Synthesis by Enzymatic Catalysis VI—An Investigation of Chemical Modification of HRP by Fourier Transform Infra-Red Vibrational Spectroscopy

Horseradish peroxidase was chemically conjugated on its carbohydrate moieties with short aliphatic chains (C₈ and C₁₆). An analytical method using FT.IR spectroscopy was developed to analyze this alteration in enzyme structure. This method is non-destructive, and can be applied directly to samples of the reaction mixture. More general applications of this technique are described and discussed.     

Lipopolysaccharides from thermosensitive mutants of Escherichia coli K12 (author's transl)]

Thermosensitive mutants of Escherichia coli K12 were grown at 30 degrees C and 40 degrees C. The serologic properties and the composition of their lipopolysaccharides were investigated. The inhibition of hemagglutination by the lipopolysaccharides of various mutant strains was tested against the anti-E. coli K12 CR34 system. An inhibition was observed with all the mutants but one, CR34 T83 which had no inhibitory effect. Chemical analysis of lipopolysaccharides and mass spectrometric analysis of their methylated derivatives indicated the presence of the same components in the various lipopolysaccharides: glucose, galactopyranose, galactofuranose, heptopyranose and heptofuranose. However the 2,3,4 tri-O-methyl glucose is missing in the lipopolysaccharide of the mutant T83. This result agrees with the absence of a substituent on the 6-position of the non-reducing core-terminal glucose. The lipopolysaccharide of the T83 mutant has the complete core-type of E. coli K12. The relations of mutations with modifications of the composition of inner and outer envelopes in various mutants are discussed.     

On the control of septation in Escherichia coli.

Mutants of E. coli defective in cell septation (ftsA to ftsG, conditional thermosensitive mutants isolated by Ricard and Hirota) were studied with respect to their membrane protein composition, murein hydrolase activities and rates of synthesis of murein and phospholipids. Three classes of mutants have been distinguished: 1) those affected in both murein and phospholipid synthesis; 2) those affected in either murein or phospholipid synthesis and 3) those affected in neither of these parameters. Overall murein hydrolase activities, after activation, is of the same order in all the mutants screened. In addition to soluble products of murein splitting, we have found insoluble products that appear to be in dynamic equilibrium with the murein of the sacculus. Endogenous levels of cyclic adenosine 3',5'-monophosphate measured after blocking septation showed no variation. This suggests that the cyclic nucleotide is not involved in the metabolic control of septation.     

Specific interaction between VH and VL regions of human monoclonal immunoglobulins

Preferential reassociation of immunoglobulin H and L chains was investigated using a method of competitive hybridization. A model was built in which two monoclonal light chains, one autologous, L_A, and one heterologous, L_B, were competitively reassociated with a given monoclonal heavy chain, H_A. A total of 12 human myeloma proteins were used with known isotypic, allotypic and variability subgroups: 44 distinct combinations were studied by competitive hybridizations. It was found that a preferential reassociation occurred between the complementary H and L chains that were associated in the native molecule in 80% of the cases. It was clearly established that the subgroups had no influence on the preferential reassociations that seem, therefore, to rely exclusively on individual (“idiotypic”) structural differences. It was shown that, although H and L chains had been fully reduced and denatured, the same degree of preferential reassociation was observed after the chains had been reoxidized and refolded. These experiments suggest, therefore, that the observed preferential reassociations are the consequence of an antigen-independent selection process that must have taken place during the differentiation of the antibody-forming cells.     

An amphibian two-domain ‘big’ neurophysin: conformational homology with the mammalian MSEL-neurophysin/copeptin intermediate precursor shown by trypsin-Sepharose proteolysis

A ‘big’ frog (Rana esculenta) neurophysin, encompassing sequences homologous to mammalian MSEL-neurophysin and copeptin, has been passed through a trypsin-Sepharose column in order to compare its conformation with that of the two-domain intermediate precursor isolated from guinea pig. Whereas the polypeptide possesses 8 arginine residues, only two cleavages were observed located in a putative inter-domain sequence (at Arg-94 and Arg-114). Because free vasotocin has been isolated from the frog, it is assumed that pro-vasotocin has a three-domain conformation similar to that of pro-vasopressin but processing in amphibians involves only one step rather than two steps as in mammals.     

Molecular mechanism of beta-adrenergic receptor blockers with intrinsic sympathomimetic activity

beta-Adrenergic receptor (beta AR) blocking agents with intrinsic sympathomimetic activity (ISA) can induce modest increases in beta AR-stimulated activity, such as rate and force of contraction in cardiac tissue. The molecular basis for this activity has been elusive. Previous studies have suggested that these compounds do not stimulate cyclic AMP (cAMP) formation even though activation of adenylate cyclase is the generally accepted mechanism for beta AR promotion of target cell response. In the current studies, we show that several beta AR antagonists with ISA (dichloroisoproterenol, pindolol, and celiprolol) stimulate cAMP accumulation five-, two-, and threefold, respectively, in S49 lymphoma cells, but only if cells are simultaneously incubated with the diterpene forskolin. The KI values observed for inhibition of isoproterenol-stimulated cAMP accumulation or of beta AR [( 125I]iodocyanopindolol) binding for each of the beta blockers with ISA were comparable in magnitude to their respective EC50 values for forskolin-potentiated cAMP accumulation. The forskolin-potentiated responses of these compounds were abolished by the beta AR-antagonist propranolol. These results indicate that the ISA of beta-blocking drugs most likely results from a modest beta AR-mediated stimulation of adenylate cyclase activity. The results further suggest that treatment of target cells with forskolin provides a means to define partial agonism at receptors that are linked to stimulation of adenylate cyclase.