354 DISCUSSION AND PRELIMINARY REPORTS

ACKNOWLEDGMENTS

I am grateful to Dr. S. Brenner for suggesting the principle of this technique, and to Dr. M. Cohn for instruction in the use of indolyl-galactoside. This work was supported by grant number GM- 14426-01 from the National Institutes of Health.

REFERENCES

1. KELLENBERGER, G., ZICHICHI, M. L., and WEIGLE, J., J. MoZ Biol. 3,399-408 (1961).

2. ZICHICHI, M. L., and KELLENBERGER, G., Virology 19, 450-460 (1963).

8. ZISSLER, J., Virology 31, 189 (1967); GOT- TESMAN, M. E., and YARMOLINSKY, M. B., J. Mol. Biol., in press; GINGERY, R., and ECHOLS, H., in preparation.

4. WOLLMAN, E. L., Compt. Rend. Acad. Sci. 257, 4225-4227 (1963).

6. SIGNER, E. R., BECKWITH, J. R., and BRENNER, S., J. Mol. BioZ. 14, 153-166 (1965).

E. R. SIGNER Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts ORlS9

Accepted July 24, 1967

Studies on the Arginose Activity of Shope

Papilloma: Possible Presence of lsozymes

Rogers et a?. demonstrated an increased level of arginase activity in tumors induced by Shope papilloma virus on the skin of domestic rabbits and cottontail rabbits (1). They suggested, on the basis of physico- chemical and immunochemical data, that the information for the synthesis of arginase was derived from Shope papilloma virus (SPV). Their data showed that papilloma arginase differed from rabbit liver arginase in molecular weight, sensitivity toward ca- navanine, Mn++ activation, and immuno- logical behavior. Their findings led Beaty

et al. to investigate a related virus, rabbit kidney vacuolating virus (2), and Passen et al. to use arginase as a biochemical marker (9.

Recently, Orth et al. (4) reported that arginase from papilloma and liver were iden- tical in kinetic behavior and immunological properties, suggesting that the genetic infor- mation for the arginase produced in the

SPV-induced papilloma may reside in the rabbit genome rather than in the viral DNA. This view was supported by the finding that the properties of arginase in dimethyl- benzanthracene (DMBA)-induced papilloma are very similar to those of the enzyme in SPV-induced papilloma and liver. Our data, arising from assays of arginase in cutaneous papillomas and in normal rabbit skin, con- firms the data of Orth et al. (4).

Arginase was partially purified from rab- bit liver and SPV-induced papillomas ac- cording to the method described by Green- berg (5), and was assayed in the presence of 212 pmoles/ml of arginine and 40 rmoles of Tris buffer, pH 8.0, for 10 minutes at

FIG. 1. K, values of arginase from SPV-induced papilloma (above) and the normal rabbit liver (below). The experimental conditions were essen- tially the same as those in Table 1 except that the enzyme preparation was purified by alcohol pre- cipitation and extraction (1). 19% ethanol-acetate buffer extract was used as the enzyme solution. Approximately 40 pg of protein was employed in a total volume of 1.0 ml.

DISCUSSION AND PRELIMINARY REPORTS 355

TABLE 1

A~~GINASE ACTIVITY IN PAPILLOMATOUS TISSUES OF RABBIT SKIN INDUCED BY SHOPE PAPILLOMA VIRUS

AND DIMETHYLBENZANTHRACENE (DMBA)u

Tissues 17 24

Days after virus inoculation

31 38 45 52 76

Shope pailloma (RN-1807) 4.28 4.55 5.7 9.0 4.65 - (RN-1793) - 6.02 4.55 7.2 5.3 2.8 -- (RN-1810) 2.62b 5.45 4.8 3.76 2.5 2.16 (RN-1803) - 1.75 2.4 1.18 - -

(RN-1811) - 2.85 2.84 0.90 -

l.lc 0.82C (T-179)d - - - - 4.05

DMBA papilloma (pooled)e - 6.09 - - - - (RN-2011)’ - - 4.23 - - - - (RN-1815)b - - 4.85 - - -

