Purification and Properties of a Thermo-labile Antigen from Baker’s Yeast Cells

A thermo-labile antigen (TLA) on the yeast cell surface was isolated from a yeast cell autolyzate and purified to a homogeneous state by chromatography on an immunoadsorbent affinity column. The molecular weight of TLA was about 1.45 x 10⁵ on SDS-polyacrylamide gel electrophoresis and about 1.5 x l0⁵ on gel chromatography on Sephadex G-200. The TLA contained 74.5% protein and 25.5% sugar. It was characterized by high contents of glycine, glutamic acid, serine and aspartic acid. Half-cystine, methionine, histidine and arginine were not found. The sugar moiety was composed of galactose, mannose, N-acetylglucosamine and fucose. The antigenic determinant of TLA was distinct from that of cell wall mannan in the Ouchterlony immunodiffusion test. No precipitin line against anti-TLA serum was observed, when TLA was heated at 90°C for 10 min. Oxidation with periodate had little effect on antigenicity, but digestion with Pronase or treatment with protein denaturants resulted in loss of the antigenicity. These results suggest that the protein moiety plays an important role as the antigenic determinant of TLA. Moreover, the antiserum specific to TLA agglutinated fresh yeast cells, and the distribution of TLA was apparent on the yeast cell surface by immunofluorescence staining. These findings suggest that TLA molecules were exposed on the outer surface of the yeast cell wall.     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

Limited trypsin cleavage distinguishes MHC and thymus-leukemia antigens.

A simple technique is presented for the identification of particular cell membrane antigens. The method employs labeled membrane antigens that are isolated immunospecifically and subjected to limited trypsin digestion followed by polyacrylamide gel electrophoresis in detergent. A large "core" peptide is produced by proteolysis of murine thymus-leukemia antigens (TLA) and from antigens of the major histocompatibility complex (MHC). The tryptic cores from H-2K and H-2D are regularly distinguishable from the thymus-leukemia antigens (TLA) by gel electrophoresis in one dimension. This chemical distinction is particularly important in the analysis of antigen mixtures isolated with antisera specific for beta 2 microglobulin. These techniques have been used to identify thymus-restricted beta 2 microglobulin-associated antigens on cell membranes from mouse, man, guinea pig, and monkey. In appropriate inbred mouse strains, these are the TLA and it is proposed that in the three other species examined they may be analogues, although not necessarily homologues, of TLA. The broad species distribution of these thymus-restricted cell membrane antigens suggests that they are involved in the differentiation of thymus-dependent lymphocytes (T cells).     

Identification of immunologically distinct antigens in pseudorabies virus

Antigenic proteins of pseudorabies viruses (PrV) are poorly understood. Proteins from purified PrV and membrane proteins from these viral infected cells, therefore, have been studied by antigenic analysis, using virus neutralization and agargel immunoelectrophoresis tests and by polyacrylamide gel electrophoresis, respectively. The study of crossed immunoelectrophoresis against specific antiviral serum antibodies revealed four immunologically distinct antigens involved in PrV. According to their electromobilities, these four immunologically distinct antigens were designated as Ag 1, Ag 2, Ag 3 and Ag 4. The study of dodecyl sulfate-polyacrylamine gel electrophoresis of a membrane-bound but detergent solubilized viral antigenic complex from PrV infected cells also demonstrated the involvement of four glycoprotein antigens. By interpolations of relative mobilities between known protein markers, the molecular weights of these four glycoproteins were estimated to be 61,500, 68,000, 75,000, and 88,000. Results from two dimentional immunoelectrophoresis seemed to be concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis. This report, therefore presents results, which strongly suggest antigenic similarities in the virion of PrV and cellular membrane glycoproteins of cells infected by this agent. The molecular weight of these four immunologically distinct antigens, Ag 1, Ag 2, Ag 3 and Ag 4, are presumed to have the following molecular weights of 88,000, 75,000, 68,000 and 61,500, respectively.     

Studies on the rabbit proacrosin-acrosin system

A single molecular form (Mr = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 117;21-11728) was initially converted by autoactivation into an acrosin (Mr = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities (Rm = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands (Rm values of 0.395-0.412 and 0.497-0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the Mr 34,000 form of acrosin. This property was not shown by Mr 68,000 acrosin. Initial acrosin (Mr = 68,000) was activated by divalent cations such as Ca2+ and Mg2+. The enzyme was inhibited by Zn2+, Fe2+, Hg2+, and sulfhydryl blockers such as 5,5'-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn2+ is involved in acrosomal stabilization. The initial rabbit acrosin (Mr = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.     

