On the arginase of the shope papillomas

I>ISCUSHION ANI) PREI,IRIINAI~Y tivc at low dilution (undiluted through 1: 8) with 4-5 units of het’erologous tumor antibody (Table 1). Bovine 3 tumor antisera also reacted specifically with virus-infected MDBK cells. Specific complement-fixing antigen was det’ected at dilut’ions of 1:321 : 128 within24-48 hours post-infection. It was possible to distinguish viral from T and tumor antigens based on the ext’reme labiliby at, 5G” of the latter antigens, 5 minutes of heating being sufficient to inactivate all activity (Table 2). It should be noted that none of bhe tumor antisera tested reacted with heated (56”) infected cell preparations, supporting the view that t,hese sera do not contain viral antibody; however, more extensive tests will be necessary to est’ablish the lack or presence of viral structural elements in these tumors. There is no doubt’, however, that the tumors induced by bovine adenovirus type 3 do contain a new antigen distinguishable from the major viral antigens. REFERENCES 1. HUEBNER, R. J., ROXVE, W. P., TURNER, H. C., and LANE, W. T., Proc. S&l. dead. Sci. U.S. 50, 379-389 (1963). 2. HUEBNER, R. J., Perspectives Viral. 4, 142-163 (1965). 3. DARBYSHIRE, J. H., JENNINGS, A. R., OMAR, A. R., DAIVSON, P. S., and LAMONT, P. H., Salure 208, 307-308 (1965). 4. DARBYSHIRE, J. H., Matwe 211, 102 (1966). 5. MADIN, S. H. and DARBY, N. B., hoc. Sot. Expll. Biol. Med. 98, 574-576 (1958). 6. GILDEN, It. \‘., BEDDO\\., T. G., and H~EBNER, It. J., in preparation. 7. HULL, R. N., JOHNSON, I. S., CULBERTSON, C. G., REIMER, C. B., and WRIGHT, H. F., Science 150, 1041-1046 (1965). 1
729

I:EPOI:TS

On the Arginase

of the Shope

Papillomas

It has been well established by Rogers et al. (1-3) that the tumors provoked by the Shope papilloma virus 011 the skin of the cottoruail and the domest)ic rabbit contain high levels of I,-arginase (t-arginine ureohydrolase, ES 3.5.3.1). These workers rcported that the papilloma arginase appears to be absent from t’he normal rabbit skin and that it differs from the other rabbit arginases. They therefore concluded t(hat the genetic informat8ion of t,he papilloma arginase does not belong to the rabbit chromosomes but is carried by the Shope virus. The observation of Rothberg a11c1Van Scott (,G)that’ some arginnse act’ivity can bc detected in extracts of the normal rabbit epidermis in the absence of any viral infection prompted us to compare the properties of n-arginase from papilloma wit,h L-arginnse from rabbit liver. The data presented in this paper show that the arginases extracted from various papillomas are, in many respects, related t’o the enzyme extracted from t’he domestic rabbit liver and strongly suggest’ that the genet,ic determinant of the tumor enzyme is borne by t,he rabbit chromosomes. Rabbit Liver and Shape Papillomas Kinetic properties. In their comparat,ivc studies of t,he papilloma and rabbit liver arginases, Rogers and Moore (3) emphasized t,he difference in sensitivity of these enzymes to the activation by i\lnC&. hccording t,o their results L-arginase from the liver is activated by manganous ions whereas Larginase from papilloma is not activated by manganous ions. However, our observations on partially purified preparations of liver n-arginase (610 U/mg protein) and of papilloma Larginase (45 Ir/mg prot,cin) previously treated with et’hylenediamine sodium tetraacetat,e (EDTA) indicate that llnClz activates both enzymes to nearly the same extent; furthermore both preparat,ions are stabilized by 1\In++ ions against t’hermal inactivation (Fig. 1). III addit)ion, liver and papillomn nrginases exhibit the same

