VOL. XXIV NO. II THE JOURNAL OF* ANTIBIOTICS 785

EFFECT OF TUNICAMYCIN ON THE SYNTHESIS OFMACROMOLECULES IN CULTURES OF CHICK

EMBRYO FIBROBLASTS INFECTED WITHNEWCASTLE DISEASE VIRUS

Akira Takatsuki and Gakuzo Tamura

Laboratory of Microbiology, Department of Agricultural Chemistry,The University of Tokyo, Bunkyo-ku, Tokyo, Japan

(Received for publication May 24, 1971)

The effect of tunicamycin on the synthesis of macromolecules in culturesof chick embryo fibroblasts was investigated by measuring the incorporation

of radioactive percursors into acid-insoluble product after infection or mock-infection with Newcastle disease virus (NDV). Tunicamycin had slight or no

effect on incorporation of uridine, thymidine and choline. Protein synthesisproceeded to some extent depending on concentrations of tunicamycin. In-corporation of glucosamine and glucose was greatly affected by the antibiotic

at low concentrations. Mechanism of action of tunicamycin against NDVmultiplication is discussed in relation to membranesynthesis.

Tunicamycin is a potent antiviral antibiotic containing glucosamine^. It inhibits

multiplication of NDVwhen it is added during viral one-step growth cycle2), and theantiviral activity is partially reversed by some aminosugar derivatives.3) Tunicamycinhas also antimicrobial activity and induces various morphological changes in micro-

organisms.4) The effect of tunicamycin on the synthesis of macromolecules has beenstudied using cultures of chick embryo fibroblasts (GEF) infected or mock-infectedwith NDVas the first step in elucidation of the action mechanism of the antibioticon NDVmultiplication.

Materials and Methods

Virus and cell culture : The Miyadera strain of NDVand primary monolayer culturesof CEFwere employed.Preparative methodswere the same as reported previously.5)

Effect of tunicamycin on the synthesis of macromolecules : Macromolecular synthesisin cultures of CEFwas followed by incorporation of radioactive precursors into acid-insoluble product. Duplicate samples were treated with tunicamycin and, at designatedtime intervals, chilled in melting ice to stop incorporation of radioactive compounds.

Sufficient 50 % trichloroacetic acid (TCA) was added to make the samples 10 % in respectto TCA after freezing-and-thawing three to five times in a dry ice-acetone bath. Thesamples were stored in an ice-water bath for more than 3 hours. Cold TCA-insolubleprecipitates were then collected on Millipore filters and washed three times with cold10% TCA (2ml each). The filters were placed into glass scintillation vials and driedat 80°C. Ten milliliters of scintillation liquid containing 6g PPO and 0.4g dimethyl-

POPOPper 1,000 ml toluene were added to each vial. Radioactivity was counted usinga liquid scintillation counter (Hitachi-Horiba, Tokyo). Some modifications of the methodswill be described in the text or legends.

786 THE JOURNAL, OF ANTIBIOTICS NOV. 1971

Sucrose density-gradients centrifugation : Ribonucleic acid (RNA) was extracted by

the sodium dodecyl sulfate (SDS)-phenol method and precipitated with three volumes ofethanol at -20°C as described previously.5) Precipitates were collected by centrifugation,.redissolved in phosphate buffered (0.05 m, pH 6.7) saline (0.1 m), and loaded on preformedsucrose density-gradients (5-^15w/v %, 4.5 ml per tube) containing the same buffer. Aftera 6 hour-run in a swinging bucket rotor (RPS 40) at 130,000xg using a preparative

ultracentrifuge Model 65P, Hitachi, fractionation was carried out drop-wise by puncturingthe bottom of the tubes. Bovine serum albumin (200 jug) was added to each fraction {ca.0.15 ml) and cold 10 % TCA-insoluble radioactivity was counted as described above.

Chemicals : Tunicamycin was prepared according to the method reported previously1)and the same lot of the antibiotic was used throughout these experiments. Uridine-uC(uniformly labeled ; specific activity, 492 mCi/mM), uridine-5-3H (specific activity, 5.0 Ci/him), thymidine-6-3H (specific activity, 25.3 Ci/mM), choline chloride-methyl-14C (specificactivity, 54 mCi/mM), D-glucose-14C (uniformly labeled ; specific activity, 261 mCi/mM), D-glucosamine-l-14C (specific activity, 55.6 mCi/mM) and carrier-free 32P were purchasedfrom the Radiochemical Centre, Amersham, England. 14C-Amino acids mixture was-

obtained from Dai-Ichi Chemicals, Tokyo, Japan. Actinomycin D was a gift from Merck,.Sharp and Dohme International, New York, U.S.A.

