Pyridine nucleotide stimulation of lipid phosphorylation

BIOCHEMICAL

Vol. 32, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PYRIDINE NUCLEOTIDE STIMULATION J. F. Erbland,+ University Biochemistry

ReceivedJuly

(Friedkin

Conover

et.

can show high

This

anomaly

of lipid

cles is tute

the

al,

incubation

incorporation

et.

of high

1949,

Kennedy,

al.,

Marinetti

et.

further

study

the

energy

mammalian

1963,

were

donor

phosphate

1953,

into

energy

production

the

et.

1967).

localization

compound,

on substrate

al,

phosphorylation.

cellular

phosphate

one

and the

phosphorylation

phosphorylation.

a long

search

found

with

that

a critical

NADH (or

NAD+ or NADH. cofactors

studies

NAD+>. The pattern

such as Mg "'

and on the

cell

particles

on rat factor

In these of lipid

liver

necessary systems

for

lipid

and cell

also

with

parti-

phosphorylation

NADP+ or NADPH cannot

labeling

FDP, CMP and is which

homogenates

32 Pi is

dependent

substi-

dependent

on the

time

are present.

Supported by a USPHS grant HE 02063 from the National Heart Institute. Abbreviations: LEC, lecithin, PE, phosphatidylethanolamine, PA, phosphatidic acid, MPI, monophosphoinositide, GPX, a glycerolphosphatide tentatively identified as phosphatidylglycerolphosphate, 32Pi, radioactive orthophosphate, DNP, dinitrophenol, FDP, fructose diphosphate. Present address:

al.,

common phospha-

of lipid

to determine

et.

mitochondria)

Erbland

the nature

high

have met with

and Hawthorne,

these

1964,

the common

Marinetti

microsomes,

al,

into

systems

Galliard

of 14 C-glycerol

1962,

of this

of 32Pi

free

(homogenates,

us to investigate

objectives

coenzyme

for

Garbus

incorporation

et. led

we have

on other

*

level

oxidative After

level

systems

phosphorylation,

dependency versus

1960,

in these

(Marinetti

The major

high

and Lehninger,

al.,

However,

tides

tt

and M. Chatterjee

School of Medicine and Dentistry Rochester, New York

such as LEC and PE in cell

success

1963).

of Rochester Department,

to demonstrate

phosphatides

1957,

G. V. Marinetti

PHOSPHORYLATION*

22, 1968

Attempts

little

OF LIPID

t tt

Monsanto Research, Everett, Mass. Nattermann-Arzneimittel Export GMBH Co. Eupenerstr. 159a-161 Cologne-Braunsfeld NEST GERMANY

of

BIOCHEMICAL

Vol. 32, No. 5, 1968

Materials

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Methods

ATP, ADP, CTP, CMP, NAD+, NADH, NADP+, NADPH were chemicals

Inc.,

FDP from

A from Nutritional Y-~*P-ATP

Biochem.,

International

Chemical

homogenates

incubations

1967).

on Watman

were were

carried

SG 61 silica

was spotted

on the

gel

silica

out were

paper

solvent

solvent

radiograms

were made on Kodak rapid

one week.

The radioactive

was diisobutyl

the radioactivity

in the

the total

by 50 to get

from

tables

are

total

previously

(Erbland

in a shaking

water

at 37c C.

bath

by two dimensional

Mann

of each incubation

(20 x 20 cm).

Ascending

process were

chromatography

a Packard for

Liquid

each lipid

medium volume cpm in the

lipid

film.

The exposure

component

was

cut out,

and

20 ul

cpm must

Auto-

time

counter.

in the

the

(v/v>.

(v/v).

delineated,

Scintillation

was 1.0 ml, per

65:25:4 40:25:5

carefully

Air

system

acid-water X-ray

et.al,

chromatography

20 ul aliquots

spots

in

incubation the

1 demonstrates

of 32 Pi into

lipids

the

of rat

labeling

requires level

labeling

of LEC and PE is labeling

NADH (or

The aliquot.

be multiplied

200 mg of wet weight

CMP in this slotser

of a number

extent. NAD+).

However, Labeling

stimulated

liver.

dependent

that

of GPX.

stimulation with

hours

incubation.