730

I>ISCUSSION

AND

1’I:ELIMINAIIY

I:El’ORTS

2 4 6 Minutes at 60°C

Minutes ot 60°C

8

IO

FIG. 1. Thermal inactivation of papilloma (PA) and liver (LA) arginases from domestic rabbit. PA (45 U/mg protein) and LA (610 U/mg protein) partially purified according to Schimke (5) are filtrated on Sephadex G50,equilibrated with2 X 10-lMTrismaleate-NaOH buffer,pH$.4,supplementedwith 1 X lO+ iV EDTA. The enzymes are kept in l,his buffer at, 4” for a week and dialyzed before use against 2 X 10-l Tris maleate-NaOH buffer pH 7.4. Inactivation is started by diluting the enzymes int,o the same buffer previously heated at GO”, in the presence or in the absence of 5 X 10-a 31 MnClz [concentration of

protein in the activation mixture 50 pg/ml (LA) and 250 pg/ml(PA)]. Samples are immediat>ely cooled at 0” and residual activity assayed. Assay medium: 50 pmoles of L-arginine HCl, 7.5 Fmoles of MnC12 or none in i X 10m2IV glycine-NaOH buffer pH 9.6, in a total volume of 5 ml.The reaction is started at 37” by addition of appropriate dilution of enzyme, and aliquok of 1 ml are sampled each 5 minutes during

20 minutes [specbrophotomet,ric assay of urea following the method of Ceriotti and Spandrio (6)]. Initial rates are expressed

in micromoles

of urea liberated

per minute.

Inactivation

in the presence of 5 X IO-3 in the absence of assay in the presence of 1.5 X 1OP dl MnClz

M MnC12; assay in the presence of 1.5 X 10-a 1U MnClz (V: LA; V: PA). Inactivation MnCl?: assay iu the absence of MnCl? (0: LA; 0: P-4).

(A:

LA; A: PA);

nlichaelis corkant for arginine (1.9 Z!Z 0.5 X 1O-3 ilZ) and t.he same dissociation constant for t.he competitive inhibitor L-ornithine (Ki = 1.2 & 0.5 X lo+ 11). I”inally, the pH opt,imum has also been found to be

the same for both enzymes: pH 9.G in glycine-SaOH buffer. In1nw2.oloyical pope&es. According t’o t’he observation

of Rogers and lUoore, the papil-

loma arginase does not show any antigenic similarity &h the rabbit liver enzyme, and antibodies directed against the tumor enzyme are present in the serum of rabbit’s infected

by the Shope virus.

However,

Icig.

2 shows that the serum of a sheep immunized against purified hcpatic rabbit arginase does precipitate

the nrginase

extractred

from

the

Shope pnpillomas; this finding suggests a similarity of st,ructure bet,ween t’he liver nrginases. The different mtl papillonm points of ecp~ivalencc observed in the titration curve of each enzyme preparation suggest, that although both arginascs possess sevcrnl structural properties in common,

they might not, be identiwl

molcculw.

Moreover, we have looked for the presence of antibodies, directed against t’he papilloma enzyme, in the serum of rabbits massively inoculated by the Shope virus. Among 10 rabbits carrying papillomas from 40 days to one year after their inoculation with the virus, none of the rabbits exhibited any detectable precipitating activity against the papilloma enzyme. Presence of L-Aryinase in Nomal Rabbit Epidermis and in Papilkwa Provoi~ecl by the 9 ,lO-Dimethyl,L-benxanthracene (DMBA) Table 1 shows that in the extra,& of rabbit ear epidermis an appreciable activity of L-arginase can be detectfed although the specific activit,y of the enzyme is much lower than in the ext’racts of papillomas obtained aft,er intracpidermal inoculation of the Shope virus. The same t#able also indicates that t,he specific act,ivity of an extract of papillomas provoked by repeated applications of DJIBA on the car skin of the domestic rabbit (7) is almost-~the same as the specific activit,y of

