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A REEXAMINATION OF THE STRUCTURE OF THE

IMMOBILIZATION ANTIGEN FROM PARAMECIUM AURELIA E. STEERSJR. AND R. H. DAVISJR. Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism. and Metabolic Diseases, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 6 April 1976)

Abstract--l. The purification of the immobilizing antigen (i-antigen) of serotype A from strain 51.7 s from Partonecium aurelia has been reexamined. 2. A contaminant protease which is activated by the sulfhydryl reagents 2-mercaptoethanol and dithiothreitol (DDT) was separated from "purified" preparations of the i-antigen by ion-exchange chromatography on DEAE-Sephadex. 3. Under the conditions usually employed to reduce disulfide bridges, this contaminant protease degraded the i-antigen into fragments averaging between 20,000 and 35,000 daltons. 4. Control samples of bovine serum albumin were similarly degraded by this material under identical conditions to those used for the i-antigen. 5. When i-antigen was purified by the procedure described herein, little or no change in molecular weight was observed following reduction with dithiothreitol as demonstrated on Na dodecyl sulfate containing polyacrylamide gels.

held together by disulfide bonds. From sedimentation experiments, Steers (1965) proposed a minimum unit Paramecium aureliu, a single celled protozoan, has of 35,000 daltons for a 310,000 dalton molecule and been shown to undergo immobilization and eventual Jones (1965) a 16,000-80.000 dalton minimum subunit death when incubated in homologous antisera made on the basis of a 250,000 dalton protein. Sommerville against a protein located on the surface of these ani- (1970) used gel filtration on Sephadex G200 and mals. This protein, termed the immobilization antigen reported a mol. wt of approx 40,000 for the i-antigen (i-antigen), is particularly interesting due to the un- following reduction with mercaptoethanol. In a separusual mechanism controlling its expression (Preer, ate approach involving the immunological analysis of 1969). Although animals of a particular strain have heterozygous animals, Finger et al., (1966) reported been shown to be capable of expressing as many as the existence of at least six subunits in the i-antigen 12 different serotypic i-antigens, only one antigenic on the basis of the appearance of heterologous serotype is expressed at any given time by any one animal. typic recombinant i-antigens. The model of the i-antigen quaternary structure Thus, animals which are genotypically identical, may be phenotypically different. The nuclear gene respon- that has emerged from these studies is that of two sible for the expression of a particular i-antigen is (Jones, 1965; Finger et al., 1966) or three (Steers, 1965) under the control of the cytoplasmic state of the ani- identical protomers, each of which is further divided mal, which in turn is influenced by the various en- into three non-identical polypeptide chains. The vironrnental conditions that the animal may experi- entire structure is held together by inter and intra ence. The expression of a particular i-antigen may disulfide bonds. Allen (1967) expressed these nine or be induced in the laboratory by a variety of methods six subunit models by the formula A3B3C3 or such as variation of temperature, nutritional state or A2BzC2. Subsequent studies by Reisner et al. (1969a, 1969b), salinity of the growth medium. While the phenomenon of induced transformation of serotypes is well Reisner & Rowe (1969) failed to confirm that the documented (Preer, 1969), the mechanism responsible i-antigen was composed of subunits. These investifor the activation and/or repression of the various gators concluded that the i-antigenis a single polyspecificity loci is not known. In fact, the function of peptide chain of 300,000 daltons, and as such is the this protein in paramecium is unknown. It is obvious largest known monomeric globular protein, This controversy was resolved recently by Hansma that some knowledge of the i-antigen molecule itself and a comparative analysis of various i-antigens will (1975). The standard procedures used to purify greatly aid in the understanding of the mechanism(s) i-antigen was shown to produce a preparation which involved. contained a sulfhydryl-activated protease. This proPhysico-chemical studies of the i-antigen have sug- tease activity was suggested to be either an inherent gested a large fibrous protein of 250,000-300.000 dal- integral part of the i-antigen or a co-purifying contons, devoid of nonproteinaceous moieties. Reduction taminant. The results reported here confirm the presence of and alkylation experiments by Steers (1965) and Jones (1965) led to the conclusion that the i-antigen was a sulfhydryl-dependent degradative activity in "puria protein molecule composed of multiple subunits fied'" preparations of i-antigen. This degradative acti195 INTRODUCrION

'~it'~ can ~ separated i?om such preparations b~ ionexchange chromatograph>. The i-antigen further purified on DEAE-Scphadcx appears to exist as a single pot~peptide chain of 3(~:~).CX~ daitons as stated b~ Reisner et cd. ~[969ai. MATERIALS A N D Mt'.I-HODS Paramecium a~reli,, Labs lee. Costa Me~t. ( A . lncubat!o:~ eio,:~:edcd for 18 2a hr after which the ,~,,~, v:ere ~ bsc:~.ed :i~4! ~,}',-t,> graphicai!y recorded. Optical density measurements were m~;du "~ith at Zeiss Model PMQ II spectropi'~,~ometer. RESL LI'S

The standard procedures !preer. !~591 used to purify i-antigen, gives a product which appears 'pure" bv various physico