Lysosome disruption by a free radical-like component generated during microsomal NADPH oxidase activity

Vol. 48, No. 6, 1972

BIOCHEMICAL

LYSOSOME DISRUPTION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BY A FREE RADICAL-LIKE

COI5PONENT

GENERATED DURING MICROSOMAL NADPH OXIDASE ACTIVITY

Kuo-Lan

Biochemistry

Section,

Oklahoma

Department

of Biochemistry

University

of Oklahoma

Oklahoma

Chen and Paul

B. McCay

Medical

Research

and Molecular Health

Foundation

Biology,

Sciences

Center,

and

College

of Medicine,

Oklahoma

City,

73104

Received

June 12, 1972 Revised

August

4,

1972

Summary The oxidation of NADPH by liver microsomes produces a --in vitro factor which causes the release of hydrolases from lysosomes. The factor appears to be a free radical involved in the mechanism of NADPH oxidation. The production of the factor requires a heat labile component in the microsome and is inhibited by the addition of substances which are radical-trapping agents or substrates for the drug metabolism system. These interactions suggest possible mechanisms which might promote necrosis through the release of lysosomal enzymes --in vivo, especially in animals deficient in dietary radical-scavenging components. Oxidation

of NADPH by normal

resembling

those

production

of a highly

rapid

hemolysis

factor in

is

existing

port

The production

actively

stopped

disappeared a free

oxidized

with

inhibitors

with

introduced for

itself

by microsomes. or by mild

a very brief

component

by the enzyme system

deficient

rats

and depressed

alterations

which

occur

NADPH-dependent

electron

only

was shown

in animals

1412

to have

in microsomes

supplemented

was the

factor

the properties

The production

enhanced

trans-

when NADPH was

of the microsomes,

half-life. is

system

When the enzyme activity

heating

The factor

of initiating This

occurred

the

by the

(1).

lipid

during

conditions

was capable

into

the

under

to be accompanied

which

of the factor

immediately.

radical

factor

responsible membrane

microsomes

was shown

transient

apparently

(2).

being

--in vivo

of erythrocytes

the microsomal

liver

with

of this from higher

tocopherolthan

of

BIOCHEMICAL

Vol. 48, No. 6, 1972

normal

levels Since

of the vitamin. lysosomes

endoplasmic ping oration

are often

reticulum,

agent

in certain

in close

and because

in animal

lysosomal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

diets

hydrolases

lack

results

tissues

proximity of a suitable

trap-

structural

by increased

we studied

of the

radical

in intracellular

characterized

(3),

to elements

the effect

deteri-

activity

of the

of acid

factor

on lysosomes.

Methods Lysosomes Leighton

were

et al.

(4)

isolated as modified

microsomes

were

microsomal

NADPH oxidase

properties

of a free

McCay (1). iron,

isolated

This

incubations a final

were concentration

Protein assays

lysosomal

determined

the method amounts

aration

(1).

by the method were

et al.

(11);

and were

o-nitrophenol

after

of the (8).

microsomes

which

was

was assayed in

terms

by of

produced,

in each lysosome

0.1% Triton

factor.

was measured

expressed

with

at

The

sulfatase

and p-nitrophenol

treatment

the

systems

et al.

phosphatase

of hydrolases

inhibits

after

generation

H-galactosidase

acid

Mnilf

to the reaction

aryl

and

of ADP and

(7).

of Lowry

(10);

Maximum activity

used by Pfeifer

as follows:

of Roy (9);

The

exhibiting

Therefore,

further

Liver

(6).

factor

reaction

et al.

of nitrocatechol,

was measured

the

Mn2+ was added

hydrolases

of Fukuzawa

respectively.

for

of 10m3 M to stop

to Sellinger

reported the

of

(5).

concentrations

factor

out,

by the procedure

according

produces

cytoplasmic

radical-like

was determined

for

and Carubelli

was the same as that

a cofactor

carried

by the procedure

as previously

which

contains

of the

tissue

by Tulsiani

system

radical

being

the production

liver

and washed

system

the latter

from

prep-

X-100.

Results Incubation of enzymically acid

phosphatase

of lysosomes oxidizing activity,

with

NADPH results while

in

the control

1413

are

a progressive system

in the process increase

(containing

of free no NADPH

Vol. 48, No. 6, 1972

showed

very

(Fig.