Results

1 Effect on Synthesis of NDV-and CEF-RNAMonolayer cultures of GEF in test tubes were infected or mock-infected with NDV

at an input multiplicity of 10 plaque-forming units (PFU) per cell. Actinomycin D(5.0 /zg/ml) was added to stop cellular DNA-dependent RNAsynthesis after 5-hourincubation of infected cells at 39°C, and cell sheets were re-fed with fresh mediumFig. 1. Anti-NDV-activity of tunicamycinadded at various times after theinfection.Monolayer cultures of CEFin test tubes (1 ml/tube) were infected with NDV at an inputmultiplicity of 10PFUper cell. Tunicamycin(0.5jug/ml) was added at various times afterth.6 infection, and effect of the antibiotic onvirus multiplication was followed by titrationof hemagglutinin. The titration method wasthe same as repoted previously,5) and virusproduction was expressed as hemagglutininunits (HAU)per ml.

added at 7 hour

<>L

added at5 hour addedat Ior3hour

7 8 9 \O ll 12 13 14 15 16 17

Hours after infection

Fig. 2. Effect of tunicamycin on NDV- andCEF-RNA synthesis.

RNA synthesis in NDV-infected (10 PFU/cell) and mock-in-fected cultures of CEF in test tubes (1 ml/tube) was assessedby the incorporation of 14C-uridine (0.5 ^Ci/ml) into acid-in-soluble product. NDV-RNA synthesis(A) was revealed by theinhibition of CEF-RNA by actinomycin D (5. 0 jug/ml). CEF-RNAsynthesis(B) in mock-infected cells was followed in the absenceof actinomycin D.

7 A. NDVRNA 7 B- CEFRWA

g - /control'

O Q /x5,,"s: / 0.25ug/m/^ 0.25,ug/m!gL k

°~ /A ^/ /a

%5' /^lOpg/rr.l |3 / ///^

o ^^ o / ///

jffi Mock infected ^^

nl̂g-t-̂-;-*-r~t 01/å ..0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7'-

Hours Hours

VOt- XXIV NO. II THE JOURNAL OF ANTIBIOTICS 787

containing actinomyein D (5.0 //g/ml), uC-Uridine (0.5 juCi/ml) and tunieamycin at 1hour after the addition of actinomyein D, when CEF-RNAwas nearly completelysuppressed (Fig. 2 A, mock-infected).

NDVmultiplication was inhibited immediately on addition of tunieamycin (0.5juglxnl). whenever the antibiotic was added during the viral multiplication cycle asrevealed by titration of hemagglutmin (Fig. 1). But viral RNA synthesis proceeded

normally or was affected slightly in the presence of tunicamyein (Fig. 2 A).GEF-RNA synthesis was affected slightly by tunieamycin and proceeded at a

constant rate for a prolonged time (Fig. 2 B).

NDV^RNAlabeled with ^C-uridine was separated by centrifugation (10,000xg,

Fig. 3. Synthesis and RNase-resistance of NDV-RNA

Cultures of CEF in test tubes (1 ml/tube) were infected with NDV(10PFU/cell) and treated withactinomycin D (5.0 fig/ml) at 5 hours after the infection. Infected cultures were re-fed with prewarmedfresh medium containing actinomycin D (5.0//g/ml) and 14C-uridine (0.5 //Ci/ml) at 6 hours after theinfection. Tunicamycin was added at the same time with the radioactive uridine. At designated timeintervals duplicate samples were chilled in an ice-water bath. After freezing-and-thawing four timesdestructed cell debris were separated into soluble and pa,rtieulate fractions by centrifugation (10, 000 xg,15 minutes), and each was divided into two fractions. One was treated with RNase (100fig/ml, 37°C, 30minutes) and precipitated with TCA,and the other was precipitated with TCAwithout RNase-treatment.Total radioactivity in particulate (A) and cytoplasmic (El) fractions and RNase-resistant radioactivityin both fractions (A' and B/) were counted as described in Materials and Methods.