Hemocyanins in spiders, IV[1]. Subunit heterogeneity of Eurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains.

The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.     

Purification and Some Properties of Exo-type Fucoidanases from Vibrio sp. N-5

Three fucoidanases were purified from Vibrio sp. N-5 by ammonium sulfate fractionation and chromatography with DEAE-Toyopearl 650 M, Sephacryl S-300 HR, and chromatofocusing. The purified enzymes gave a single band on disc polyacrylamide gel electrophoresis. The molecular weights of the enzymes, E-1, E-2, and E-3 were 39,500, 68,000, and 68,000, respectively, by SDS polyacrylamide gel electrophoresis and 158,000, 68,500, and 67,500 by gel filtration. The enzymes hydrolyzed gagome-fucoidan to give small oligosaccharides containing sulfate as main product.     

Isolation and partial charcterization of a cytochrome b complex and cytochrome oxidase from yeast mitochondria.

A cytochrome b complex and cytochrome oxidase have been purified 14- and 20-fold respectively from yeast submitochondrial particles by a simple procedure involving their spontaneous precipitation from a deoxycholate extract. The recovery of both proteins was almost quantitative. The specific heme contents were 11 and 8 nmoles/mg protein for the cytochrome b complex and cytochrome oxidase respectively and both were spectrally pure. Sodium dodecyl sulfate gel electrophoresis resolved the cytochrome b complex into seven distinct subunits with molecular weights 42,000, 33,000, 27,500, 23,000, 15,500, 13,000 and 10,500. Cytochrome oxidase contained five bands with molecular weights 42,000, 26,500, 21,000, 14,000 and 10,500.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Cell surface proteins of encapsulated Streptococcus cremoris: identification and immunochemical characterization

Cell surface proteins of two slime-forming, encapsulated Streptococcus cremoris strains, MLS96 and T₅ from the fermented milk product viili, were extracted with the non-ionic detergent Triton X-100. The isolated protein antigens were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immuno-blotting with antisera produced against whole Strep. cremoris cells. When protein profiles of these strains were compared, seven prominent polypeptides were found common to both and were recognized by both antisera. Five of these polypeptides with molecular weights of 70000, 54000, 50000, 47000 and 40000 were identified as cell wall components. The remaining two polypeptides with molecular weights of 42000 and 26000 are being studied further in connection with slime formation for which modified Triton X-100 extraction provides a suitable method for isolation of the surface-associated antigens of lactic streptococci.     

Two-dimensinal gel electrophoresis of rat liver nuclear washes,nuclear matrix,and hnRNA proteins

The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis. The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction. In the nuclear washes and chromatin, we observed five classes of proteins: (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; (c) proteins of 94,000, 25,000, and 20,500 mol wt specific to the nuclear washes; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix; and (e) two proteins of 68,000 mol wt present only in the final chromatin. The major 65,000- 75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the second major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA- bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNp particles and vary in the degree to which they share different intracellular compartments.     

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.Abbreviations GTase glucosyltransferase - FTase fructosyltransferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PAG-IEF polyacrylamide gel-isoelectric focussing - PAS periodic acid-Schiff     

Preparation of h and m antigens ofHistoplasma capsulatum free of heterologous antigens

Salt gradient elution of crude histoplasmin on CM-Sepharose CL6B at pH 3.0 was used in essentially a one-step procedure to isolate the h, m, and non-m antigens ofHistoplasma capsulatum and free them of any c antigen common to other pathogenic fungi. The h antigen fraction was pure; the isolated m and non-m antigens contained less than 0.1% of the c antigen present in the original preparation. The residual c antigen in these fractions was removed by affinity chromatography on Affigel-10 coupled to aBlastomyces dermatitidis fungal antiserum. The final preparations of h, m, and non-m were pure when tested by immunodiffusion (ID), sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), or electrofocusing. The antigens were serologically stable at neutral pH for at least three months at 10°C. The relative molecular weights, isoelectric points, and amino acid composition of the antigens are described.     

Human alpha-1-antichymotrypsin: purification and properties.

