Incorporation of labeled orthophosphate into nucleoside diphosphates

BIOCHEMICAL

Vol. 18, No. 4, 1965

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INCORPORATION OF LABELEDORTHOPHOSPHATE INTO NUCLROSIDEDIPHOSPHATES* Bert Winter and Robert S. Bandurski Department of Botany and Plant Pathology Michigan State University East Lansing, Michigan ReceivedDecember

29, 1964

We wish to report fraction

on the presence of an enzyme in a particulate

of tobacco leaves which catalyzes

inorganic

phosphate

ability,

into ADP, UDP, CDP and GDP but has little,

to incorporate

attention

is directed

phosphates

Cl4 -labeled

or no

ADP or ATP into RNA. In addition

to the non-enzymatic

by impurities

P3'-purification

the exchange of P32-labeled

labeling

in P32-orthophosphate,

of nucleoside

and a procedure

for

described.

Methods and Results: Enzyme

Equal weights

preparation

and homogenizing

medium (0.04 M, NH4OH, 0.05 g Tris-free

FJmercaptoethanol) pH approximately

of tobacco leaves,

were ground in a mortar 8.6, was filtered

through

cheese cloth

and centri-

paper,

pellets

were suspended in 75 ml of a solution

H Tris,

pH 7.4,

at 50,000 X g. natant

fluid

were collected

then centrifuged

The slurry,

through

The resultant

centrifuged

*Supported, of Health,

was filtered

pellets

The

of 0.005 g MgCl2, 0.05

and centrifuged were discarded

for 2 hours at 105,000 X g.

for 25 minutes and the superThe pellets

and suspended in 4 ml of the above solution.

P32 purification diphosphates

fluid

at 105,000 X g for 2 hours.

0.005 g mercaptoethanol

sand

base and 0.005

and pestle.

The supernatant

fuged for 10 minutes at 7,000 X g.

quartz

Incubation

of unpurified

leads to incorporation

P32-Pi with nucleoside

of radioactivity

into

the diphos-

in part, by a research grant from The National Institutes United States Public Health Service, AM-05916. 512

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL

is adsorbable

RESEARCH COMMUNlCATlONS

phates.

The radioactivity

tographs

with ADP, GDP and UDP plus CDP using 0.1 E phosphate

6.8/ammonium sulfate/n-propanol solvent

(Pabst,

1956).

onto charcoal

(100:60:2)

The labeling

and co-chromapH

as the chromatographic

is temperature

and time dependent

and has a pH optimum at 5.0. The impurity carrier-free

is eliminated

HCl concentration

heated to 100° for 10 minutes. is passed through

neutralized After

0.1 N.

Following

Pi and HCl and This mixture

dilution

After

the first

neutralization

and last

is eluted

is

Assay

to pH 7 the P32-Pi remains useable for

Owing to the presence of phosphatases

cedure was used to obtain and to demonstrate

that

such as that described the resultant

the specific all

the supernatant

fluid

After

by filtration

with a total elutions,

To a reaction

CDP and ADP are coincident.

stirring

pH 5 and

for 10 minutes,

and washed with 25 ml of 10 ml of H20.

of 5 ml of ethanol-l?H40H-H2O

and the combined supernatant

to Whatman 3 MM paper and subjected pH 3.3.

mixture,

To 0.9 ml of

pH 5 and, finally,

and adsorbed onto a filter

buffer,

pro-

of the diphosphates

was added 3.6 ml of 0.1 g phosphate,

H20, 15 ml of O.OlM phosphate,

using 3 successive

the following

removed by centrifugation.

was collected

was eluted

labeling.

1, was added 0.55 ml of E HC104 and

100 mg of acid-EDTA washed charcoal. the charcoal

activity

4 were labeled.

for Fig.

precipitate

with

10% of the radio-

6 weeks at 4O and does not cause non-enzymatic

evaporated

It

to pH 7 and adsorbed to a 0.5 gm column of Dowex l-X8-Cl.

activity.

charcoal

is

to 0.02 x HCl the

washing the column with H20, the radioactivity

at least

of

a 0.5 gm column of Dowex 50-X4-$.

4 ml of 0.05 g HCl, discarding

citrate

To 3 millicuries

P32-Pi are added 3 pmoles of unlabeled

water to make the final

solution

as follows.

paper disk.

separates

For most assays,

513

this

(50:1:49)

solutions,

The disk was stuck

to electrophoresis

This procedure

The

using 0.01 3

UDP and GDP, but was sufficient.