A . P a r t i c i p a t e B . C y t o p fa s m ie

7C o nt ro l O. I u g / m l

6

CMo

2

'o. ¥ pg/ml

0.2 5 u g /m l

^ ^ c o n t ro l

0.25 ijg/ rnl

D_o*- " 4

-r jｫn 0 .5 jjg/ ml

0.5jug /m l

-2 3 r - - ^ i

a .oCD

2

o

I

o ^ - 3 4 5 62 3 4 17

H o u rs H o u r s

2 5 r A '. p a r t ic u la t e B '. c y t o p la s m ic

0 . 1 u g / m l

'CMb 2.0^

X^Q_u l. 5

-rj

0 .25 u g / m l

C o n t ro l

01o 0 .1 u g /m la 1. 0

o<DQ _

0. 25 u g/ ml

u2t 0 .5 0 . 5 u g / m l / / S * 0. 5 jug/m l

o

0 I 2 3 4 5V /蝣 _ - I Ufr p V . . i17 0 1 2 3 4 5 / / -

17H o u r s H o u r s

788 THE JOURNAL OF ANTIBIOTICS NOV. I97t

15 minutes) into two fractions, /. £., mem-brane-bound and cytoplasmic, after freez-ing-and-thawing four times. Each frac-

tion was divided into two portions. Onewas treated with 100 ^g/ml ribonuclease A(RNase) for 30 minutes at 37°C and addedto cold TCA,and the other was added toTGA without treatment. Membrane-bound and cytoplasmic NDV-RNAweresynthesized at nearly the same rate in thepresence of tunicamycin (0.1~0.5 //g/ml)as in the controls for at least 5 hours

(Fig. 3 A and B). Adifference was detect-ed in the rate of syntheses of mem-

brane-bound and cytoplasmic NDV-RNA.Membrane-bound NDV-RNA synthesisincreased linearly and cytoplasmic virusRNA exponentially. RNase treatmentrevealed significant differences in the state

of newly synthesized NDV-RNAbetweentunicamycin-treated and control cultures(Fig. 3 A' and B'). Total NDV-RNA

synthesis proceeded to similar extents inthe presence of tunicamycin as in the

control for the first 5 hours (Fig. 3A andB), but treatment with RNase solubilizedNDV-RNAsynthesized in the presence oftunicamycin more extensively than in thecontrol, especially at a drug concentrationof 0.5 ^g/ml. These results indicate thatthe NDV-RNAsynthesized in the presenceof tunicamycin is not associated with viralcapsid protein or encapsulated incom-pletely at high drug concentrations. A

preliminary experiment suggests the latterpossibility applies (unpublished observation).

Extracted NDV-RNAlabeled with 3H-uridine was analyzed by sucrose density-gradient centrifugation or methylated albumin-Kieselguhr (MAK)column chromato-graphy. Profiles of the sucrose-density-gradients with NDV-RNA did not revealsignificant differences between the control and tunicamycin-containing samples (Fig.4), and similar results were obtained by MAK column chromatography (Fig. 5).

These studies reflect the sum of viral RNAsynthesized in infected cells, and analysisof the various fractions of NDV-RNAis now under study with fractionated viral

Fig. 4. Sucrose density-gradient profiles ofNDV-RNA synthesized in the presenceand absence of tunicamycin.

Monolayer cultures of CEF in Petri dishes (9 cmin diameter, 15 ml/dish) were infected with NDV(lOPFU/cell) Four dishes were used for eachsample At 5 hours after the infection cell cul-tures were re-fed with fresh medium (5 ml/dish)containing actinomycin D (5 0 jug/ml) Newlysynthesized NDV-RNAwas labeled with 3H-uri-dine (5 /iCi/ml) for 5 hours (6~11 hours after theinfection). Tunicamycin was added at the sametime with the radioactive uridine. RNA wasextracted by the SDS-phenol method and preci-pitated with ethanol. Collected precipitates weredissolved in 1 ml phosphate buffered saline, and0 2ml of the solution was analyzed by sucrosedensity-gradient centrifugation (5~15 w/v%, 4 5ml, 130,000xg, 6 hours).