1).

labile.

little

increase

Enzymic

oxidation

Mild

plete

loss

like

BIOCHEMICAL

warming

of this

component.

on the

release

system

in which

release

occur.

hydrolase

release,

this is

factor

radicals.

not

II

shows

the addition

rats

before depressed versely, plemented tection perhaps,

of the

in

X-100

release

sacrificing

the animals,

rats

against is

incubation

the

considerably

donating for

a period

the

release

were

of 2 weeks, release

fact

lysosomes

that

injected

the lysosomes

more susceptible

occurred

1414

hydrolases caused of with

and N-methyl all

effective

in

by these

was

substances

systems

at the end of the

hydrolases.

In addition,

with

a-tocopherol

of lysosomal

fed

(Fig.

by the

12 hours

hydrolases

system

(Fig.

2).

was Con-

an a-tocopherol-sup-

an even greater

from animals to lysis

That

of reacting

hydrolases

active

cause

The protection

in the NADPH oxidase

hydrolase

have

hydrolases.

of fully

of

has

(1).

diphenylamine

to duplicate

were

system

would

capable

lysosomal

did not

so by the addition

santoquin,

of the lysosomal

the microsomes

during

doing

hydrolase

lysosomal

of 1 x 10e3 M were

donating

diet

from

which

the

to NADPH alone.

reported

of the

or substances

of Triton

resulted

if

systems

that

microsomes

been

in

no activation

lysosomes

the release

concentration

the release

incubation

for

by microsomes

significant

by the microsomal

has already

prevented

due to inactivation

since

of the

the radical-

Only

in essentially heated

produced

agents

at a final

preventing

does

Incubation are

Table

was occurring

exposure

was responsible

release

hydrolases.

heat

in com-

to produce

lysosomal

containing

radical

radical-trapping

aniline

did

a factor

shown as follows.

free

are

that

of a free

hydrolase

if

nor

results

of NADPH oxidation

NADPH resulted

Systems

ability

enzyme

is markedly

one minute)

and their the effect

lysosomal

microsomes

(65 O for

different

NMPH oxidation

activity.

properties

I shows

Omitting

of this

of NADPH by liver

enzyme activity

of three

Evidence

in the activity

of microsomes

Table

hydrolase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3).

degree

of pro-

More significant,

deficient radical-like

in a-tocopherol factor

BIOCHEMICAL

Vol. 48, No. 6, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Progress curve of release of acid phosphatase from lysosomes exposed to microsomal NADPH oxidase activity. The reaction systems contained lysosomes (Lys), (0.3 mg protein/ml reaction system); microsomes (Mic), 1.0 mg protein/ml reaction system; NADPH (where indicated), 0.3 mM; ADP, 0.4 mM; Fe 3+, 0.012 mM; 0.015 M Tris-HCl buffer, pH 7.4; sucrose 0.7 M. Incubation was carried out at 37'.

Table I. Release of lysosomal Components added to incubation mediuma

hydrolases by microsomal NADPH oxidase activity. B-Galactosidase Acid phosphatase Aryl sulfatase activity activity activity umoles product/l0 min/mg protein

Lysosomes

(Lys)

1.91

.49

.05

Microsomes

(Mic)

1.18

.ll

.oo

3.82

.84

007

2.06

.53

.ll

25.74

1.93

.61

1.62

1.08

.08

6.32

.a7

.29

Lys + Mic Lys + NADPH (0.3

umole)

Lys + Mic + NADPH Lys + Mic

(A)b

+ NADPH

Lys + Mic + NADPH + Mn2+

Lys + Triton X-100 29.41 3.40 1.33 aThe incubation medium, quantity of components added and conditions of incubation were as described in Fig. 1. Incubation time was 45 min. bMic (A) = Microsomes heated at 650C for 1 min before addition to the reaction system.

than

are normal Because

studies

the effect

may have been

by the Triton with

lysosomes

lysosomes

of this dependent

injection which

(Fig.

procedure, were

isolated

3). factor

on the

lysosomes

on the lysosomes similar from

1415

having

experiments rats

using

used in been were

these

isolated carried

a discontinuous

out sucrose

Vol. 48, No. 6, 1972

Table

II.

Components incubation

BIOCHEMICAL

AND BIOPHYSICAL RESEARCh COMMUNICATIONS

Effect of free radical-scavenging agents on the release of hydrolases by microsomal NADPH oxidase activity. added to Acid phosphatase Aryl sulfatase mediuma umoles activity umoles activity umoles product/l0 minlmg

Lys + Mic Lys + Mic + NADPH

lysosomal g-Galactosidase pmoles activity protein

3.82

.58

.13

24.41

1.60

.73

Lys + Mic + NADPH + Santoquin (1 umole)

6.18

.66

.13

Lys + Mic + NADPH + N-methylaniline

4.41

.62

.27

.85

.13

(1 pmole)

Lys + Mic + NADPH + Diphenylamine (1 umole)

10.29

Lys + Triton X-100 29.41 3.15 1.07 aThe incubation medium, quantity of components added, and conditions of incubation were as described in Fig. 1 except as indicated. Incubation time was 45 min.

