Conversion of diglyceride to triglyceride by rat liver microsomes: A requirement for the 105,000 × g supernatant

BIOCHEMICAL

Vol. 58, No. 1, 1974

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CONVERSION OF DIGLYCERIDETO TRIGLYCERIDEBY RAT LIVER MICROSOMES: A REQUIREMENT FOR THE 105,000 x g SUPERNATANTl Eric R. Manley2, Harold B. Skrdlant,

Elizabeth

Hansbury and Terence J. Scallen3

Departments of Chemistry and Biochemistry University of New Mexico Albuquerque, New Mexico 87131 Received

March

21,1974

This paper describes a requirement for the 105,000 x g supernatant of rat liver for the synthesis of triglyceride from diglyceride and palmityl coenzyme A by rat liver microsomes. ATP and magnesium chloride are also required. The incorporation of both ]l-14C]-palmityl coenzyme A and [l-l%]diolein into triglyceride has been observed. The 105,000 x g supernatant has no enzymatic activity for this reaction when incubated in the absence of microsomes. The supernatant contains a soluble, essential protein which is nondialyzable, heat sensitive, and destroyed by trypsin. Net synthesis of triglyceride has been demonstrated by chemical analysis. SUMMARY:

Work in this eral

substrate

rat liver,

laboratory

specific

over the last

proteins,

as having essential These soluble

present roles

in the microsomal biosynthesis

(l-5).

proteins

sterols

by the microsomal membranes, and it

operate

as carriers

are required

for water insoluble lipid

have chosen to study is the acylation

years has implicated

sev-

in the 105,000 x g supernatant4

sterols

phenomena might occur in other

several

substrates.

biosynthetic

of

for the synthesis

has been proposed that

of diglyceride

of

they

We reasoned that pathways.

of

similar

The reaction

we

to form triglyceride.

MATERIALSAND METHODS Chemicals:

[1-14C]-Diolein

CoA from New England Nuclear; from Sigma; Silicar

Science Laboratories;

tories,

London, Ontario.

3. 4. Copyright All rights

palmityl-CoA,

CC-7 Special

Applied

1. 2.

was obtained

from Dhom; [1-14C]-palmityltrypsin,

from Mallinckrodt;

and 1,2-diolein

and trypsin 1,2-dipalmitin

inhibitor from

from Serdary Research Labora-

This research was supported by NIH Grant AM 10,628-08. This work is part of a thesis being presented to the University of New Mexico in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Reprint requests should be addressed to T.J. Scallen, Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131. Abbreviation used: Slo5, 105,000 x g supernatant. 0 1974 by Academic Press, of reproduction in any form

Inc. reserved.

229

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

SlO5 and Microsome Preparation: 300 g) were sacrificed

by decapitation.

washed in ice cold potassium (0.1 mM).

The livers

cold buffer

were blotted

After

any residual

was removed with a pipet. fer,

fitting

This washing procedure

were carried

37' under an atmosphere of nitrogen phosphate

buffer

dioxane-propylene

5-10 ~1 of sodium acetate Extractions: form-methanol

a brief

buffer

Incubations

(7.5 ml).

were added as described After

fraction

was repeated

at

twice more.

in liquid

nitrogen,

loss of activity. shaker at

volume of 2 ml of potassium EDTA (0.1 mM).

Diglyceride

Palmityl-CoA

were stopped by the addition chloroform

was

was added in

The residue by silicic

of 1:2 chloro-

(2.5 ml) and water

procedure

the chloroform

was isolated

SlO5)

in fresh buf-

out in a Dubnoff

glycol.

in the extraction

centrifugation

(constituting

(0.1 M, pH 6.0).

Subsequently

under a stream of nitrogen. glyceride

in a final

(0.02 M, pH 7.4) containing

added in S-10 ~1 of 2:l

removed from the

and recentrifuged

separately

at -80' without

incubations

was centrifuged

was resuspended

Teflon pestle,

and they can be stored indefinitely All

The homo-

was discarded,

was carefully

pellet

washed microsomes were frozen

Incubations:

the pellet

were

at 105,000 x g for 60 minutes to remove

The microsomal

105,000 x g for 60 minutes. SlO5 and triply

Teflon pestle.

The clear middle fraction

homogenized with a tight

EDTA

at 20,000 x g for 15 minutes.