746

rapid

labelof lipid

NADH alone

Higher stimulates

as effective

whereas

but

The labeling

CMP also

CTP is not

of GPX, PE and MPI is l-2

2.

incorporation

stimulate

NADH and Mg++.

on CMP as shown in Table

Labeling

and requires

alone

the marked

of GPX occurs both

on the

by FDP when NADH and Mg++ are present.

of PE and MPI but not system.

of agents Mg++ ions

homogenates.

of PE and MPI requires

of LEC is

labeling

effect

liver

of PE and MPI to a small

LEC is

Corp.,

and Discussion

Table

the

oligomycin

Corp.,

ketone-acetic

lipid

determined

given

high

antimycin

and Engineering

was chloroform-methanol-water

The second

ing

Kodak,

as described

separated

paper. gel

The first

was employed.

Results

Science

and Nuclear

prepared

The lipids

was the gas phase.

Since

Eastman

from P-L Bio-

Lab.

Liver

counts

DNP from

32 Pi from Nuclear

Biochemical,

from

Research

Schwartz

obtained

labeling

as of

BIOCHEMICAL

Vol. 32, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Incorporation

of

32 Pi Into

1 Lipids

of Rat Liver

cpm - 120 min. Additions (all 10 mM)

GPX

incubation

PE -

LEC

MPI

None

190

68

61

0

Mg++

111

378

58

63

FDP

174

24

24

0

NADH

5293

169

63

0

NADH + Mg++

7218

1664

36

FDP t Mg++ NADH f Mg++ FDP

(100 mg wet weight

liver,

of CMP on the

68

72

0

1753

2695

187

125 mM sucrose,

Table Effect

Homogenates*

Incorporation

1388 0 399

45 uC 32Pi)

2

of 32 Pi Into

cpm - 60 min.

Lipids

of Rat

Liver

incubation

System

GPX

-PE

LEC

MPI

Control

385

1550

80

110

CMP

1mM

370

2200

280

160

CMP

10 mM

325

1450

195

180

*

(100 mg wet weight

liver,

125 mM sucrose,

44 pc 32Pi)

747

Homogenates*

10 mM NADH, 10 mM MgC12,

10 mM FDP,

BIOCHEMICAL

Vol. 32, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of NADH Concentration

on the

3 Incorporation

Rat Liver

k

of NADH

-PE

0

158

225

55

43

5

2931

800

65

613

10

4742

962

112

1010

15

4160

541

72

706

20

4160

470

94

660

(100 mg wet weight

in a liver optimal

3 shows

liver,

the

homogenate. concentration

In order

to elucidate

tors

of oxidative

tion

was examined.

systems

of Mg

tt

stimulatory

one can assume that

was supported

by the

coenzyme in Table

GPX, PE, and MPI by DNP and cyanide GPX by antimycin These the electron

data

A is give

transfer

also

of NADH the effect

is

of oxidation

the

same action

was undergoing 4.

The marked

evident.

10 mM.

The

to be 10 mM.

and of uncouplers

this data

found

action

NAD+ has essentially

phosphorylation

of NADH is

and FDP was also

the

50 pC 32Pi>

on lipid

concentration

phosphorylation Since

10 mM MgC12,

of NADH concentration

The optimal

of

-MPI

125 mM sucrose,

effect

Lipids

incubation

-GPX

Table

This

Pi Into

Homogenates*

cpm - 30 min. Concentration h&i)

32

of

of inhibi-

phosphorylaas NADH in these

oxidation-reduction. inhibition

The inhibition

of labeling of labeling

of

apparent.

evidence

that

chain

and that

the NADH effect high

748

energy

is

in part

phosphate

is

of

mediated

through

involved.

This

BIOCHEMICAL

Vol. 32, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of Inhibitors Lipids

on the

4 Incorporation

of Rat Liver

of 32Pi

Homogenates*

cpm - 30 min. Inhibitor Exp.

1

Exp.

none

2

Into

incubation

GPX

-PE

MPI

1312

160

138

DNP

0.1 n-84

71

74

0

CN-

1.0 T&l

59

90

0

none

3010

**

**

DNP

0.1 mM

194

**

**

CN-

1.0 mM

2-l

**

**

10 Pg

127

**

**

A

Antimycin

(100 mg wet weight

liver,

125 mM sucrose,

10 mM NADH; 10 mM MgC12,

50 ?.lc 32Pi)

**

(These

was supported lipid

lipids

not

by our finding

analyzed

that

in this

oligomycin

experiment.)

also

gave a strong

inhibition

of

phosphorylation. The data

very

low

inhibition greatly

for

in these of lipid reduced,

FDP may be mediated in

were

the

lecithin systems labeling or entirely

are not given if

FDP is

not

in Table added.

by DNP, cyanide eliminated

by FDP enhancing

4 since However,

cytoplasm.

