Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.     

Cloning of chromosomal beta-lactamase genes from Yersinia enterocolitica

Two beta-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum beta-lactamase I ('A') was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) beta-lactamase II ('B') was cloned from a strain of the O:5b serotype. The properties of the beta-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.     

Trypsin-SBTI interaction in reverse micelles. A slow intermicellar exchange-dependent binding

Solubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule-containing reverse micelles slows down by a thousand times and the limiting-step in the exchange, the fusion, by 10(6) times. Both reverse micellar size (omega 0 = [water]/[surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given.     

Structure-function analysis of the human integrin VLA-4 (alpha 4/beta 1). Correlation of proteolytic alpha 4 peptides with alpha 4 epitopes and sites of ligand interaction.

The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti-alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.     

Extreme divergence of a novel wheat thionin generated by a mutational burst specifically affecting the mature protein domain of the precursor.

A new type of neutral thionin (type V), specifically expressed in developing wheat endosperm, has been found to be encoded by a set of single-copy genes located in the long arms of chromosomes 1A, 1B and 1D, within less than 10,000 base-pairs of those corresponding to the highly basic type-I thionins. Divergence between types I and V has occurred through a process of accelerated evolution that has affected the amino acid sequence of the mature thionin but not the precursor domains corresponding to the N-terminal signal peptide and the long C-terminal acidic peptide. This process involved a deletion and a non-synonymous nucleotide substitution rate equal to the synonymous rate in the thionin sequence.     

Kinetics of a model of autocatalysis, coupling of a reaction in which the enzyme acts on one of its substrates.

A global kinetic analysis is presented of a model of an enzyme autocatalytic process, to which a reaction is coupled, in which the enzyme acts upon one of its substrates. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we determine the corresponding kinetic equations for several particular cases which are characterized by certain relations between the rate constants. Finally, a kinetic data analysis is proposed for one of these particular cases. It can easily be extended to any of the other cases.     

Glucosylenamines as glycosyl acceptors: synthesis of gentiobiosylenamines.

The preparation of 2,3,4-tri-O-benzyl- (3), 2,3,4-tri-O-acetyl- (4), and 2,3,4-tri-O-benzoyl-N-(2,2-diethoxycarbonylvinyl)-6-O-trityl-beta- D-glucopyranosylamine (5) is described. The reaction of 3-5 with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide yields 2,3,4-tri-O-benzyl- (9), 2,3,4-tri-O-acetyl- (10), and 2,3,4-tri-O-benzoyl-N-(2,2-diethoxycarbonylvinyl)-6-O-(2,3,4,6-tet ra-O- acetyl-beta-D-glucopyranosyl)-beta-D-glucopyranosylamine (11), respectively. 2,3,4-Tri-O-benzyl- (6), 2,3,4-tri-O-acetyl- (7), and 2,3,4-tri-O- benzoyl-N-(2,2-diethoxycarbonylvinyl)-beta-D-glucopyranosylamine (8) are also described.     

Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.

The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.     

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.