A ControlI8S

B 025pg/ml C 05pg/mFI8S

28S 4S

I8S

28S I4S

^Ir

0 (0 20 30 40 0 10 20 30 40 0 10 20 30 40

Bottom Fraction number Top

YOU. XXIV NO. II THE JOURNAL OE'/AN?J6fOfid! 789

Fig. ,5. ,Effect of tiinicamycin on NDV-RNAsynthesis a§ analyzedby MAKcolumn chromatography.

NDV-RNAwas labeled and extracted according to the same method as Fig. 4 except 32P-ph6sphate(50juCi/ml) was used instead of 3H-uridine. Monolayer cultures of CEFin four Petri dishes were usedfor the preparation of RNA. A half of the RNApreparation (1ml) was loaded on MAKcolumn (25x300 mm). MAKcolumn ehromatography was carried out as described previously. 5)

-Control - Tunicamyc/n (0.5jug/ml)

0.2- I /\ à" A T2.000 .

i / T' Ml *-o å  "à"' .. i ll " ! " Q_

Jo.i-/i r 1] "A / f "li00°°-

0 20 40 60 80 0 20 40 60 80 100

; 'Fraction number Fraction number

RNA, such as membrane-bound,cytoplasmic and capsid-associatedRNA.

Analysis of CEF-RNA synthe-sized in the presence of tunicamycinby sucrose density-gradient centri-fugation reveals some abnormalities.At 4 hour of treatment, significantdifference was not detected in thesucrose density-gradient profiles bet-ween the control and tunicamycin-

treated samples (Fig. 6 A, B and C).But at 24 hour of treatment theprofiles reveal that RNA lost itsnative size and radioactive peakswere detected at lighter regions, andthis tendency was more significant

at a drug concentration of-0.:5 ^g/mlthan at.0.25^g/ml (Fig. 6 D, E andF). Degradation of RNA in thepresence of tunicamycin was observedwith Bacillus subtilis.^ Pulse-chaseexperiments are required for a pre-

Fig. 6. Sucrose density-gradient profiles ofCEF-RNA synthesized in the presence andabsence of tunicamycin.

Monolayer cultures of CEF in Petri dishes (9cm indiameter, 15 ml/dish) were re-fed with fresh medium(5 ml/dish) containing 3H-uridine (5 /*Ci/ml). Treatmentwith tunicamycin (0.25 or 0.5 fig/ml) was started at the

same time. After4 (A, B and C) or 24 hours (D, EandF) of incubation at 39°C, RNAwas extracted by theSDS-phenol method. Two dishes were used for eachsample. Labeled RNAwas analyzed by sucrose density-gradient C5-15 w/v%, 4.5 ml) centrifugation (130,000xgy6 hours).

A^, Control JQ. a25^q'/m\ C. 0.5jjg/m!

å å "" '28$" |I8S 3- I|4,S 6 , V-2 k . 2 I

'(INIf

0t=Z^I , i i Ol "V ,-å å .'. O-l-i 1 1 1 0 10-20 3040 0 10\g'p';3040 0 10 20 30 40 Bottom Fraction \numbef Top

riseå analysis of the nature of the CEF-RNAdegradation.2 Effect on GEF-DNASynthesis

Effect of tunicamycin on the synthesis of CEF-DNAwas;rassiesse;d bytjncorporationof 3H-thymidine into acid-insoluble product. Inhibition of the incorporation by

7iQ THE JOURNAL OF ANTIBIOTICS NOV. 1971

Figv 'e.

5 r D. Control E. 0.25jjg/ml E o.Spg/ml28S

I

4- 4r 4r

I8S3å  I 3* 3"

^. to l*>

2 1 '2 ^ I

x " x x

:^ S 20- 0- Q.° O 1 O /

2å  2- J[ 2-X 3: å  Jlf X /

°p [Q20304Q °0 10 203040 °0 1020 304(

sP^o^ Fraction number T.

Fig. 7. Effect of tunicamycin on CEF-DNAsynthesis.Monolayer cultures of CEFin test tubes (1 ml/tube")were treated with tunicamycin. DNA synthesis was

followed by counting 3H-thymidine incorporated into acid-insoluble product. Radioactive thymidine (1. 0 juCi/ml) wasadded at the same time with tunicamycin.

3.5"

à"Control3,0 *- /

/ <>0.25jig/m]

k 2.0- /à" / A<X5jjg/ml

1 1.5- / //y P10pg/m1

0.5- i/Wo

tunieamyein was significant only athigh antibiotic concentration (Fig.7).