Human alpha-1-antichymotrypsin has been purified to homogeneity by the following sequential steps--(a) ammonium sulfate fractionation; (b) chromatography on Cibacron Blue Sepharose at pH 7.0; and (c) chromatography on SP-Sephadex C-50 at pH 5.5. The inhibitor has a molecular weight near 68,000 and contains approximately 26% carbohydrate alpha-1-Antichymotrypsin has an amino-terminal arginine and a carboxy-terminal glycine. It also has some homology with alpha-1-PI based on amino-terminal sequence analysis of both proteins. Complexes of alpha-1-antichymotrypsin with human chymotrypsin and human leukocyte cathepsin G are stable in sodium dodecyl sulfate and have molecular weights near 90,000 suggesting 1:1 complex formation on a molar basis between inhibitor and enzyme.     

A novel glycoprotein that binds to hyaluronic acid

Hyaluronic acid binding protein (HBP) has been purified to homogeneity from normal rat brain by using Hyaluronate-Sepharose affinity chromatography. It appears as a single band in non-dissociating gel electrophoresis. The molecular weight of native protein, as determined by gel filtration is found to be 68,000 daltons, and has a single subunit of molecular weight approximately 13,500 as determined under denaturing conditions in polyacrylamide gel electrophoresis, indicating that this protein is apparently composed of five identical subunits. Amino acid analysis shows the purified HBP to be rich in glycine and glutamic acid content, and is distinct from fibronectin, link proteins, and gelatin binding proteins which are known to bind to hyaluronic acid. This protein is further characterised as sialic acid containing glycoprotein.     

Studies on the Molecular Weights of Esterase and Peroxidase Isozymes of Maize

The molecular weights of esterase and peroxidase isozymes of maize seedlings were directly determined by improved polyacrylamide gradient gel electrophoresis. The different isozyme bands developed in polyacrylamide slab gel electrophoresis (uniform gel) were identified in polyacrylamide gradient gel electrophoresis by means of isozyme variants. The molecular weights of esterase isozymes E1, E2, E3F, E3S, a, b, c, named according to isozyme patterns in uniform gel, are <20000, 35200, 33000, 38500, 29900, 28500, 34000 doltons respectively. The molecular weights of peroxidase isozymes PX4F and PX4S are 131000 and 149000 doltons respectively. According to the band location in uniform gel and in gradient gel, some biochemical properties of the isozyme bands and relationships between the isozyme bands were analyzed. The possible errors in the determination of smaller molecular weight isozymes are discussed.     

Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated.     

Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

Purification and properties of galactokinase from Saccharomyces cerevisiae.

Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully induced for the production of the galactose metabolizing enzymes. The purification was accomplished by:(a) ammonium sulfate fractionation, (b) streptomycin sulfate precipitation. (c) DEAE-cellulose chromatography, (d) hydroxylapatite chromatography, and finally (e) Bio-Gel A-0.5 m gel filtration. The resulting preparation of galactokinase was judged to be at least 95% pure by the following criteria: (a) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) ultracentrifuge analysis, (c) nondissociating polyacrylamide gel electrophoresis, and (d) Bio-Gel A-0.5 m gel filtration. The purified enzyme preparation was used to determine the Km values for the two substrates, galactose and ATP, which were found to be 0.60 and 0.15 mM, respectively. Vmax was also determined and found to be 3.35 mmol/h/mg. This corresponds to a turnover rate of 3350 molecules of galactose phosphorylated/min/enzyme molecule. The effect of pH on the galactokinase-catalyzed phosphorylation of galactose was determined; the results showed the pH optimum of the reaction to be in the range of pH 8.0 to 9.0. The enzyme is highly specific for galactose since galactokinase did not appear to phosphorylate any of the other sugars tested at a rate greater than 0.5% of the rate of galactose phosphorylation. Amino acid analysis was performed on the enzyme preparation and the results were used to calculate the partial specific volume (v) of 0.736. The NH2-terminal sequence was determined for the first 3 residues. The molecular weight and subunit composition were determined by ultracentrifugation and polyacrylamide gel electrophoresis under dissociating and nondissociating conditions. The data obtained indicated that galactokinase is a monomeric protein of molecular weight 58,000.     

Immunodiffusion Analysis of Yaba Poxvirus Structural and Associated Antigens

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.