An NADP-dependent L-glutamate dehydrogenase from chloroplasts of Viciafaba L

Vol. 32, No. 4,196B

BIOCHEMICAL

AN NADP-DEPENDENT L-CLUTAMATE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DEUYDROCERASE FROM CHLOROPLASTS OF VICLA

FABA L. Rachel Department

Received

Chlorella

becon

cells

photosynthesis.

acids

during

oration

oE nitrogen

glutamate

in whole

Ritenour

et al

Eaberii

unable

to detect

this

the

oE --Vicia L-glutamate

times

ions

England.

(1967)

will

stimulate

greater

rate

concluded

activity This

in the

reports

which

appears

of glutamate

in vivo

aauaonium incorporation

(1959)

would

have oE 14c02 would

dehydrogenase

oE Zea mays and We were

activity formation

also

fractions

Eor an NADP-

to be localised

seem likely

has

incorporation

L-glutamate

the evidence

in the direction and it

skeleton

enzyme in chloroplast

The synthetic

of 2-oxoglutarate

carbon

enzymic

leaves

system.

production

the mode oE incorp-

to the mitochondria. of this

paper

but

of amino-

of incorporation

A likely

that

steady-

in isolated

and Moses et al

by NAD-dependent

dehydrogenase lamellar

demonstrated

1958)

the

cells.

during

Formation

produced

(1959)

enzyme was conEined

faba.

been

photosynthetically

in whole

C fixed

1960).

and Whatley,

Chlorella

signiEicant

in the chloropiast six

York,

CO2-fixation

and Kirk,

has also

oE 2-oxoglutarate

Setaria

is

oE York,

32% oE the total

Holm-Hansen

annnonium

dependent

R. Kirk

during

Bassham

Capindale

into

that

leaves

for

photosynthesis

investigated.

be by &nation

from

labelled

(Smith,

(Rosenberg,

little

but

rapidly

in vitro

chloroplasts

into

University

and may account

state

reported

of Biology,

and Paul

July 16, 1968

Amino-acids

been

M. Leech

exclusively

oL this than

enzyme in the

to be responsible

Eor

in chloroplasts.

Abbreviations : NAD - nicotinamide-adenine dinucleotide (free acid); NADP nicotinamide adenine dinucleotide phosphate (disodim salt); NADH2 - nicotinamide adenine dinucleotide reduced (disodiun salt); NADPH2 - nrcotinamide adenine dinucleotide phosphate reduced (tetrasodianu salt); Tricine - Tris (hydroxymethyl) methyl glycine.

685

Vol. 32, No. 4, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALSANDMETHODS: Plants of Vicia faba L. var. as previously Phillips

described (Ridley and Leech, 1968) under warm white fluorescent

Reflectalites

Freshly-harvested

(colour 29), 2500 foot candles with a 16 hour-day.

leaves were homogenised in phosphate buffer

O.lM, pH 6.8 containing:-

sorbitol

sodium isoascorbate (0.1%). weight.

'The Sutton' were grown

(0.33M), NaCl (0.1X),

The ratio

(KH2P04/Na2HP04)

MgC12 (0.1%) .and

of mediumto leaves was 2.5:1 by

A leaf homogenate was prepared according t,p the method of Walker

(1964) and fractionated

by differential

centrifugation

as: follows:

LEAF HOMOGENATE I FILTEREDTHRO' 4 layers 25~ nylon bolting 8 layers muslin I E FILTRATE CENTRIFUGED15 sec. 5400 x g

r

SUPERNATANT I CENTPIFUGED 20 min. 2000;E;;T

silk

DI p enriched fraction

SLJPERNATANT = S2 I 2 mitochondria enriched fraction All

operations were carried out at O°C and as rapidly

Pl and P2 were separately

as possible.

resuspended in 10 ml. tricine-NaOH buffer

The pellets (O.O2M,

pH 8.0) and h,omogenisedin a Ten Broek ground glass homogeniser. Examination by phase contrast all

microscopy showed that this hypotonic medium broke virtually

the chloroplasts. Enzyme assays given under Tables I and III;

chlorophyll

determined by

the method of Arnon (1949). RESULTS: The total

activities

hydroganase in (Pl), Table I, Assay I.