3 Effect on Protein SynthesisProtein synthesis in cultured

CEFwas affected to a large extentby tunicamycin than cellular RNAand DNA syntheses (Fig. 8).

To investigate the nature of

the effect of tunicamycin on proteinsynthesis, NDV-infected or mock-infected CEF were fractionatedaccording to the scheme shown in

Fig. 9. Some differential inhibitionof protein synthesis was detectedas shown in Table 1. Membraneancl extracellular secreted proteinsynthesis was more sensitive totunicamycin inhibition than eyto^

plasmic protein synthesis in mock-infected GEF. Infection with NDVdecreased amino acid incorporationinto all fractions. Treatment ofNDV-infected GEF with tunica-mycin decreased incorporation to

about 50%of the control as forcytoplasmjc and secreted protein

Fig. 8. Effect of tunicamyein onprotein synthesis.14C-Amino acid mixture (0.1 //Ci/ml) wasadded to cultures of CEFin test tubes (1 ml/tube), and protein synthesis was assessedby counting acid-insoluble radioactivity.

r"2~- --3"' 4

YOU- XXIV NO. II THE JOURNAL OF ANTISIOTJOS ?m

Table 1, Effect of tunicamyein on 14C-amino acids incorporation into cytoplasmic,membrane and secreted proteins

F r a c tio n sN D V

in fe c tio n

T u n ie a m y e in(0 .5 ju g /m l)

tr e a tm e n t

R a d io a c tiv it y

T o ta l C P M x l O "3 %

3 1 . 9 1 0 0 1 0 0蝣III4 - 2 1 . 1 6 8 6 8

C y to p la s m ic

2 4 . 6 1 0 0l l . 9 4 9+

+

7 73 7

38. 2 1 0 0 1 0 0II+ 2 0 . 9 5 5 5 5

e m D ra n e

++

3 0 . 2 1 0 02 6 . 0 8 7

7 96 8

8 . 0 1 0 0 1 0 0

+ 4. 8 6 0 6 0S e c r e te d

++

5 . 3 1 0 03 . 0 5 7

6 63 8

Monolayer cultures of CEPin test tubes were infected (10 PFU per cell) or mock-infected with NDV.After a 2-hour adsorption period at 4°C, cell sheets were fed with the mediumcontaining one-tenth concentra-tion of lactoalbumin hydrolysate and incubated at 39°C for 5 hours. The same medium containing 14C-aminoacids mixture was added to each tube, and tunicamycin-treatment was started at the same time. At 12 hourof treatment at 39°C, cell cultures were fractionated according to the method shown in Fig, 8. Three tubeswere used for each determination.

fractions. Incorporation intothe membrane fraction wasaffected to a lesser extent thanthe other fractions. NDV is

knownto mature at the sur-face membrane and obtainsits envelope from the plasmamembranq during budding

from it.6) Altered membranesynthesis is induced by other

viruses.7~"*Q) The resistance ofmembrane protein synthesisin the presence of tunica-

myein in NDV-infected CEFsuggests some relation withthese observations.

4 Effect on GholineIncorporation

Choline is a lipid abundantly found in the plasma membranes of animal cells.n)In contrast to ammoacid incorporation, choline uptake was stimulated after infectionwith NDV(Fig. 10). Tunicamycin present in concentrations up to 1.0jug/ml had noeffect at all on the incorporation.

§ Effect on Sugar Incorporation

Glucosamine is knownto be incorporated only into the membranefraction ancl

Fig. 9. Fractionation of cultures of CEF labeled withuC-aminoacids for determination of effect oftunieamycin on protein synthesis.

Whole culture in test tubedecantation

MediumCell sheet

washed with phosphate buffered salinecombined+50% TCA

(final 10 %)Precipitate(secreted protein)

Washed cell sheet+2 ml phosphate buffered salinefreezing-and-thawing 5 times

centrifugatipn (lQ,QQQ g, 15 min.)