DISCUSSION

AND PR.ELIM1NAR.Y

731

REPOR.TS TABLE

ARGINASE EAR

ACTIVITY EPIDERMIS

APPLICATION THE

SHOPE

OF

DMBA papillomas Shope papillomas

Argmose

units added (x10-‘)

the extracts of Shope papillomas. In addition all these arginase activit.ies possess the common property of being activated by RlnCl,. Since high levels of L-arginase are observed in the absence of Shope virus, we conclude that the structural gene of the papilloma arginase does not belong to the virus. The most likely interpretation is either that the Shope virus “induces” the expression of genetic information carried by the rabbit, or that the virus directs the

1 EXTRACTS

PAPILLOMA

DMBA

OR

BT

OF RABBIT

PROVOKED INOCULATION

BY OF

VIRUS”

Epidermis

2. Precipitation of the Shope papilloma arginase (PA) and liver arginase (LA) of domestic rabbit by the serum of a sheep immunized against the rabbit liver enzyme. Purified LA, 10,000 units, mixed with 50% v/v of Freund adjuvant is injected intradermally to a sheep in two series of 4 weekly injections at 1 month interval; a booster injection of 10,000 units is given intravenously 10 days after the last injection. Prior to use, the serumisdialyzedagainst1X10-2MTris-HC1 buffer, 14 X 10-l M NaCl at pH 7.4. The serum (0.6 ml) is mixed with variable amounts of partially purified LA (1,050 U/mg protein) or PA (100 U/mg protein) diluted in 0.6 ml of 1 X lO+M Tris-HCl pH 7.4,1 X 10m2M MnCl?, 14 X lo-‘M NaCl. Incubation 1 hour at 37”, then 18 hours at 4”. After centrifugation the arginase activity is assayed (see Fig. 1) in the presence of 1.5 X 1e3 M MnClz in both the supernatant and the precipitate washed with 1 X 1OV M Tris HCl, 5 X lop3 121MnCI?, pH 7.4. 0, .:PA; A:LA.

CRUDE

AND

Sample

FIG.

IN

Assay with MnClz Assay without x0 prein- PreincuMnC12 cubation bation (a) (b) Cc) 0 .3 0.2 0.05 0.2

0.04 0.02 0.9 1.1 0.5 0.9

0.08 0.05 2.5 1.9 1.4 1 .9

a Separation of epidermis from dermis is obtained by incubation of normal skin from rabbit ears iu 0.3% trypsin (Difco) in Tyrode’s solution for about 30 minutes at 37” (8). Crude extract,s are prepared bygrindingina mortar the tissues in a few milliliters of 1 X lo+ il/ Tris-HCl buffer, pH 7.4. The preparations are centrifuged 30 minutes at 40,000 g and filtered on Sephadex G 50 equilibrated with the same buffer. These extracts are assayed in absence of MrClp (aj, or in presence of 1.5 X 10m3M MrCL, with (c‘) and without (b) preincubat,ion at 60” for 60 minutes in the presence of 5 X 10m3M MnCli. Assay for proteins following the technique of Lowry st al. (9). Specific activities are expressed in micromoles of urea liberated per minute and per milligram of protein.

preferential growth of part’icular strains of epidermal cells with a high arginase cont,ent. The mechanism of this interesting aspect of the problem is not clear, but we feel that the evidence presented strongly suggests that the genetic informat’ion for L-arginase produced in papillomas during infection by Shope virus is carried by the rabbit, not by the virus. ACKNOWLEDGMENTS This work was supported by the “D~lBgation GBnerale B la Recherche Scientifique et Technique,” Contrat no 61 FI< 60, Comiti: Cancer et LeucBmie. REFERENCES S., ,Vature 183, 1815-1816 (1959). 2. ROGERS, S., 1m “The Molecular Basis of Neoplasia,” pp. 483-4!15,. TJniv. of Texas Press, Austin, 1962. 3. ROGERS, S., and ~IOORE, M., J. Ezpll. Med. 117, 521-542 (1963). 1.

ROGERS,

732

I~ISCI!SSION

ANI) PI
I~EPOI:T8

The Ellen strain (9) of \‘-2 virll$ \WS IW(YI except, where ot herwisc indic:;tt cd. \‘irrls 5. SCHIMXE, 1:. T., 2. ~aining 2% serum and 0.15 “c NaHCO:{. small volumes of HBSS since only 0.125-