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A lectin, Crenomytilus grayanus (CGL), was purified from sea mussel C. grayanus by affinity chromatography on acid-treated Sepharose 6B and following gel filtration on Sephacryl S-200. Molecular weight of the lectin obtained was determined by SDS-PAGE to be 18,000, independent of the presence or absence of beta-mercaptoethanol. CGL was found to agglutinate all types of the human erythrocytes together with mouse and rabbit. In hemagglutination inhibition assays, N-acetyl-D-galactosamine, D-galactose, and D-talose were the most potent inhibitors among the monosaccharides tested. Out of the oligosaccharides containing nonreducing terminal D-galactose, melibiose, and raffinose were found to be strong inhibitors. On the other hand, among the glycoproteins, asialo-BSM was the best inhibitor. The hemagglutinating activity of CGL was independent of the divalent cations Ca2+ and Mg2+. Significant CGL activity was observed between pH 8-10.

Immunochemical and physico-chemical properties of two oncofetal antigens - carcinoembryonic antigen (CEA) and pregnancy-specific P,-glycoprotein (SP-1, PSPG) have been studied. The spatial organization of the protein portion of SP-I and CEA preparations, as revealed by spectroscopic evidence, has been discussed with respect to their antigenic activity. We found that the SP-1 and CEA protein portion consists mainly of a P-structural type. The antigenic determinants of both preparations were shown to be topographic. It seems, that the orientation of the CEA carbohydrate chains relatively to the protein core plays an important role in the formation of the antigenic sites. SP-1 preparations were found to be capable of splitting p- nitrophenylphosphate, y(32P)-ATP and 4-nitrophenyl-5'-TMP. SP-1 was also shown to effectively hydrolyse (3H)-polyuridilic acid and heatdenatuted r3H)-DNA of embryos of marine urchin S. inrermedius. Enzymic hydrolysis proceeds from 3' end of the DNA chain by exonuclease's mechanism. Peculiar glycoproteins, termed as oncoprecipitins have been found in the extracts of various marine invertebrates. The following oncoprecipitins have been isolated and characterized: crustacin from hermit crab Pagurus prideauxii, cyprein from cowrie Cyprea capufserpentis recognizing CEA and oncoprecipitin A from ascidian Didemnum fernatum interacting with SP-1. The interaction pattern of CEA and its oncoprecipitins is similar to that of CEA and its antibody. The possibility of application of CEA oncoprecipitins for serologic tumour diagnosis has been demonstrated. The interaction between oncoprecipitin A and SP-1 differs from that of SP-1 with its antibodies. The effect, produced by oncoprecipitin A on HeLa-M and A431 tumour cells causes the essential alteration of their cytoskeleton. The incubation of HeLa cells with oncoprecipitin A, followed by SP-1 leads to a recurrent state of their cytoskeleton. The possible role of oncoprecipitins as substances able to monitor the appearance and elimination of neoplastic cells of invertebrates will be discussed.

Cell localization of mucin type receptors in low-differentiated human colon adenocarcinoma was studied. Several types of receptor localization were identified. It was shown that lectin CGL most intensively binds to intracellular membranes, cytoplasm, and secrete of mucin-producing tumor cells.

The possibility was examined whether weak external electromagnetic radiation can affect the parameters of enzymic reactions. It was found that, in a system involving an aqueous solution of concanavalin A, glucose, and erbium salt exposed to helium-neon laser light of a wavelength of 633 nm, epimers of glucose: allosa, mannose, and galactose are formed. The effect observed was found to represent a photoinduced enzymic reaction in which the protein dissolved in water converts the glucose associated with it into epimers by the action of electromagnetic radiation. The photoepimerase activity of concanavalin A was established for the first time.