The supernatant

microsomes.

and

the livers

and the 20,000 x g supernatant

and recentrifuged

light

mincing with scissors,

was then centrifuged

at 105,000 x g for 60 minutes.

excised

(0.02 M, pH 7.4) containing

(1 mm clearance)

was discarded,

pellet

were immediately

at 1000 x g for 10 minutes,

and the 1000 x g supernatant

microsomal

Livers

(200-

dry, weighed and suspended in 2 ml of fresh

homogenized with a loose fitting genate was centrifuged

male Sprague-Dawley rats

phosphate buffer

per gram of tissue.

Again the pellet

Adult

RESEARCH COMMUNICATIONS

layer

(2.5 ml)

of Bligh and Dyer (6). was removed and dried

was taken up in toluene; acid chromatography

the tri-

as described

below. Chromatography: CC-7 Special)

Separations

columns (1 x 5 cm).

were carried

out on silicic

Toluene was the eluting 230

acid (Silicar solvent,

and 7 ml

Vol. 58, No. 1, 1974

fractions

were

column

in Fraction

Fatty

acids

unless

the

fatty

acids

BIOCHEMICAL

collected.

was changed

3375 liquid

ester

ether-toluene

6.

fractions

in which

measurements

In the

the

case

in one fraction.

counter.

of Esters:

from

2 through

in much later

together

scintillation

removed

in Fractions

radioactivity

was determined

and Alonzo

diethyl

RESEARCH COMMUNICATIONS

completely

separately

eluted

All

Determination

triglyceride Rapport

were

were

eluted

eluted

to 1:9

Counting:

Chemical

were

were

and diglycerides

Model

esters

Triglycerides

and diglycerides

Scintillation a Packard

Cholesterol

1.

solvent

AND BIOPHYSICAL

were

Counting

net

quantitatively

synthesis by the

performed

with

efficiency

was 82%.

experiment

(Table

hydroxamate

method

of

(7). RESULTS

As shown

in Table

CoA and diolein, triglyceride

only fraction

chloride

were

label

1 when microsomes

(21.4%)

added

incorporation

of

(Flask

1).

when SlO5,

the

S105 or ATP from

fall

to low

incubation

levels

detected

when SlO5 was incubated preparations

one preparation

another,

the

aration

dialysis

chloride.

added ATP nor

ATP and magnesium

of SlO5

After

simply full

We presume

Il-14C]-diolein

2 shows the into

that

5). absence

SlO5 have

given

chloride results triglyceride

observation

incorporation

to

of magnesium activity

was

results.

required,

and with

against

In any

was required.

can be induced

by addition is

Omission

different

chloride

16 hours

6).

omission

was not

can be restored this

(Flask

of

of microsomes.

requirements for

incorporation

slightly

chloride

in the

ATP and magnesium

No enzymatic

added magnesium

by dialysis

[l-14C]-palmitylwas observed

caused

while

in the

chloride

activity

of ATP and magnesium Table

of

(3.9%)

was seen mixture

(Flask

added magnesium

neither

event,

effect

with

substantial

2, 3 and 4),

produced

With

mixture,

incubation

(Flasks

a lesser

label

fraction

the

chloride

Different

However,

triglyceride

of either back

incubated

slight

to the

into

were

in any prep-

20 volumes

of buffer.

of ATP and magnesium

due to variable

endogenous

levels

in S105. of an experiment was monitored.

231

in which Again

the

incorporation

SlO5 was required

of for

4)

BIOCHEMICAL

Vol. 58, No. 1, 1974

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 INCORPORATION OF 11-14C]-PALMITYL-CoA INTO TRIGLYCERIDEBY LIVER MICROSDMES: REQUIREMENTFOR S105, ATP AND MAGNESIUMCHLORIDE Incorporation of ]1-14C]-palmityl-CoA into triglyceride Flask

Additions

-%

nmoles

none

3.9

0.066

ATP + M&l2

4.6

0.078

S105

2.2

0.037

slos + MS12

1.9

0.032

S105 + ATP

10.7

0.182

S105 + ATP + MgC12

21.4

0.364

All lasks contained microsomes (1.9 mg protein), diolein (8.1 nmolesj and [l- 15Cl-palmityl-CoA (1.7 nmoles, 0.1 PCi) in a final volume of 2 ml of buffer. Flasks 2, 4 and 6 contained magnesium chloride (4 mM). Flasks 2, 5 and 6 contained ATP (4 mM). Flasks 3-6 contained S105 (17 mg protein).

