay.

of the

The kinetics

enzyme

from

of plant

gave

double-reciprocal

occurs

in other

synthesis

unclear, phosphate underway

formed (25).

plants

apparently

dehydrogenase in

this

kinase

laboratory

via

its

two substrates

of a sequential

However,

activity

and leaves.

with

kinase

plant

to not of high

to investigate

route

possess specific this

mechanism

(23)

glycerol

its

COMMUNICATIONS

may be significant

and seedlings,

indicative

of lipids

since

seeds

RESEARCH

there

such as adenylate

Glycerol-3-phosphate the

BIOPHYSICAL

Nevertheless,

glycerol

plots kinases,

AND

(22).

and hexokinase

kinase of entry cytoplasmic activity

(Figure

3) This

(24).

can be utilized

in

into

is

glycolysis

glycerol-3(11).

Studies

are

pathway.

ACKNOWLEDGMENT This

study

was supported

by NSF grant

PCM-8306708.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Stumpf, P.K. (1980) Biochemistry of Plants, Vol. 4. Academic, New York. Beevers, H. (1982) Ann. N.Y. Acad. Sci. 386, 243-253. Breidenbach, R., Kahn, A., and Beevers, H. (1968) Pl. Physiol. 43, 705-713. Beevers, H. (1956) Pl. Physiol. 31, 440-445. Beevers, H. (1961) Nature 191, 433-436. Lin, E.C.C. (1976) Ann. Rev. Microbial. 30, 535-578. Lin, E.C.C. (1977) Ann. Rev. Biochem. 46, 765-795. Barron, E. and Stumpf, P.K. (1962) Biochim. Biophys. Acta 60, 329-337. Cheniae, G. (1965) Pl. Physiol. 40, 235-242. Huang, A. and Beevers, H. (1975) Pl. Physiol. 55, 555-558. Hippman, H. and Heinz, E. (1976) Z. Pflanzenphysiol. 79, 408-418. Lowe, C., Harvey, M., and Dean, P. (1974) Eur. J. Biochem. 41, 347-351. Robinson, J. and Newsholme, E. (1969) Biochem. J. 112, 455-464. Good, N., Winget, G., Winter, W., Connolly, T., Izawa, S., and Singh, R. (1966) Biochem. J. 5, 467-478. Sedmak, J. and Grossberg, E. (1977) Anal. Biochem. 79, 544-552. Lowry, O.H., Rosebrough, N., Farr, A., and Randall, R. (1951) J. Biol. Chem. 193, 265-275. Bergmeyer, H.U. (1983) Methods of Enzymatic Aanlysis. Verlag Chemie, Weinheim. Huang, A. and Beevers, H. (1971) Pl. Physiol. 48, 637-641. Wilkinson, G. N. (1961) Biochem. J. 80, 324-332. Crabbe, M.J. (1984) in Microcomputers in Biology (Ireland, R. and Long, S. eds.) pp. 107-150. IRL Press, Oxford, Cleland, W.W. (1979) Meth. Enzymol. 63, 103-138. Rudolph, F. and Fromm, H.J. (1979) Meth. Enzymol. 63, 138-159. Rhoads, D. and Loewenstein, J. (1968) J. Biol. Chem. 243, 3963-3968. Ning, J., Purich, D. and Fromm, H, (1969) J. Biol. Chem. 244, 3840-3845. Gurr, M. (1980) in Biochemistry of Plants, Vol. 4. Academic, New York, pp. 205-248.

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