Figure 2. Effect of supplementing u-tocopherol to rats donating microsomes on the release of acid phosphatase from lysosomes by oxidase activity. Normal rats fed a stock ration were injected a-tocopherol (75 mg/kg body weight) 12 hours prior to preparing The conditions were as in Fig. 1. (+E) mic = microsomes isolated a-tocopherol-injected animals; other particles were from normal

gradient results

(12) were

without identical

previous with

injection those

reported

Discussion

1416

of the above.

rats

with

the liver NADPH with microsomes. from animals.

Triton.

The

BIOCHEMICAL

Vol. 48, No. 6, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 3. Effect of dietary levels of a-tocopherol on the release of acid phosphatase from liver lysosomes by microsomal NADPH oxidase activity. N = lysosomes from normal rats; -E = lysosomes from rats fed a-tocopherol-deficient diet (15); +E = lysosomes from rats fed -E diet supplemented with 50 mg% a-tocopherol. The microsomes were from livers of normal rats. The conditions of the experiment are as in Fig. 1. -EC, NC and +EC are 45 min values for control systems in which no NADPII is added.

Previous

studies

of Zalkin

tocopherol-deficient drolases

rabbits

correlated

They concluded invasion

factors

the hydrolase by tissue

such as those

diseases

for

Evidence damage

whose

studies

cause

multiple

noted

also.

lesion

that

hydrolases structure

indicated

that

chromosome reported

release released

(3).

primary

release

radical-like could

be

or deficiency

of lysosomal

lysosomal structural

of macrophage

activity

in cells

by Allison

hy-

peroxidation.

absorption

--in vivo

Other

1417

lysosomal

lipid that

NADPH oxidase

intracellular

that

from

in tocopherol

from

degeneration

possible

was provided

breaks.

of free

tissue

was a consequence

is

the intracellular

to cell

Pearce

it with

muscle

of muscle

damage resulting

indicates

the initial

that

an excess

increase

associated

by triggering

vivo.

contains

showed

the development

investigation

responsible

severe

that

initiated

Our current

--in

with

et al.

hydrolases can cause

and Patton DNAase release

abnormalities of lysosomal

(13) could were

enzymes

Vol. 48, No. 6, 1972

may occur

BIOCHEMICAL

in some myopathies

AND BIOPHYSKAL

and followed

RESEARCH COMMUNICATIONS

by macrophage

invasion

(14).

Acknowledgement The assistance thanks.

The work

National

Institutes

of Donald

Gibson

was supported

in this

by grants

study

AM-08397

is

acknowledged

and AM-06978

with from

the

of Health.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Pfeifer, P.M. and McCay, P.B., J. Biol. Chem. 246, 6401 (1971). McCay, P.B., Poyer, J.L., Pfeifer, P.M., May, H.E. and Gilliam, J.M., Lipids 5, 297 (1971). Zalkin, H., Tappel, A.L., Caldwell, K.A., Shibko, S., Desai, I.D. and Holliday, T.A., .I. Biol. Chem. 237, 2678 (1962). Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S. and DeDuve, C., J. Cell Biol. 2, 482 (1968). Tulsiani, D.R.P. and Carubelli, R., J. Biol. Chem. 245, 1821 (1970). May, H.E. and McCay, P.B., J. Biol. Chem. 243, 2288 (1968). Poyer, J.L. and McCay, P.B., J. Biol. Chem. 246, 263 (1971). Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem. 193, 265 (1951). Roy, A.B., Biochem. J. 2, 12 (1953). Sellinger, O.Z., Beaufay, H., Jacques, P., Doyen, A. and DeDuve, C ., Biochem. J. 74, 450 (1960). Fukuaawa, K., Suzuki, Y. and Uchiyama, M., Biochem. Pharmacol. 0, 279 (1971). Ragab, H., Beck, C., Dillard, C., Tappel, A.L., Biochim. Biophys. Acta 148, 501 (1967). Allison, A.C. and Patton, G.R., In: Lysosomes in Biology and Pathology, Dingle, J.T. and FellTH.B., eds. Vol. 2, p. 627, American Elsevier Publ. Co., N.Y., N.Y. (1969). Pearce, G.W., Ann. N.Y. Acad. Sci. 138, 138 (1966). Schwarz, K., Proc. Sot. Exp. Biol. & Med. '7, 818 (1951).

1418