The effect of solution ionic strength, calcium ion concentration, and temperature on spatial structure of cyprein was examined by CD, UV, and fluorescence spectroscopy. The secondary structure of the cyprein molecule was calculated from CD spectra, and the prevalence of the beta-structure (85%) was shown. An irreversible conformational transition in the range 55-60 degrees C was found, which reduces the binding activity of cyprein in interaction with carcinoembryonic antigen (CEA) and anti-cyprein antibodies. In the latter case, the binding activity was reversibly restored. Cyprein was shown to be a calcium-binding protein. Binding of calcium by cyprein and increasing the ionic strength of solution affect only tertiary structure of the protein. At an ionic strength of solution close to physiological conditions, calcium-bound cyprein shows maximum binding to CEA and anti-cyprein antibodies. It was shown by difference UV spectroscopy that cyprein does not interact specifically with the monosaccharides of the carbohydrate chains of CEA: fucose, mannose, galactose, and N-acetylglucosamine.

A molecular form of CEA non-binding Con A (CEAnC) was isolated from colon adenocarcinoma metastases in liver as a fraction of CEA having no affinity to Con-A-Sepharose. CEAnC was shown to be immunochemically identical to CEA, but to differ substantially with regard to the amino acid and sugar composition, and structure of the sugar moiety, possibly containing non only N-, but also O-glycosyl carbohydrate chains. The antigens studied were also found to possess different spatial structures. The differences between CEA and CEAnC suggest CEAnC to be a new molecular form of CEA.

The effects of temperature and pH on the spatial structure of carcinoembryonic antigen (CEA) and its various derivatives are studied by circular dichroic spectroscopy and differential UV spectroscopy. The spatial organization of the protein portion of CEA and its derivatives as revealed by spectroscopic evidence is discussed with respect to the antigenic activity of the species studied. We conclude that the protein portion of CEA consists mainly of a beta-structural type. Using the various modifications of the sugar moiety and the protein portion, antigenic determinants of CEA are shown to possess the main conformational character of the molecule while participation of the sugars in the formation and orientation of the carbohydrate chains relative to the protein core of the antigen appear to be important.

Oncoprecipitins isolated from a number of marine invertebrates have been shown to be glycoproteins possessing a higher specificity to carcinoembryonic antigen (CEA)--similar to the interaction of antigen-antibody. The oncoprecipitin-CEA interaction was found to be substantially more specific than that of carbohydrate-containing polymers with lectins. Two oncoprecipitins, crustacin and cyprein, isolated from Pagurus prideauxii and Cyprea caputserpentis, respectively, have been characterized. The binding reaction of CEA with crustacin and cyprein failed to be inhibited with glycoproteins having sugar chain patterns closely related to those of CEA. Inhibition of the oncoprecipitin-CEA interaction with sugars was shown to be nonspecific and was observed only at sufficiently high concentrations of sugars used as inhibitors. Immunoenzymic assay of CEA content in normal and tumor tissues using CEA crustacin and CEA antiserum against CEA test systems revealed highly consistent results for both systems.

Using immunoperoxidase technique, antibodies to CEA (Ab) were compared to a glycoprotein krustacin (Kr) extracted from Pagurus prideauxii, which has an ability to precipitate specifically CEA. It was found that Kr and Ab reacted in a similar manner with embryonic and normal gastrointestinal tissues, revealing practically identical localization of the antigen in the tissues and cells. It was possible, however, to note some quantitative and qualitative differences in the distribution of antigen, which showed that Kr and Ab reacted with different determinants of the CEA molecule.

It has been shown by the methylation method that pelvecyan and sargassan are polysaccharides of similar structures with a high degree of branching of the carbohydrate chains. Fucose, xylose, and galactose are located at the nonreducing ends of their molecules, and mannose and galactose form points of branching of the carbohydrate chains of these polysaccharides.

Human embryonic prealbumin (EPA) was purified from pooled fetal sera by a combination of the salt precipitation methods, ion-exchange chromatography and electro-isofocusing. Purified EPA preparations had about 80% peptide and 20% carbohydrate including mannose, fucose, glucose, galactose, glucosamine and galactosamine. Amino acid composition of EPA may be characterized by high concentration of aspragin. glutamine acids and proline but very low concentration of methionine. By immunodiffusion in agar, EPA was found in all fetal sera and in some malignant tissue homogenates including carcinomas of ovary, breast, stomach, lung, kidney, and intestinal tract.