SupernatantPrecipitate washed with

the buffer

combined+50% TCA

(final 10 %)Precipitate(cytoplasmic protein)

Washedprecipitate(membraneprotein)

792 THE JOURNAL OF ANTIBIOTICS NOV. 1971

without modification in HeLacells12) and the same may bepresumed with CEF. Similarto choline uptake incorpora-

tion of glucosamine into acid-insoluble product was stimu-

lated after infection withNDV (Fig. ll). Treatmentwith tunicamycin significantlyinhibited the incorporation.Glucosamine uptake was

more sensitive to the anti-biotic in NDV-infected cells

than in mock-infected cells,i. e., the inhibition ratios

were 22, 44and 53% in mock-infected cells (Fig. ll A) and28, 61 and 87^ in NDV-infected cells (Fig. ll B) atantibiotic concentrations of

0.25, 0.5 and 1.0 ^g/ml, respec-tively.

When 14C-glucosamine-

labeled cells were filtered afterfreezing-and-thawing withouttreatment with TCA, it wasfound that incorporation ofglucosamine was inhibited

more extensively (72 % inhibi-tion at 0.5jug/ml) than ob-served with whole cells (Fig.12 A).

A similar inhibitory effectof tunicamycin was observedduring the incorporation of14C-glucose (Fig. 12 B).

Discussion

Tunicamycin exerted negli-gible effects on the incorpora-tion of 14C-uridine (Fig. 2), 3H-thymidine (Fig. 7), 14C-choline(Fig. 10) and 14C-amino acids(Fig. 8), but it significantly

Fig. 10 Effect of tunicamycin on incorporation of choline

into membranefraction.Cell sheets of CEF in test tubes (1 ml/tube) were infected (10 FFU/cell) or mock-infected with NDV.14C-Choline chloride (0 1 ^Ci/ml)was added at 4 hour after the infection, and tunicamycin treatmentwas started at the same time. At designated time intervals duplicatesamples were chilled in an ice-water bath After freezing-and-thaw-ing four times destructed cultures were filtered and membrane frac-tion was collected on Millipore niters. Radioactivity on the niterswas counted as described in Materials and Methods

A Mock infected B. NDVinfectedà" Control

o 0 25pg/ml

£, 0.5 pg/ml

o (.0 pg/ml

6" °- /

T5" / " /O / /x. I I

& / /A - /

0L^-I 1 1 1 1-_i i 1 IZ_j 1 i i i i i r

0 1 2 3 4 5 6 7 8 0 / 2 3 4 5 6 7 8

Hours Hours

Fig. ll. Effect of tunicamycine on incorporation ofglucosamine into acid-insoluble product.

Cultures of CEF in test tubes (1 ml/tube) were infected (10PFU/cell) or mock-infected with NDV, and 14C-glucosamine (0 25 j«Ci/ml>wasadded at 4 hour after the infection. Tunicamycin was addedat the same time with the radioactive glucosamine. Acid-insolubleradioactivity was counted after destruction of cell sheets by freezing-and-thawing four times

26r A Mock infected r B NDV infected

Control*

*? / 0 25jug/mf2 . Control - / P

J /O2.5pg/m\ / /

j. / /O5pq/m\. y / O5^g/mfl

/// /^ 'Ojjg/ml " / y^ ^s^

/^^^rr^^^ ' /X ^^^ l Opg/mt'

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9

Hours Hours

VOL. XXIV NO. II THE JOURNAL OF ANTIBIOTICS 793

inhibited the uptake ofglucosamine and glucose(Fig. 12). A similar effectof the antibiotic was ob-served with Bacillus sub-tilis.^ Tunicamycin wasfound to induce variousmorphological changes ofgram-positive bacteriaand yeasts.4) The results

obtained with animal andmicrobial cells suggestthat similar tunicamycin-sensitive processes of cellenvelope synthesis takeplace in both types ofcells. Reversal of theantiviral activity of tuni-camycin by some amino-sugar derivatives^ sug-gests that the sensitivestep is somehow associatedwith glycosidation.

NDV is known to mature at the cell surface and some components of the virusparticle are synthesized there.6) The stimulated incorporation of choline and glucosamineafter infection with NDV (Figs. 10 and ll) might be related with the characteristicmaturation process of the virus.

Glycoproteins have attracted interest lately in relation to virus multiplication,7>8)malignant transformation13^1^ and synthesis of secretion proteins16). Appropriate in-

hibitors of such events are rare at present, and tunicamycin might becomea useful toolin such investigations.