Normal skin (pooled)0 0.7 0.77 0.77 0.85 0.60 0.40 -

a Unit = pmoles urea formed/mg protein./10 minutes. The number of the donor rabbit is given in parentheses in column 1 except when tissues from several rabbits were pooled. Conditions: Arginine 212 pmoles, Tris buffer 40 @moles, pH 8.0, and approximately 2 mg of the 20,000 rpm supernatant of papilloma or skin homogenate act,ivat,ed in the presence of 5 X 10e3 M MI? maleate were incubated for 10 minutes. The reaction was stopped by the addition of 1.0 ml of 87v/, acetic acid. Urea formed was determined by the diacetylmonoxime method (6).

b Average of 5 papillomas. c Average of 2 regressing papillomas. d San Juan rabbit (8) papilloma induced by SPV. e San Juan rabbit papilloma induced by DMBA. Tissue samples from 5 rabbits were pooled. 1 Domestic rabbit papilloma induced by DMBA 90 days after treatment. g Four specimens of normal skin were pooled.

37”. The urea formed was determined by present in the first peak was found to be the diacetylmonoxime method (6). Table 1 homogeneous on the basis of analytical cen- shows the levels of arginase in SPV-induced trifugation studies. Detailed studies of the papillomas of domestic rabbits in DMBA- isozymes and their immunological proper- induced papilloma of San Juan and domes- ties, using a highly purified preparation of tic rabbits and in normal rabbit skin. the enzyme, are in progress.

Both liver arginase and papilloma arginase were found to have Km values close to 2.9 X 10m2 M for L-arginine under the experimental conditions employed, and to require pre- incubation with Mn++ for maximum activity (Fig. 1). Arginase from liver and papilloma showed identical behavior on a CM cellulose column (Fig. 2), the elution profile indicd ing the possible presence of arginase iso- zymes. Our data agree with those of Bascur et al. (7), who demonstrated isozymes for arginase from human liver. It is not clear at this stage of our investigation whether the two peaks obtained by CM-cellulose column chromatography are due to different physi- cal states of the same protein. The protein

ACKNOWLEDGMENT

This work was supported in part by grants from the National Cancer Institute (USPHS CA-08698) and from the Jane Coffin Childs Memo- rial Fund for Medical Research. We wish to thank Drs. C. A. Evans and A. Rashad of University of Washington, Seattle, for the gift of papillomas from the San Juan rabbits; and Miss Yoshimi Fukushima for her able technical assistance.

REFERENCES

1. ROGERS, S., and MOORE, M. (1963), J. Exptl. Mecl. 117, 521-542.

2. BEATY, L. E., and HODES, M. E. (1966), Rac- teriol. Proc. p. 111.

3. PASSEN, S., and SCHULTZ, R. (1965), I’irology 26, 122-12e

356 DISCUSSION AND PRELIMINARY REPORTS

r

280 0. D.

0.1

Tube number(3 ml)

0 5 10 15 20 25 30 Tube number0 ml.)

FIG. 2. Carboxymethyl-cellulose column chromatography of the arginase from SPV-induced papilloma (above) and normal rabbit liver (below). CM cellulose (0.72 meq/lOO g) was equilibrated with 0.0005 M Tris buffer, pH 7.4, and packed in a column (16 X 190 mm). Approximately 8 mg of the protein from the 19% ethanol-acetate buffer extract (1) was applied on the column. It was washed with the starting buffer (0.005 M Tris buffer, pH 7.4); after 45 ml of washing, it was eluted with 0.2 M KC1 (7).

4. ORTH, G., VIELLE, F., and CHANGEUX, J. P. (1967)) Virology 31, 729-732.

6. GREENBERG, D. M. (1965), In “Methods in Ensymology” Vol. II, p. 368. Academic Press, New York.

6. FRIEDMAN, H. S. (1953). An&. Chem. 25, 66% 664.

Y. BASCUR, L., CABELLO, J., VELIZ, M., and GON- ZALEZ, A. (1966) Biochim. Biophys. Acta 128, 149154.

8. THOMSEN, J. J., and EVANS, C. A. (1964). Lab. Animal Care 14, 155-160.

PAUL S. SATOH T. 0. YOSHIDA

YOHEI IT0 Laboratory of Viral Oncology Research Institute Aichi Cancer Center Chikusa-ku, Nagoya, Japan

Accepted July 27, 1967