TABLE 2 CONVERSIONOF Ii-14C]-DIOLEIN

TO TRIGLYCERIDE

Incorporation into Flask

of [1-14C]-diolein triglyceride

%

Additions

nmoles

1

none

1.0

0.010

2

palmityl-CoA

6.7

0.067

3

S105

25.9

0.259

4

SlO5 + palmityl-CoA

25.4

0.254

All flasks contained microsomes (1.9 mg protein), ATP and magnesium chloride (1 nmole, 0.1 I.&X) in a final volume of 2 ml of (4 mw, and [l-14C]-diolein buffer. Flasks 3 and 4 contained SlO5 (17 mg protein). Flasks 2 and 4 contained palmityl-CoA (10 nmoles).

triglyceride chloride again,

synthesis by this

by microsomes.

The requirement

system was the same as shown previously

when incubated

in the absence of microsomes,

matic activity. 232

for ATP and magnesium in Table 1.

S105 exhibited

Once

no enzy-

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 3 TRYPSIN TREA'IMENTOF LIVER S105 Incorporation of [l-14C]-palmityl-CoA into triglyceride Additions

Flask 1

-%

nmoles

none

3.9

0.066

5105

26.3

0.447

Slo5 + trypsin

inhibitor

11.3

0.192

Slo5 + trypsin inhibitor

+ trypsin

1.5

0.025

All incubations and preincubations were carried out at 37' under nitrogen. Flask 1 received no preincubation. The Slo5 in Flask 2 was preincubated alone for 60 min. The SlO5 in Flask 3 was preincubated alone for 30 min at which time trypsin inhibitor (5 mg) was added and the preincubation was continued for an additional 30 min. In Flask 4 SlOs and trypsin (5 mg) were preincubated together for 30 min at which time trypsin inhibitor (5 mg) was added and the preincubation was continued for an additional 30 min. At the end of the preincubation period, microsomes (1.9 mg protein), ATP and magnesium chloride (4 mM), dipalmitin (7 nmoles), and [l-14C]-palmityl-CoA (1.7 nmoles, 0.1 i&i) were added to all flasks. The final incubation volume for all flasks was 2 ml. All flasks were then incubated for 60 min.

TABLE 4 NET SYNTHESISOF TRIGLYCERIDEFROM[1-14C]-PAWTYL-C~A Triglyceride

Triglyceride Incubation

nmoles

nmoles -

AND DIOLEIN

[1-14C]-palmityl-CoA incorporated

synthesized -%

-nmoles

-%

1

569

---

---

---

---

2

791

222

34.4

191

29.6

Each incubation consisted of five identical flasks. All flasks contained microsomes (1.9 mg protein), SlO5 (17 mg protein), and ATP and magnesium chloride (4 mM) in a final volume of 2 ml of buffer. Incubation 1 contained no added substrate. Incubation 2 contained a total of 645 nmoles of [1-14C]palmityl-CoA (0.645 Ki) and 405 nmoles of diolein. Evidence for the protein ble for the activation heat and trypsin 5 minutes,

of microsomal

treatments.

SlO5 lost

nature

of the component present triglyceride

synthesis

When heated in a boiling

a substantial

was obtained

water bath

amount (50-75%) of its

233

in S105 responsi-

capacity

(95')

by for

to activate

Vol. 58, No. 1, 1974

the conversion

of diglyceride

shows the results While trypsin it

is clear

BIOCHEMICAL

that

triglyceride

to triglyceride

of an experiment

inhibitor

synthesis

totally

tion thesis

but rather

Incubation

that

Table 3

with trypsin.

reaction

(Flask 3),

of S105 to activate

incorporation

an experiment

of label

palmitate

was designed

added diolein

no exogenously

added substrate.

2 as determined corresponded

for the

of such an

and added Il-C14]-palmityl-CoA.

in Incubation

Incuba-

The net syn-

chemically

closely

was due

with fatty

to test

Table 4 summarizes the results

or 34.4% of the added palmityl-CoA)

(222 nmoles

with the value deter-

(191 nmoles or 29.6% of the added [l-14C]-palmityl-CoA).

