Purification and characterization of macromomycin-I, a protein antibiotic containing a non-protein chromophore

Vol. 94, No. 3, 1980 June

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 769- 776

16. 1980

PURIFICATION

Department +Permanent

Received

AND CHARACTERIZATION OF MACROMOMYCIN-I, A PROTEIN ANTIBIOTIC CONTAINING A NON-PROTEIN CHROMOPHORE ?; Jan M. Woynarowski'+ and Terry A. Beer-man

Roswell Park Memorial Institute, of Experimental Therapeutics, Buffalo, New York 14263, U.S.A. Address: Department of Pharmaceutical Technology and Biochemistry Technical University of Gdansk, 80-952 Gdansk, Poland

April

21,198O

Summary One-step chromatography of crude macromomycin on DEAE-Sephacel yielded two forms of the antibiotic. The more active form of the drug contained a non-protein chromophore. Apoprotein and chromophore components of this drug form were separated. The apoprotein was found to be inactive and the chromophore only slightly active against Sarcina lutea, while recombination of both components resulted in a partial recovery of the activity. In contrast, recombination of chromophore and apoprotein did not restore the ability of intact macromomycin to nick isolated DNA. Macromomycin, molecular growth

an antitumor

weight

of around

of several

cellular

bacteria

DNA (5)

damage cellular inhibition

(1,

6, 7).

of identical

composition,

but

the

studies

method

we describe biotic

that

from crude chromophore

was found

DNA (6-8).

tumor

to induce

The drug's

to inhibition

a

was shown to inhibit

cultured

(2),

with

cells

(3)

breaks

in

ability

to

of DNA synthesis

and

Recently,

it

molecular

weight,

degrees

was reported

that

by several the drug

isoelectric

point

to as auromomycin)

different exists

in

and amino

acid

contains

a 350 nm

(8). on the mode of action

to obtain a new,

The drug

to various

one (referred

chromophore

For our simple

free

protein

(5).

has been purified

two forms

absorbing

in vivo

DNA seems to be related

Macromomycin

(1).

Macromomycin

as in cell

growth

is an acidic

daltons

tumors (4).

as well

of cell

procedures

12,000

experimental

and Gram-positive

antibiotic,

purified

one-step preparation

containing

of macromomycin,

and better

procedure

which

of macromomycin form of the drug

defined

drug.

allows

isolation

and also

we needed In this

enables

from the one that

a

paper,

of the antiseparation lacks

of

chromophore.

0006-291X/80/110769-08$01.00/0

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Cop.vrighi ~2 1980 righrs of reproduction

by Academrc Press, Inc. in an-v form reserved.

Vol.

94, No.

3, 1980

The former,

BIOCHEMICAL

most

macromomycin-I (MCR-II).

likely

corresponding

(MCR-I)l, In addition,

of MCR-I to explore activity

AND

while

BIOPHYSICAL

RESEARCH

to auromomycin,

the

latter

we separated

is

is

referred

of both

designated

as

to as macromomycin-II

the chromophore

the contribution

COMMUNICATIONS

from

components

the protein

to the

part

biological

of the antibiotic. MATERIALS

AND METHODS

Crude macromomycin (NSC 11170105) was provided by Developmental Program Chemotherapy, of the National Cancer Institute. All steps of the isolation procedure were done at S'C in partial or complete darkness. Crude material (1.5 g) was applied on a column (3 x 80 cm) packed with DEAE-Sephacel (Pharmacia, Sweden), and equilibrated with 10 mM Tris-HCl, pH 7.9. The column was eluted (80 ml/hr) initially with the equilibrating buffer followed by elution with a two-step linear gradient of NaCl concentration (O-10 mM NaCl in 250 ml and lo-20 mM NaCl in 2000 ml, respectively). Elution of protein was monitored by absorbtion at 280 nm (Gilson UV monitor), and the salt gradient was determined by measuring conductivity of the collected fractions. Antibacterial activity against Sarcina lutea was measured by ____a disc method (4). Drug damage to isolated DNA was assayed by measuring conversion of Form I PM2 DNA into Form II based on the loss of fluorescense of nicked PM2 DIiA-ethidium bromide complex in alkali (9). PM2 DNA was incubated with drug for 30 min at 37", and results were expressed as decrease of fluorescense in treated samples normalized with respect to the fluorescense Cytotoxic activity of MCR-I and MCR-II against HeLa of control samples (10). and L1210 cells was determined as described previously (5). The active by ultrafiltrafractions were pooled as indicated in Fig. 1, concentrated The amount of protein in tion on Amicon P-10 membrane and stored at -2O'C. MCR-I and MCR-II preparations was found to be 8 mg and 17 mg, respectively, for calibration as estimated by Lowry method (11) using bovine serum albumine Separation of MCR-I into a 350 nm chromophore and protein components curve. was done by extraction of freeze-dried material with methanol as described for neocarzinostatin (12). UV spectra were recorded on a Cary 118 spectrophotometer. RESULTS AND DISCUSSION Preliminary that

the drug's

cell

growth

associated (Fig.