The spatial structure features of intact and deglycosylated carcino-embryonic antigen (CEA) have been studied by circular dichroism. Raman and UV-spectroscopy methods in order to elucidate a pattern and localization of CEA immunodominants. The temperature-induced changes in the spatial structure of the protein moiety were compared with data on the CEA immunochemical activity estimated by EIA procedure. A conformational transition was found within the 55-75 degrees range that produced irreversible alterations in the tertiary structure, while the secondary structure could be restored after lowering the temperature to 20 degrees C. Spectral studies of intact and deglycosylated CEA demonstrated that immunochemical activity, at least partly, was associated with the tertiary structure of the CEA protein portion.

Temperature-, ionic strength-, calcium ion- and pH-dependence of spatial structure of crustacin have been studied using CD and fluorescent spectroscopy. Secondary structure of crustacin was estimated by CD spectra. An irreversible conformational transition of crustacin's protein moiety connected with the loss of CEA-binding activity has been found at ca. 50 degrees C. Crustacin is shown to be calcium-binding protein, stability of the native crustacin conformation being markedly enhanced by calcium ions (1 mM Ca2+ shifted up the transition temperature by approximately 10 degrees C). Calcium binding and ionic strength increase led to alteration of both secondary and tertiary structures of crustacin. The highest CEA-binding activity was observed for the calcium-bond form of crustacin. A lack of specific interaction of crustacin with some saccharides was shown. Interrelation between conformation and immunochemical activity of crustacin is discussed.

Antigenic determinants of carcino-embryonic antigen (CEA) were spatially located using N-bromosuccinimide modification of tryptophan residues both in native (acetate buffer solution) and unfolded (guanidinium chloride solution) molecule of the antigen. Modification of exposed tryptophan residues failed to alter CEA antigenic activity and conformation of its protein portion as shown by CD spectroscopy. On the contrary, modification of buried tryptophan residues induced conformational changes of CEA protein portion connected with a considerable loss of its antigenic activity. It was shown that CEA antigenic activity depends on spatial structure of its protein moiety.

The majority of topographic antigenic determinants of the carcinoembryonic antigen (CEA) representing a glycoprotein were shown to contain sugar residues of the CEA carbohydrate chains. Periodate oxidation of CEA followed by reduction afforded a corresponding CEA derivate (CEA-POR), which retained 3% antigenic activity of CEA. In secondary and tertiary structures CEA-POR was proved to be close to CEA pre heated to 80 degrees C. Formation of borate complexes with sugar residues of CEA decreased the CEA antigenic activity to 5% while a but did not affect the spatial structure of its protein core.

Crustacin from Pagurus prideauxii and cyprein from Cyprea caputserpentis have been shown to be glycoproteins with molecular masses 36 +/- 1 and 44 +/- 1.4 kDa, containing 3 and 18% of carbohydrates, respectively. Their amino acid and monosaccharide composition have been determined. Crustacin or cyprein interact with CEA more specifically than carbohydrate-containing polymers with lectins. Carbohydrates unspecifically inhibit this interaction at rather high concentrations. Association constants of CEA-crustacin and CEA-cyprein complexes are 0.6.10(8) M-1, respectively. Besides, ELISA showed that antibodies against CEA bind antibodies to crustacin and cyprein.

Carcinoembryonic antigen (CEA) isolated from metastases of colon adenocarcinoma was subjected to deglycosylation with liquid hydrogen fluoride. The protein fraction obtained (PF CEA) was used for to prepare monospecific antiserum to PF CEA. Comparative studies of CEA, PF CEA, and non-specific cross-reacting antigen (NCA-1) have been carried out using monospecific antisera. Circular dichroism spectra of CEA and PF CEA have been studied. The data obtained suggest that some immunodominant regions of CEA are topographic, and their formation needs a specific conformation of the macromolecule, which is stabilized by the sugar moiety.

58 human tumors have been studied immunohistologically with the aid of CEA-specific precipitin--crustacin (Cr). The results were in general similar to those which were achieved with anti-CEA-antibodies. But there were some differences. The reaction with Cr in the cytoplasm was mostly diffuse, while the reaction with Ab was localized usually in the peripheral parts of the cytoplasm. The reaction with Cr has a more restricted character and is more expressed in ovarian tumors and less expressed in gastric and colonic tumors than the reaction with Ab.