The two choleragen protein constituents were isolated and characterized. Protein I has a molecular weight of approximately 54 000. It consists of subunits of approximate molecular weight 10 000. Protein I1 with molecular weight of approximately 32000 is cleaved by 2-mercaptoethanol into two fragments, protein 11, (N-terminal Asx, M , = 25000) and protein 11, (N-terminal Ser, M , = 7000).Proteins 111 and 112 could be recombined by oxidation to yield protein 11. Upon treatment of choleragen with 2-mercaptoethanol protein 11, precipitates quantitatively. The remaining protein consisting of proteins I and II,, was quantitatively precipitated by ganglioside GGtetl. Of the separated choleragen subunit proteins, only protein I and not protein I1 complexed specifically with ganglioside GGtetl. The isolated proteins I and I1 were considerably less toxic in the skin test but almost full toxicity was recovered after mixing the two proteins I and 11. Antisera against protein I and protein 11 revealed no immuno-cross reactivity between the two proteins. Both antisera inhibited the biological effects of choleragen in the skin and ileal loop tests. A molecular model for the constitution of choleragen is proposed.The pathogenic effects of cholera bacteria in vivo are caused by the action of a secreted toxin upon the mucosal cells. This cholera exo-entero-toxin, choleragen, induces major irreversible membrane permeability changes including the massive outflow of fluid into the intestine. Choleragen also acts on numerous other cell types by way of stimulation of adenylate cyclase (for review see [3]).The work of van Heyningen and collaborators first identified ganglioside to be the cell receptor for choleragen [l 1. These authors discovered that choleragen complexes very specifically with the major monosialo-ganglioside of brain, ganglioside GGtetl .In an earlier investigation it was shown that both the lipophilic and the hydrophilic moiety of the glycolipid contribute to the strength of the binding [Z]. It was also reported that the precipitation of choleragen with ganglioside GGtetl leads to the formation of an apparently high-molecular-weight aggregate of one of the two protein subunits of the toxin. Theother protein subunit, however, showed a less tight binding to the toxin-ganglioside complex and could be readily dissociated by detergents.The ganglioside complexing ability does not appear to be directly related to the toxic activity of the choleragen in vivo. This is shown by the fact that ganglioside is also bound to choleragenoid, which itself is nontoxic and consists only of one type protein of the original choleragen. Furthermore, gangliosidoides i.e. analogues of the ganglioside GGtetl, display comparable binding properties to choleragen, but in contrast to the natural ganglioside still do not inhibit its toxicity in vivo.In order to study the role of the subunits of choleragen in the binding to ganglioside GGtJ as well as choleragenicity, the two proteins were separated and isolated in pure form.The two choleragen prot...
The stereochemistry of the enzyme mediated chalcone-flavanone isomerisation has been investigated. Cyclisation of 4,2',4'-trihydroxychalcone catalysed by either one of the two chalconeflavanone isomerases isolated from mung bean seedlings (Phaseolus aureus Roxb.) leads to the (-)(2 5)-7,4'-dihydroxyfiavanone.When 4,2',4'-[a-2H]trihydroxychalcone is the substrate, deuterium is located preferentially in the equatorial position at C-3 of the flavanone as was shown by nuclear magnetic resonance measurements. Conversely, when the cyclisation is carried out in 2H20 with the unlabeled chalcone, deuterium is preferentially located in the axial position at C-3 of the flavanone. The mechanistic implications of these results are discussed. In experiments described in this paper the absolute configuration at (3-2 was determined by circular dichroism measurementswith 7,4'-dihydroxyflavanone which had been obtained with two isomerase enzymes from mung bean seedlings (Phaseolus aureus Roxb.) [3]. We also investigated whether the cyclisation to the 7,4'-dihydroxyflavanone represents a formal cis-or trans-addition to the double bond of the chalcone (I). The idea of this experiment is the following. When a chalcone deuterated