biological

and strand with

scission

column step)

1 Abbreviations: tris(hydroxymethy1) covalently closed

(inhibition

peak,

while

that

other

the active

as isolated

components

antibiotic

salt

on DEAE-Sephacel

showed

of HeLa and ___Sarcina

as well

of crude

and a two-step revealed

macromomycin

of cellular

Chromatography

(longer

the second

of crude activities

one protein

1A).

ditions for

fractionation

gradient material

were

under with

DNA) were inactive

improved

a shallow consisted

lutea

conslope

of at

MCR-I, macromomycin-I; MCR-II, macromomycin-II; Tris, aminomethane; DTT, dithiothreitol; Form I PM2 DNA, duplex DNA. duplex DNA; Form II PM2 DNA, nicked circular

770

Vol.

94, No.

BIOCHEMICAL

3, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

2

a2 ,-------

_______ s l-r Y I

0. I ,% 0

1.0

0.S ELUTION WUJYE

IL1

i3 F 0.04 !i 2 0.02

IO

E i! e f -20I!3vi - IO

Fig. 1. Chromatography of crude MCR on DEAE-Sephacel columns. Panel A: 300 mg sample was fractionated on a 1.5 x 25 cm column eluted at 52 ml/h with 10 mM Tris, pH 7.9 and a gradient of NaCl as shown in the figure. Shaded area indicates fractions which inhibited the growth of HeLa and Sarcina lutea cells and nicked cellular DNA in HeLa cells. Panel B: 1.5 g sample was fractionated on a 3 x 80 cm column as described in Materiais and Methods. Solid circles show activity of the collected fractions (at 2 ul/disc) against Fractions were pooled as indicated by ~Sarcina --lutea shaded areas and designated MCR-I (shoulder) and MCR-II (main peak). Insert shows elution profiles of MCR-I and MCR-II on a Sephadex G-50 column (1.5 x 75 cm) eluted with 10 mM Tris, pH 7.9 at 20 ml/hr. An arrow indicates the elution volume of neocarzinostatin (NCS).

least

two

eluted

substances

as

(Fig.

a broad

peak

Fractions

corresponding

indicated

in

Fig.

respectively. phoresis on both

Sephadex close

A similar

IBand

acrylamide

G-50

(Fig.

value

the

the

further

molecular

and

at the

referred

(not

The

conditions

shoulder

NCR-II

gel 1B).

these

shoulder

and

elution of

Under

a distinct

are

MCR-I

15%

to

with to

Both in

1B).

elution

volume weight

of

to

banded shown)

main

at and

volumes

771

around

17-19 peak

were

MCR-I

and

the

same

position

when of

analyzed MCR-I

was

1B). as

MCR-II, upon by

and

(Fig. pooled

estimated

electro-

chromatography

MCR-II

[MW 10,700 12,000

was

mM NaCl

as

neocarzinostatin of

macromomycin

were

(13)]. pre-

Vol.

94, No.

3, 1980

Fig.

2.

viously

W spectra

for

the for

spectra

(1)

isoelectric

above

300 nm.

shifted

with

In contrast,

the presence

of MCR-I is spectrum lacking mycin

very

macromomycin

(1,

macromomycin

(and

data

thus

presumably

of previously

probably

identical It

same drug

rather

and without

than

with

the

substances

two different

712

chromophoreas macromobut by

preparation

likely

of

should

that

as well much of the

preparations

con-

Auromomycin

chromophore.

the same protein

both

the

to auromomycin

seems very

was obtained

light

isolated

same microorganism

a substance

contains

spectrum

(8) while

the crude

is

at 350 nm

from that

showed

of MCR-I

to previously

the

W

no absorbtion

referred

indicate

In this

MCR-II

centered

auromomycin

Our results

MCR-I)

forms

of t&c

spectrum

with

8).

The entire

for

of the drug

(8).

as was re(1,

2).

band

reported

antibiotic.

macromomycin

broad

chromophore.

Substances

on "macromomycin"

a mixture

and auromomycin

an additional

the

8).

as the chromophore-lacking published

is

have been isolated

contains

to be 5.4

(Fig.

pH 7.9.

focusing

a maximum at 280 nm and almost

to that

resembles

procedures.

Isoelectric

significantly

of non-protein

similar

and auromomycin

(8).