These data demonstrate

conclusively

ATP + magnesium chloride) diglyceride

in this

to exchange of labeled

1 contained

of triglyceride

mined isotopically

microsomes.

the ability

the possibility

of triglyceride.

2 contained

inhibitory

destroyed

acids of endogenous triglyceride,

experiment.

by liver

(Flask 4).

In order to eliminate

net synthesis

RESEARCH COMMUNICATIONS

in which SlO5 was incubated

was slightly

trypsin

not to net synthesis

AND BIOPHYSICAL

that

this

system (microsomes + S105 +

causes the net synthesis

of triglyceride

from

and palmityl-CoA. DISCUSSION

We have demonstrated

a requirement

for a soluble

105,000 x g supernatant

(5105) which is required

tyl-CoA

to triglyceride

and diglyceride

we have shown a requirement thesis

has been monitored

either

[l-14C]-palmityl-CoA

either

substrate,

incubated

there

by rat

by incorporation

liver

of label

or ]l-14C]-diolein is no enzymatic

complete system has been demonstrated

ticles

as their

microsomes.

activity

associated

by chemical

source of enzymes.

234

of palmiIn addition

Triglyceride

into triglyceride

Net synthesis

when they used chicken

in liver

liver

syn-

using

as the source of label.

Weiss, Kennedy and Kiyasu (8) did not report reaction

present

for the conversion

for ATP and magnesium chloride.

in the absence of microsomes.

ment for this

protein

Using

with S105 when

of triglyceride

analysis

of ester

a soluble

protein

by the linkages. require-

20,000 x g sedimented par-

Vol. 58, No. 1, 1974

BIOCHEMICAL

Smith, Sedgwick, Brindley by both rat liver conversion

(10) observed a soluble mitic

and cat intestinal

protein

requirement

phatidate

into

the protein

phosphohydrolase

Johnston,

Rao, Lowe and Schwarz

for the incorporation

triglycerides.

responsible

(EC 3.1.3.4),

for SlO5

mucosa microsomes in the

acid to glycerides.

acid or L-a-glycerophosphate

in both papers was that

RESEARCH COMMUNICATIONS

and Hiibscher (9) noted a requirement

mitochondria

of phosphatidic

AND BIOPHYSICAL

of either

The conclusion

for stimulation

which converts

pal-

drawn

was L-cr-phos-

phosphatidic

acid to

diglyceride. Such an explanation

is not possible

for the system which we have described

since phosphatidic

acid is not the substrate.

that

one or more proteins

SlO5 contains

synthesis

of triglycerides

It is possible

which operate

from water insoluble

that there

are substrate

sent in SlO5 which are essential strates

An alternative

for the synthesis

by microsomal membranes.

posal exists

for the synthesis

pounds (4).

Further

experiments

In fact

as carriers

precursors specific

explanation

by liver

soluble

are in progress

for the microsomes.

proteins

pre-

of most water insoluble

evidence compatible

of many different

is

with this

types of water insoluble to test

this

sub-

procom-

hypothesis.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Scallen, T.J., Schuster, M.W., and Dhar, A.K. (1971) J. Biol. Chem. 246, 224-230. Scallen, T.J., Srikantaiah, M.V., Skrdlant, H.B., and Hansbury, E. (1972) FEBS Letters 25, 227-233. Seetharam, B., Gavey, K.L., Srikantaiah, M.V., Hansbury, E., and Scallen, T.J. (1973) Circulation 48, Suppl. IV, IV-253. M.V., Seetharam, B., Hansbury, E. and Gavey, Scallen, T.J., Srikantaiah, K.L. (1974) American Society of Biological Chemists Symposium on Metabolism of Membrane Lipids, Fed. Proc., in press. Seetharam, B., Hansbury, E., Srikantaiah, M.V., and Scallen, T.J. (1974) Fed. Proc., in press. Bligh, E.G., and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. Rapport, M.M., and Alonzo, N. (1955) J. Biol. Chem. 217, 193-198. Weiss, S.B., Kennedy, E-P., and Kiyasu, J.Y. (1960) J. Biol. Chem. 235, 40-44. Smith, M.E., Sedgwick, B., Brindley, D.N., and Hiibscher, G. (1967) European J. Biochem. 5, 70-77. Johnston, J.M., Rao, G.A., Lowe, P.A., and Schwarz, B.E. (1967) Lipids 2, 14-20.

235