at the u-position is cyclised stereospecifically to the flavanone without deuterium exchange, deuterium will appear in either the axial or the equatorial position a t C-3 of the flavanone. Conversely, when the reaction is carried out in 2H20 deuterium must appear in the opposite position at C-3 of the flavanone. METHODS Synthesis of 4,2',4'-[a-2H]Trihydroxycha2cone ( I I I )300 mg of 7,4'-[3-2H]bisbenzyloxyflavanone [6] were dissolved in 8 ml of absolute dioxane and stirred with 5°/0 Na02H in 2H,0 for 3 h a t room temperature. At the end of this time thin layer chromatography (on silica gel with benzene-isopropanol-methanol, 96 : 6 : 1, v/v/v) showed 4,4'-bisbenzyloxy-2'-hydroxychalcone (RF = 0.89) to be the only product. After addition of ether to the reaction mixture alkali was removed by washing with 2H,0 and subsequently with H,O. After removal of ether the dried residue was stirred for 2 h at room temperature with 1 g of BBr, and 30 ml of methylene chloride. The solvent and BBr, were removed in wacuo and H,O was added. The reaction product was extracted with ether and after the ether solution had been washed with water to neutrality, it was applied to 16 sheets of Whatman 3MM and chromatographed with 50°/, methanol. The zone with RF = 0.40 was eluted from the paper with methanol and the chalcone recrystallized from methanol-water. [6] were dissolved in a small amount of methanol and treated for 10 min with 2 N NaOH. After acidification with H,SO,, the solution was extracted with
Purification of human pregnancy-specific beta1-glycoprotein (SP1) and antigenically related proteins of sub-human primates (chimpanzee, rhesus monkey, cynomolgus and baboon) was achieved by means of an immunoadsorbent technique. The immunoglobulins of a rabbit antiserum to human SP1 were isolated on DEAE-cellulose and coupled to CNBr-activated Sepharose. This immunoadsorbent was used to bind human SP1, respectively monkey proteins immunochemically related to SP1 from placental extract fractions. After extensive washing the proteins were eluted by an acidic glycine buffer. Contaminating serum proteins could be removed by chromatography on hydroxyapatite columns. With this method it was possible to obtain SP1 and the antigenically related proteins of monkeys in good yield and in highly purified form. The proteins thus isolated from human and sub-human primate placentae were compared in their physicochemical and immunochemical properties. The amino acid and carbohydrate compositions of human SP1 and rhesus SP1 have been determined. In a biological test certain inhibitory effect of human SP1 on the mixed leukozyte culture (MLC) could be demonstrated.
We have found that a maleimidobenzoyl spacer attached to OH-4' of the rhodosamine moiety of rhodosaminylanthracyclinone-type anthracyclines is most suitable for the attachment of these drugs to carriers, providing important advantages: The spacer is selectively and most readily introduced into the rhodosamine moiety of the drugs, is stable enough for proper handling of the derivatives, and can easily be attached to thiol groups of carrier systems such as reduced monoclonal antibodies. The anthracyclines can be liberated from the conjugates by mere hydrolysis, requiring neither hydrolytic enzymes nor acidic pH. Liberation of the drugs can, moreover, be affected by the presence of the appropriate substituents Z on the phenylene ring of the spacer, thus allowing slowed or enhanced liberation of the cytostatically active drug. The corresponding p-maleimidobenzoyl derivatives of beta-rhodomycin I, N,N-dimethyldaunorubicin, and rodorubicin have been attached to thiol groups of the hinge region of reduced monoclonal antibody BW 494/32, directed against a pancreatic cancer associated glycoprotein antigen, resulting in MoAb BW 494/32 conjugates, carrying 4.8-6.8 mol of cytotoxic residues/mol of MoAb. Rodorubicin was similarly attached to MoAb BW 575/931/2, directed against a small cell lung cancer associated antigen and to MoAb BW 431/26, recognizing an epitope detectable on carcinoembryonic antigen. The results provide evidence that the newly developed method of coupling of anthracyclines to the hinge region of monoclonal antibodies may be of broader use.
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