COMMUNICATIONS

in 1 n&l Tris,

the maximum of the UV spectrum

of MCR-II

separate

taining

differ

to 267 nm and there

indicating

and MCR-II (---)

macromomycin

spectrum

RESEARCH

of MCR-I and MCR-II

chromophore-lacking

protein

BIOPHYSICAL

and auromomycin

point

of MCR-I and MCR-II

a typical

AND

of MCR-I (-)

macromomycin

indicated ported

BIOCHEMICAL

as chromophore-lacking be considered

antibiotics.

as two

Vol.

94, No.

3. 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Hela

~100 F m F z E 50 g E LA 0 0.1

1.0 IO

:

100

q/ml

W/ml

Biological and biochemical activities of BICR-I (-•-) and MCR-II Fig. 3. (-o-). Left panel shows growth inhibition of HeLa cells; middle panel, growth inhibition of Sarcina lutea, and right panel, ability to nick isolated PM2 DNA.

Using lacking the

the

extinction

macromomycin

(8),

chromophore-containing

indicates

form.

MCR-II

of --Sarcina

that

Though

spectively

(ID5o

leukemia

at 2-10

against

values). cells

HeLa cells

not

vert

Form I PM2 DNA to Form II

also

cut

drug with confirm

the maximal

levels

of 4-8

(Fig.

those

published

The biological were

reported

found

that

posure

ug/ml

activity

MCR-I loses

to normal

for

15-20%

laboratory

3).

These

3) was 2.9

data

auromomycin

light of its light,

activities

against

antibacterial

to completely at 0.7

was only

of macromomycin

activity

773

MCR-II

40% conversion

at

consistent

form

sensitive

re-

con-

pg/ml.

in general,

even when the sample

activity

cultured

and macromomycin

and temperature

in

and 9.4 ngfml,

found

are,

1)

was needed Cytotoxic

of the chromophore-containing

and biochemial

to be strongly

(Fig.

observed

(Fig.

MCR-I inhibited

effect

50% of the effect

3).

previously

by itself.

MCR-I was able

could

profile

rig/disc

effect

85% of the

elution

were

80% of

of MCR-II

20-50

activities

with

about

while

(Fig.

shown).

PM2 DNA, but

the higher

the activity

a similar

of about

activity

properites

Similar

(data

contains

the biological

rig/disc,

and chromophore-

MCR-I consists

MCR-II

of MCR-I,

to produce

of MCR-I and MCR-II

while

has antibiotic

lutea

the case of MCR-II

L1210

we estimated

by the presence that

growth

of auromomycin

drug,

chromophore-lacking be enhanced

coeffecients

(6-8)

and

of the drug. preparations (1,

after

was kept

7).

We

3 hr exin

ice.

Vol.

94, No.

3, 1980

BIOCHEMICAL

AND

I

BIOPHYSICAL

I

250

I

RESEARCH

COMMUNICATIONS

I

300 350 WAVELENGTH (nm)

40(

Fig. 4. UV spectra of MCR-I chromophore in methanol (-*-), in 1 mM Tris, pH 7.9 (---) and spectrum of MCR-I apoprotein in 1 mM Tris, pH 7.9 (-). Concentrations were adjusted to correspond to 200 ug/ml of MCR-I.

It

has been

contains

zinostatin, from apoprotein employed

the

spectrum

that

MCR-I.

above

at 265-270 4).

A very

with

350 nm than

in

phore

(12).

than

in water

In addition, (Fig.

to be 667 daltons, In order

4).

in the biological Mass spectroscopy

activity

The molecular

experiments

shows

be separated

material

(12).

the UV spectra

We

of meth-

The spectrum

of the

of chromophore we studied

774

at shorter for

absorbs

weight

performed

MCR-I.

In contrast,

has a maximum at 345 nm, a

by preliminary

were

could

the case of intact

was reported

of MCR-I,

which

neocar-

a maximum of 275 nm and considerably

MCR-I chromophore

the role

antibiotic,

of MCR-I.

absorption

spectrum

as suggested

to explore

4

material

nm and increasing similar

Fig. fractions

of MCR-II,

protein

of freeze-dried

of the methanol-soluble

(Fig.

2

extraction for

another

chromophore,

and methanol-insoluble

absorption

shoulder

that

a non-protein

by methanol

resembles

lower

recently

the same protocol

anol-soluble latter

reported

wavelengths

neocarzinostatin

chromo-

more strongly

in methanol

of MCR-I chromophore mass spectroscopy and apoprotein their by Dr.

ability Pawel

seems data2. components

to inhibit Kolodziejczyk.

Vol.

94, No.