Vol. 18, No. 4, 1965

BIOCHEMICAL

When it was desired

to separate

Pi, which moves only slightly 1956) with isobutyric

CDP and ADP and to remove traces

acid/ammonia/water

pR 4.3 (57:4:39)

of electrophoresis.

method and radio-autography,

diphosphates

RESEARCH COMMUNICATIONS

ahead of UDP, chromatography

out at 90' to the direction sional

AND BIOPHYSICAL

were approximately

it

was demonstrated

equally

the UV absorbing

areas were cut out,

HCl for spectral

determination

labeled.

(Pabst, was carried two-dimen-

that

all

4

For quantification,

counted and then eluted

of purity

and Rf values of the diphosphates,

By this

of

and quantity.

with l

The mobilities

under our conditions,

are shown

below: Compound ADP CDP GDP UDP

Mobility

Time Course function

of time,

(cm.2sec '%olt'l) 8.7 x 10'5 8.7 x 10'5 12.4 x 10'5 16.5 x 10-5

The rate of incorporation is shown in Fig.

1.

Rf 0.44 0.38 0.23 0.20 of P32-Pi into GDP, as a

This experiment

was done by

0

0

30

60

90

120

150

180

Incubation Time (minutes) as a function of time. Each tube contained in 10. Na Acetate, pH 7.4, 50; MgC12, 4; EGle:f T$~-~~?~~~* !H47~~geac~; P32-potassium phosphate 5 (spkifit activity 5,~70'c/mpmole/min'l); enzyme, 0.2 ml ani water to a total volume of 0.55 ml. Reaction terminated and diphosphates isolated as described in text. Fig.

1.

Reaction

rate

Vol. 18, No. 4, 1965

BIOCHEMICAL

one-dimensional tion

is linear

this

time,

electrophoresis

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

and only GDP was measured.

with time up to 30 minutes

Pi and diphosphatas

and then levels

are not limiting

The reacoff.

At

nor has the enzyme

been inactivated. pH optimum

The rates

of incorporation

and ADP plus CDP are shown in Fig. at approximately

the same rate

2.

of P3*-Pi

All

for all

into UDP, GDP,

4 diphosphates

I-? concentrations

are labeled tested.

The

pH optimum is about 7.4.

10 r a !2

9

"D f0 8 ? a .r( N' ma !

* 7 6 5 4 3

i2 1 " 6.5

7.0

7.5

8.

0

6.5

7.0

PH

7.5

8.0

0-

PH

6.5

7.0

7.5

8.0

PE

Fig. 2. Reaction rate as a function of pH. Conditions as for Fig. 1, except the pH was adjusted to the indicated value, and incubation was for 30 min.

Enzyme concentration centration intervals

is shown in Fig.

Reaction 3.

employed, the reaction

rate as a function

Over the range tested, is a linear

centration.

515

function

of enzyme conand the time of enzyme con-

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL

I

6.

60'

0

0.04

4

incubation

0.08 0.12 Enzyme (ml)

RESEARCH COMMUNICATIONS

0.16

0.20

0

120'

0.04

incubation

0.08

0.12

0.16

0.20

Enzyme(ml)

Fig. 3. Reaction rate as a function of enzyme concentration. Incubation conditions were as for Fig. 1, except enzyme concentration, as indicated. The optical density of the enzyme, prepared as described in the text, is 76 and 48 at 260 and 280 mu respectively. Non-reactivity were performed under varied three di-

of 8-Cl4 -ADP and ATP A large number of experiments

in which Cl4 -ADP or ATP were incubated conditions

of time and presence or absence of the other

or triphosphates.

the perchloric

The total

acid insoluble

This is negligible

with the enzyme

pellet

radioactivity

incorporated

into

ranged from zero to 0.02 mumoles.

compared to the phosphorolysis

reaction.

Discussion: The enzyme preparation P32-Pi into

nucleoside

not synthesize similar,

diphosphates,

polynucleotide

has been obtained

Alternatively

our preparation

phosphorylase-like tissues

described,

catalyzes

but under our conditions,

1963 and Hilmoe and Heppel,

of does

from C14-ADP. An enzyme, which may be from yeast (Grunberg-Manago, could be related

enzymes previously

(Brummond et al.,

incorporation

described

1957; Kessler

1963).

to the polynucleotide in plant

and animal

and Chen, 1964; Harris,

1957).

References: Anonymous, Pabst Laboratories, Circular OR. 10 (1956). Bruannond, D. O.,Staehelin,M.,and Ochoa,S.,J.Biol.Chem.,~,835(1957). Grunberg-Manago, M., Progr. Nucleic Acid Res., 1, 93 (1963). Harris, H., Proc. Royal Sot. (London) Ser. B, 158, 79 (1963). Hilmoe, R. J., and Heppel, L.A., J. Am. Chem. Sot., 79, 4810 (1957). Kessler, B. and Chen, D., Biochim. Biophys. Acta, 80, 533 (1964). 516