Acknowledgement

This investigation was financially supported by grant-in-aid from Tokyo Biochemical ResearchFoundation, Kaiun Mishima Memorial Funds and WaksmanFoundation of Japan.

References

1) Takatsuki, A.; K. Arima & G. Tamura: Tunicamycin, a new antibiotic. I. Isolation andcharacterization. J. Antibiotics 24 : 215-223, 1971

2) Takatsuki, A. & G. Tamura: Tunicamycin, a new antibiotic. II. Some biological propertiesof the antiviral activity of tunicamycin. J. Antibiotics 24 : 224-231, 1971

3) Takatsuki, A. & G. Tamura: Tunicamycin, a new antibiotic. III. Reversal of the antiviralactivity of tunicamycin by aminosugars and their derivatives. J. Antibiotics 24 : 232-238, 1971

4) Takatsuki, A.; K. Shimizu & G. Tamura: Effect on tunicamycin on microorganisms: Morpho-logical changes and degradation of RNAand DNAinduced by tunicamycin. J. Antibiotics,

inpress5) Takatsuki, A.; G. Tamura & K. Arima: Mode of action of xanthocillin X monomethylether

o

nmultiplicationofNewcastlediseasevirusinculturedcells. J. Antibiotics 22 : 151-160, 1969

6) Scholtissek, C.; R. Drzeniek & R. Rott; Myxoviruses, in "The Biochemistry of Viruses"(H. B. Levy, ed.), Marcel Dekker, 1969

7) Keller, J.M.; P.G. Spear & B. Roizman: Proteins specified by herpes simplex virus. III.Viruses differing in their effect on the social behavior of infected cells specify differentmembrane glycoproteins. Proc. Nat. Acad. Sci. 65 : 865-871, 1970

Fig. 12. Effect of tunicamycin on incorporation of glucosamineand glucose into membrane fraction.

Monolayer cultures of CEF in test tubes (1 ml/tube) were treated withtunicamycin. 14C-Glucosamine (0. 25 ^Ci/ml) and 14C-glucose (0. 2 juCi/ml)were added at the same time with tunicamycin. Incorporation of radio-active precursors into membrane fraction was followed by countingradioactivity held on Millipore filters after destruction of CEF by freez-ing-and-thawing as Fig. 10

12 A Glucosamlne 3r B Glucose

Confrof10- " ?

Control, /

E /_3y/ 0 5^ug/ml

0 4 - j/ Cf" I - / ^/^lOjug/mr

CD / 0.5>jg/ml 2: /^yy^\0)iq/m\

0 1 2 3 4 5 6 7 8 0 / 2 3 4 5 6 7 8

Hours Hours

794 THE JOURNAL OF ANTIBIOTICS NOV. 1971

Bubge, B, W. & J. H. Strauss, Jr.: Glycopeptides of the membrane glyeoprotein of Sindbisvirus. J. Mol. Biol. 47 :449-466, 1970Holland, J. J. &E, D. Kiehn: Influenza virus effects on cell membrane protein. Science 167 :

202«v2Q§i 1970Aghadashi, M. & E. Gold: Origin of herpes simplex virus envelope. Bacteriol. Proc. 1970 :

162, 1970

Lennarz, W. J.: Lipid metabolism. Ann. Rev. Biochem. 39 : 359-388, 1970.Reith, A.; R. Oftebro & R. Seljelid: Incorporation of 3H-glucosamine in HeLa cells asrevealed by light and electron microscopic autoradiography. Exptl. Cell Res. 59 : 167-170, 1970Burger, M. M. & A. R. Goldberg: Identification of a tumor-specific determinant on neoplasticcell surfaces. Proc. Nat. Acad. Sci. 57 : 359-366, 1967Meezan, E.; H. C. Wu, P. H. Black & P. W. Robbins: Comparative studies on the carbohydrate-

containing membrane components of normal and virus-transformed mouse fibroblasts. II.Separation of glycoproteins and glycopeptides by Sephadex chromatography. Biochemistry 8 :2518-2524, 1969

Molnar, J.: Glycoproteins of Ehrlich ascites carcinoma cells. Incorporation of [14C]gluco-samine and [14C]sialic acid into membrane proteins. Biochemistry 6 : 3064-3076, 1967

Eylar, E. H.: On the biological role of glycoproteins. J. Theoret. Biol. 10 : 89-113, 1966