New crystalline derivatives of bovine liver rhodanese

Mitochondrial bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase) has been crystallized in the form deprived of the transferable sulfur. The essential condition for crystallization was the removal of oxygen. Crystals of the sulfur-free enzyme are isomorphous with the previously characterized crystals of the sulfur-substituted enzyme. The new crystal species can react with either thiosulfate or selenosulfate to form the catalytic intermediate and, subsequently, with cyanide to form the corresponding product. Furthermore, the enzyme active site can be alkylated by iodoacetic acid.     

Studies on stimulus-response coupling in human neutrophils. II. Relationships between the effects of changes of external ionic composition on the properties of N-formylmethionylleucylphenylalanine receptors and on the respiratory and secretory responses

Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na+-free, high-K+ buffered solution. The results demonstrate that: (a) the variation of Na+ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na+-free, high-K+ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na+ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.     

Characterization of ouabain-resistant mutants of a canine kidney cell line, MDCK

Madin-Darby canine kidney (MDCK) cells were mutagenized and variants resistant to 10, 160, and 2000 times the ouabain lethal dose for wild type cells selected. The phenotypes were stable in the absence of selection. The frequencies with which variants were recovered were consistent with genetic alterations being responsible for drug resistance. It was shown that 50% of the (Na+, K+)-ATPase activity present in mutant cells had a higher Kd for ouabain than normal while 50% remained wild type for ouabain binding. Wild type MDCK cells were measured to have 2 X 10(6) ouabain binding sites per cell with a Kd for the drug of 0.6-1.0 X 10(-7) M. The novel (Na+, K+)-ATPase activities in the mutants demonstrated Kd values for ouabain of 10(-5) M, 3 X 10(-4) M, or 3 X 10(-3) M for the different mutant classes tested. The rate of synthesis of the (Na+, K+)-ATPase as well as the total amount of enzyme per unit of cell protein was unaltered in the mutants. Comparison of the alpha subunit of the enzyme, known to contain the ouabain-binding site, by sodium dodecyl sulfate-gel electrophoresis did not reveal any difference in the size of this subunit in mutant versus wild type cells.     

"In vitro" effect of CCl4, PG and EDTA on GSH levels at different time of incubation of rat liver homogenates

During CCl4-induced lipid peroxidation GSH content in total homogenate from rat liver falls very rapidly in the first 30 min. of incubation "in vitro". CCl4 does not enhance the decrease in total glutathione (TG) during the incubation time, so GSH loss is mainly due to its oxidation to GSSG. On the contrary PG and EDTA, two substances decreasing lipid peroxidation rate, are able to decrease GSH oxidation, without affecting TG content. At 25 degrees C EDTA and PG completely prevent GSH decrease at pH 7.4, while at pH 6 PG affords only a partial prevention. At 37 degrees C both compounds are able to limit GSH decrease at a large extent. Lipid peroxidation seems to have a great importance in the kinetics of GSH decrease and GSSG formation, at least "in vitro". It is noteworthy that PG which inhibits lipid peroxidation stimulated by CCl4 is also able to limit the high GSH loss observed in the homogenates incubated in the presence of halogeno-alkane.     

Acid phosphatase from maize scutellum: Negative cooperativity suppression by glucose

Acid phosphatase purified from maize scutellum, upon acylation with succinic anhydride, still shows negative co-operativity for the hydrolysis of glucose-6-phosphate at pH 5.4. This phenomenon is abolished by glucose, for both native and succinylated enzymes, through stimulation of the initial velocities at sub-optimal substrate concentrations. However, negative co-operativity for the enzymatic hydrolysis of p-nitrophenylphosphate at pH 5.4 is suppressed only at high concentrations of glucose. Furthermore, the hydrolysis of p-nitrophenylphosphate is noncompetitively inhibited (low affinity form of the enzyme molecule) by glucose, which suggests the existence of different substrate binding sites.     

Role of accessory cells in the induction of a secondary cytotoxic response to Moloney murine sarcoma virus-induced tumors

The role of Ia-positive accessory cells in the generation of a secondary cytotoxic response to tumor-associated antigens induced by Moloney murine sarcoma virus (M-MSV) was evaluated. Spleen cells from M-MSV-immune A.TL mice, depleted of accessory cells by anti-Iak serum plus C treatment and stimulated in secondary mixed leukocyte tumor cell culture (MLTC) with syngeneic Ia-negative A6ATL Moloney leukemic cells, failed to generate virus-specific cytotoxic T lymphocytes (CTL). CTL generation in Ia-depleted MLTC may be reconstituted by the addition of nonimmune Ia-positive spleen or peritoneal cells obtained not only from syngeneic A.TL but also from I-incompatible A.TH mice. This lack of restriction observed in accessory cell function is explained in terms of a nonspecific mechanism of CTL triggering mediated by soluble factors. In fact, IL 2 as well as supernatants obtained from I region-incompatible cultures consisting of M-MSV-immune, Ia-depleted A.TL spleen cells and A.TH Ia-positive cells, reconstituted secondary virus-specific CTL generation.     

Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.     

Purification and properties of two lectins from the latex of the euphorbiaceous plants Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge).

1. From the latex of two members of the plant family Euphorbiaceae, Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge), two lectins were purified by affinity chromatography on acid-treated Sepharose 6B followed by elution with D-galactose. 2. The lectin from E. characias is a single molecular species with Mr 80 000, made up of two identical subunits with Mr 40 000, and is a glycoprotein containing 11% carbohydrate. 3. The lectin from H. creptians appears as a mixture of three isolectins with Mr 140 000, consisting of four different subunits with Mr values 37 500, 35 500, 31 000, and 29 000. 4. Both lectins have haemagglutinating activity, with no specificity for human blood groups. The haemagglutinating activity is inhibited by D-galactose and by galactose-containing oligosaccharides. 5. The lectin from H. crepitans is mitogenic to human T-, but not to B-, lymphocytes. The latex of E. characias is mitogenic to T- and, to a lesser extent, to B-, lymphocytes, but the purified E. characias lectin has no mitogenic activity. 6. The lectin from H. crepitans, but not that from E. characias, inhibits protein synthesis by a rabbit reticulocyte lysate.