749

labeling

we later

and antimycin

by addition substrate

lecithin

found

phosphorylation

that

in homogenates

of 10 mM FDP. during

This

is the

is action

glycolysis

of

BIOCHEMICAL

Vol. 32, No. 5, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATtONS

It was found that y- 32P-ATP can replace NADH+ 32Pi for labeling of GPX either

in homogenates or in isolated mitochondria.

primary donor of phosphate for phosphorylation ATP did not replace NADH+ 32Pi for labeling system (Chatterjee,

Marinetti

Hence ATP appears to be the However, y- 32p-

of this lipid.

of PE in a microsomal-cytoplasm

and Pettit).

The following phosphorylation

observations have also been made: (a) ADP inhibits lipid 32 from Pi in homogenates. (b) Lipid phosphorylation in a micro-

somal-cytoplasm system requires NADH, Mg'+ and either pyruvate or FDP. (c) in homogenatesfrom 14C-glycerol

The

labeling

of lipids

or NADHt

However, whereas the major phosphatide labeled with l4cis LEC, the major lipids labeled with 32Pi are PE and GPX. (d) When

glycerol

requires either ATP t Mg++

Mgtt.

pyruvate and NADHare added to a homogenateor microsomal-cytoplasm system a yellow fluorescent olized

NADH-pyruvate complex is rapidly

(Chatterjee,

Marinetti

formed and is then metab-

and Pettit).

Chemical and enzymatic studies on GPX showedthat it is an acidic glycerophosphatide which is labeled by both 32Pi and 14C-glycerol.

There are several

forms of GPXwhich we believe differ

in the number and type of fatty

esterfied

Mild alkaline

to the glycerol

moieties.

hydrolysis

acids

of GPXyields

a

water soluble phosphate ester of glycerol. This compounddoes not react with hydroxylamine or 2,4-dinitrophenyl zine but is cleaved by periodic

acid.

yields at least two lyso-derivatives. represents phosphatidylglycerol esterfied

Hydrolysis of GPX with phospholipase A Our evidence to date indicates

phosphate which has one or more fatty

to the two glycerol

that GPX acids

units.

These experiments point to a major role for NAD+-NADHin lipid tion.

hydra-

phosphoryla-

High energy phosphate required for this process is produced both by mito-

chondrial and non-mitochondrial

systems.

The non-mitochondrial

system (glycoly-

sis) can predominate when FDP is added to homogenates. The significance

of the rapid and high level

clear at present, especially

labeling of lipid

since we do not observe a similar

750

GPX is not

high labeling

of

BIOCHEMICAL

Vol. 32, No. 5, 1968

PA in these systems.

It

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is of interest

that mitochondria can synthesize GPX in

our systems and can produce acyl dihydroxyacetonephosphate

under other conditions

(Hajra et. al. 1968).

References Chatterjee,

M., Marinetti,

G. V., Pettit,

D., manuscript in preparation.

Conover, T. E., Marinetti,

G. V., Witter,

R. F. and Stotz,

Acta

E., Bioch. Biophys.

41, 264 (1960).

Erbland, J. E., Brossard, M., and Marinetti,

G. V., Biochim. Biophys. Acta 137,

23 (1967). Friedkin,

M., and Lehninger, A. L., J. Biol.

Galliard,

T., and Hawthorne, J. N., Biochim. Biophys. Acta 70, 479 (1963).

Garbus, J.,

Chem. 177, 775 (1949).

DeLuca, H. F., Loomans, F., and Strong, F. M., J. Biol.

Chem.

238, 59 (1963). Hajra, A. K., Seguin, E. B., and Agranoff,

B. W., J. Biol.

Chem.243, 1609

(1968). Kennedy, E. P., J. Biol.

Chem.201, 399 (1953).

Kennedy, E. P., Fed. Proc. 20, 934 (1961). Marinetti,

G. V., Erbland, J., Albrecht,

M., and Stotz,

E., Biochim.

M., and Stotz,

E., Biochim.

Biophys. Acta 25, 585 (1957). Marinetti,

G. V., Erbland, J., Albrecht,

Biophys. Acta 26, 130 (1957). Marinetti,

G. V., Griffith,

M. and Smith, T., Biochim. Biophys. Acta

57, 543 (1962). Marinetti,

G. V., Erbland, J. F. and Brossard, M., in "Metabolism and

Physiological

Significance

of Lipids",

Ed. by Dawson, R. M. C. and

Rhodes, D. N., John Wiley and Sons, LTD., London, 1964 pp 71-93.

751