The Relationship Between a Lysozyme‐like Enzyme and Frog Adenocarcinoma*

Abstract

The occurrence of macromolecular change accompanying differentiation in embryonic or neoplastic development is well recognized, even if poorly understood.'+ In the course of immunochemical studies on macromolecular changes related to differentiation in the amphibian,".' we have had occasion to examine the antigenic composition of the frog kidney and of the frog renal adenocarcinoma, which is believed to be of viral origin.", I" The following is a report of observations on antigenic differences between the normal and tumor kidney of the frog, on the possible identity of one of these antigens, and on some differences between tumor-susceptible and resistant f rogs.1'-*3 Materials and M e t hodsAntigens. R a n a pipiens obtained from Alberg, Vt., were used as the source of embryos, normal organs, and tumors, from which "supernatant" fractions were prepared".I5 at 0 to 4" C. by homogenization from the frozen state i n two volumes of phosphate buffered saline, pH 7.4, and centrifugation at 12,000 g for 20 minutes. These supernatants were used as injection or test antigens after adding merthiolate. Although all the renal adenocarcinomas were palpable, they were not of uniform histological character and i t was impossible to be assured of complete separation of tumor from "normal" tissues.The bacteriolytic enzyme described below was prepared from ovaries or larvae (Shumway Stages 19-23) because of the limited supply of normal kidneys. A bentonite adsorption procedure modified from the method for chicken egg white lysozyme was used."' Ovaries from two to three frogs were ground in 80 ml. of 0.85 per cent NaCI, adjusted to pH 4 to 4.5 with acetic acid and rehomogenized, frozen and thawed three times, and centrifuged at 10,000 g. The precipitate was reprocessed in 40 ml. of 0.85 per cent NaCI. The supernatants were pooled, filtered, adjusted to pH 6.8