3. 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Fig. 5. Effects of MCR-I components on growth of ~Sarcina lutea (panel A) and the nicking of isolated PM2 DNA (panel B) in absence (open symbols) Concentrations of MCR-I componand presence (solid symbols) of 1 mM DTT. ents are expressed with respect to the original concentration of MCR-I. Intact MCR-I (-o-, -RJ-); MCR-I chromophore (-V-, -T-); MCR-I apoprotein (-A-); mixture (1:l) of MCR-I chromophore and apoprotein (+). Apoprotein with DTT and the mixture of chormophore without DTT showed no activity in the PM2 DNA assay and were omitted from panel B.

the

growth

of ~Sarcina

appeared high it

that

apoprotein

as 2 vg/disc. was one order

recombination of the

(1:l)

Chromophore

Only

of magnitude

activity

slight

with

that

apoprotein

Though

of activity

contribute

recovery

of antibacterial for

a mixture

separated cutting for

components

of PM2 DNA (Fig.

chromophore

with

DNA was carried

may cause

some irreversible

out

and chrome in presence

5B).

the

extraction results

to the -___ in vivo seems to have

including

was observed

when incubation

the methanol

properties,

chromophore

recovery

though

in 20-25%

was observed

for

assay,

as

However,

stimulation

when assayed

It

MCR-I.

of intact

In contrast,

MCR-II.

inactive

in this

resulted

Similar

5B).

even at levels

some activity

than

5A).

with

mixture

of 1 mM DTT (Fig.

gations,

lower

(Fig.

practically

phore-apoprotein

of drug

showed

PM2 DNA (Fig.

activity

(up to 50% of the MCR-I activity)

of MCR-I were

tein

has no antibacterial

of MCR-I chromophore

5B).

5A) and to nick

(Fig.

of chromophore

original

activity

lutea

suggest activity

some activity

determination

that

of MCR-I. by itself.

of chromophore

775

both

changes

chromophore On the Further structure,

and apopro-

other

hand,

detailed

the investi-

are necessary

to

Vol.

94, No.

3, 1980

unequivocally activity

BIOCHEMICAL

establish

of MCR-I.

helpful

in these

in this

paper

(1,

role

Preliminary studies.

enables

phore-lacking opposed

the

characteristics

protocols

that

RESEARCH

COMMUNICATIONS

and apoprotein

in biological

hitherto

the purification

of both

macromomycin

to published

BIOPHYSICAL

of chromophore

Moreover,

isolation

forms.of

AND

presented procedure

described

chromophore-containing

and consists involve

of only

several

may be

and chromoone step,

as

chromatographic

steps

6-8). ACKNOWLEDGEMENTS The authors

excellent editing

technical this

Institute

wish

to thank assistance,

manuscript.

of Health

Grants

This

Jeff

LaDuca,

and also

Nina

Janet

Arnone,

Ruth Wright

work

was supported

CA-24906

and CA-13038.

in part

and Pat Dix

for

for

in

her

aid

by National

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Yamashita, T., Naoi, N., Watanabe, K., Takeuchi, T., and Umezawa, H. (1976) J. Antibiot., 29: 415-423. Lippman, M.M., LasterTW.R., Abott, B.J., Venditti, J., and Baratta, M. (1975) Cancer Research, 35: 939-945. Kuminoto, T., Hori, M., and Umezawa, H. (1972) Cancer Research, -32: 1251-1256. Chimura, H., Ishizuka, M., Hamada, M., Hori, S., Humura, K., Iwanage, J ., Takeuchi, T., and Umezawa, H. (1968) J. Antibiot., 21: 44-49. Beerman, T.A. (1978) Biochem. Biophys. Res. Commun., 83: 908914. Sawyer, T.H., Prestayko, A.W., and Crooke, S.T. (1979)Cancer Research 39: 1180-1184. z, W.B., Chiang, C.K., and Montogomery, R. (1978) J. Biol. Chem., 253: 3259-3264. Yamashita, T., Naoi, M., Hidaka, T., and Watanabe, K. (1979) J. Antibiot., 32: 330-339. D.E. (1974) Biochem. Biophys. Res. Morgan, A.R., and Pulleybank, Commun., 61: 396-403. Beerman, TA. (1979) Biochim. Biophys. Acta., 564: 361-371. Lowry, O.M., Rosenbrough, N.Y., Farr, A.L., and Randell, R.J. (1951) J. Biol. Chem., 193: 265-275. Napier, M.A., Holmquist, B., Strydom, O.J., and Goldberg, J.H. (1979) Biochem. Biophys. Res. Commun., 89: 635-642. Meinhofer, J., Maeda, H., GlaserFC.B., Czombos, J., and Kuromizu, K. (1972) Science, 178: 875-876.

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