(P2) and the supernatant S2 fractions The NADP-dependentactivity

chloroplast-enriched

fraction

(P2) while the activity enriched fraction.

of the NADand NADP-dependentL-glutamate de-

(A) I, is greater in the,

(Pl) than in the mitochondria-enriched

fraction

of the NAD-enzymeis greater in the mitochondria-

Assay

photometric assay i.e.

are shown in

II gives the corresponding values for the spectro-

the proportion

of the total

686

enzyme activity

of each

Vol. 32, No. 4, 1968

BIOCHEMICAL

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

cell Eraction which can be solubilized Virtually

no NADP-dependent activity

by osmotic breakage oE the organelles. can be removed by this treatment:

contrast the NAD-enzymein the mitochondria-enriched very readily

fraction

in

appears to be

These results suggested that the NADP-dependentL-

solubilized.

glutamate dehydrogenase may be firmly bound to the chloroplast lamellar system. Table I: Distribution of NAD-dependent and NADP-dependentL-glutamate dehydrogenase activity in cell Eractions from leaves oE Vicia faba L. Cell fraction (A) nano-moles 2-oxoglutarate Pl (chloropla8t enriched) P2 (mitochondria enriched) -s2 Ratio Pl/P2

(B)

nano-moles keto-acid

Pl P2

NADP-dependentenzyme aminated/20 minutes ASSAY II ASSAY I 749 47 392 17

NAD-dependent enzyme ASSAY I 336 506

II 115 763 221 0.15

ASSAY

0

1.91

2.76

aminatedlmg. chlorophyll/20 ASSAY I ASSAY II 1810 53 1960 22

0.66 minutes. ASSAY I 837 2530

ASSAY II 131 987

ASSAY I - calorimetric (Friedeman, 1943). Details given in Table III. ASSAY II - spectrophotanetric (Bulen, 1956). The resuspended pellets Pl and P2 and the supernatant S2 were centrifuged at 50000 x g Eor 30 min. and aliquota of the 8upernatantS assayed. The oxidation of reduced nitotinamide coenzyme wa8 measured at 340 nm at 25OC. Reaction medium (inu moles): NADH2 or NADPH 0.5; potassium 2-oxoglutarate (pH 7.01, 40: (NR ) SO 300; tricine NaOHbuff& (pH 8.01, 420. 0.5 ml supernatant containing td i&$&e added at zero time. Total vollnne 3 ml. The control cuvette contained the enzyme and all reagent8 except 2-oxoglutarate. This suggestion is firmly

supported by a comparison of the total

activities

oE the NADP-enzymein Pl and P2 expressed in te'rms of mg. chlorophyll

the fraction

(Table IB):

the values Eor the chloroplast-enriched fraction

seem likely

associated with P2 is located in the fragments

of lamellae present in this fraction.

hydrogenase activity

identical.

Eraction

and the mitochondria-enriched that the activity

are virtually

in

The NAD-dependent L-glutamate de-

show8 no constant ratio with chlorophyll

in the chloroplast-enriched

fraction

It would

and its presence

could be accounted for by the presence

of contaminating mitochondria. To check more rigorously is located in the chloroplast

that the NADP-dependent glutamic dehydrogenase lillaellae,

intact

chloroplasts

(Leech, 1964) and

naked lamellar systems (James and Leech, 1964), both known to be virtually

687

Vol. 32, No. 4, 1 Q68

BIOCHEMICAL

completely

freed

The intact

chloroplasts

were

Table

NADP-dependent chloroplasts

L-glutamate (2) Chloroplast

II:

from

fraction

(1) (2)

Intact chloroplas Lamellar systems

Chloroplast Incubation

from

of activity

dependent

L-glutamate

chloroplast

lamellar

The enzymic of glutamate

assay.

a measure

Intact faba L

of

of suspension.

I B).

restricted

in-

oE decrease

and lameI&

were also very

The NADP-

to a location

reaction

the

(Table

in the

in isolated

mixture

n. butanol-acetic

of the reaction

solvent

ten minutes,

III)

but

none

or with

isolated

with

and Brown chloroplast

acid-water

(4:l:l) positive,

and also

the solvent. 688

boiled

absence

tioenxyme

of

have

probably Recently

recorded

endo-

Eractions. on thin (v:v:v)

with with

is

the fraction.

(1968)

was

chloroplasts.

layers

glutamate

saturated

of .kiesel-

as solvent,

had the same Rf

and cochromatographed system

Pl

some activity

in the

reduced

was chromatographed

was ninhydrin

eluted

first

of added

and Harvey

deamination

and NADH2 or NADPH2, the

oE chloroplasts

coenxyme

reductive

in the chloroplast-enriched

in ,the absence

(1965)

and the

2-oxoglutarate

over

of coenzymes

activity

in the butanol

clearly

in both

types

Pl and P2 (Table is

of Pl with

in the absence

and when

for

in more detail

NAB&j and NABI@

glutamate

both

chloroplasts

intact

to be linear

and Santarius

G, with

and the rate

the same for

of 2-oxoglutarate

of the endogeneous

When the

the enzyme is’present

systems

is

found

studied

in the, absence

product

the

system.

was found

The small

II,

lamellar

dehydrogenase

On incubation

ammonium ions,

spot

already

amination

were

fraction.

guhr

before

and assayed.

dehydrogenase activity in (1) lamellar systems from Vicia

in Table

for’both

to the values

geneous

prepared

Change in 2-oxoglutarate (n moles/lO’) n moles/mg chlorophyll/lO’ 818 770 2910 790

per mg. chlorophyll

The levels

Heber

ruvtured

1.06 3.70

and in naked

2-oxoglutarate

found

ts

the results

chloroplasts

reaction

osmotically

were

and lamellar systems prepared as described above, conditions as Eor reaction medium A in Table III.

As seen

close

contamination,

Chlorophyll h3.)

Cell

tact

cytoplasmic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

as authentic gave

phenolic

a single H20 as

Vol. 32, No. 4, 1968

Table III:

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Activity of L-glutamate dehydrogenaae in*a chloroplast-enriched fraction (Pl) Ercm Vicia Eaba leaves measured by A. amination oE 2-oxoglutarate: B. reductive deamination'oE glutamate.

Conditions oE Assay Change in 2-oxoglutarate ( n molesimg. chlorophyll/lO' No coenzyme NADP-dependent NAD-dependent A. Decrease in 2-oxoglutarate a2 442 109 B.

Increase in 2-oxoglutarate

64

240

80

Calorimetric assay of the enzyme. 0.5 to 2.0 ml. oE resuspended pellets Pl and P2 or the supernatant 52 were incubated and the conversion of P-oxoglutarate to glutamate (Reaction mediumA) or of glutamate to 2-oxoglutarate (Reaction mediumB) measuredby the change in dicarboxyclic keto-acid conThe enzyme reaction was stopped by the addition of 4 ml. 50% centration. TCA to 6 ml reaction mixture and 2-oxoglutarate concentration measured by the method oE Friedeman (1943). With each 1 ml. sample halE the quantity oE each reagent recommendedby Friedeman Eor 3 ml samples was used and oxygenation effected by swirling the samples on a 'Whirlimix' vortex stirrer. Aliquots were taken at each separation stage and the concentration oE the 2,4 dinitrophenylhydrazone derivative assayed by optical density measurementsat 420 nm. O.D. was shown to be linear with concentration over the range 0 - 15Oug keto acid. Reaction mediumA contained (umoles): potassium P-oxoglutarate (pH 7.0), 20; NADH or NADPH2,5; (NH4)*so4, 250; tricine-NaOH buffer (pH B.O), 800. Fina 1 volume 6.0 ml. containing 2.4 mg. chlorophyll incubated at 25O in a shaking waterbath Pot 10'. Reaction medim B contained (pmoles): sodium L-glutamate (pH 7.0), 100; NADor NADP, 5; tricine-NaOH buffer (pH g.O), 900. Final volume 6.0 ml. containing 1.4 mg. chlorophyll. Other conditions as A. The rate 2-oxoglutarate

of glutamate synthesis with NADPii2was six times the rate of

synthesis under similar

conditions

(Table III).

A completely

reversed pattern was found for the NAD-dependent enzyme where the rate of 2oxoglutarate

synthesis was twice as rapid as the rate OF glutamate synthesis.

This suggests

a situation

similar

to that found in yeast (Holzer,

there appear to be two separate functional glutsmic acid oxidation of glutamate. leaves of Vicia and the latter

1966) where

enzymes, one responsible for

using NADand another which catalyses the synthesis

The present results would s.uggest the possibility faba the Eormer activity in the chloroplast

is localized

that in

in the mitochondria

lamellar system.

We are most grateful to the Agricultural Research Council (Great Britain) Eor financial support for this work. It is part of a research prograzme concerned with the biochemical self-suEEiciency of chloroplasts. 689

Vol. 32, No. 4, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Arnon, D.I., Plant Physiol., 24, 1 (1949). Bulen, W.A., Arch. Biochem. Bzphys., 2, 173 (1956). Friedeman, T.E., J. biol. Chem., 147, 415 (1943). Harvey, M.J. and Brown, A.P., Biochem. J., 105, 30P (1967). Heber, U.W. and Santarius, K.A., Biochim. BGhys. Acta, 109, 390 (1965). Holm-Hansen, O., Nishida, K., Moses, V. and Calvin,M.,J. Expt. Bot., lo, 109 (1959). Holzer, H., Biochem. J., 2, 37P (1966). James, W.O. and Leech, R.M., Proc. Roy. Sot. B., 160, 13 (1964). Leech, R.M., Biochim. Biophys. Acta, 79, 637 (196x Moses, V., Holm-Hansen, O., Bassham,J.A. and Calvin, M., J. Mol. Biol., 1, 21 (1959). Ridley, S.M. and Leech, R.M., Planta, in press (1968). Ritenour, G.L., Joy, K.W., Bunning J. and Hageman,R.H. Plant Physiol., --' 42 233 (1967). Rosenberg, L.L., Capindale, J.B. and Whatley, F.R., Nature, Lond., 181, 632 (1958). Smith, D.C., Bassham, J.A. and Kirk, M., Biochim. Biophys. Acta, .-48, 299 (1960). Walker, D.A., Biochem. J., 2, 22C (1964).

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