The use of fluorescence spectroscopy for identification of A- and B-thrombin chains during their chromatographic separation

The separation of A- and B-chains of human thrombin has been performed by gel filtration on Sephadex G-100 under the reduction of disulphide bonds with dithiothreitol. Identification of A- and B-chains has been provided by measurements of the fluorescence intensity of fractions at 310 nm and 350 nm which are near the maximum positions of tyrosine and tryptophan fluorescence, respectively. The appearance of A-chain was monitored by an increase of the ratio of Ifl310/Ifl350 greater than 2. The fluorescence spectrum of A-chain has maximum position at 304 nm, which is characteristic of tyrosine fluorescence. The fluorescence spectrum of B-chain has maximum position at 347.5 nm which corresponds to fluorescence of tryptophan residues. The identification of A- and B-chains has been confirmed by the gel electrophoresis data.     

Enzymatic properties of the isolated B-chain of human thrombin

The A- and B-chains have been isolated from the non-covalent complex of human thrombin A- and B-chains, using selective reduction of the interchain disulfide bridge. The B-chain thus isolated (de-A-thrombin) retains its conformation, which is close to the native one and thus differs considerably from the B-chain isolated from the fully reduced enzyme. Nevertheless, the proteolytic (in terms of fibrinogen clotting) and amidase activities of de-A-thrombin are markedly reduced as compared to the native enzyme and the non-covalent complex of A- and B-chains. It is assumed that the A-chain of thrombin is necessary for normal functioning of the active site of thrombin localized in the B-chain.     

Effect of partial reduction of disulfide bonds on the enzymatic activity of thrombin

It was demonstrated that partial reduction of disulfide bonds in thrombin by dithiothreitol in the absence of denaturating agents leads to a decrease of enzymatic activity with respect to fibrinogen coagulation and tosylarginine methyl ester hydrolysis. Polyacrylamide gel electrophoresis and determination of the number of SH-groups liberated in the course of reduction suggest that the observed inactivation is primarily due to the disruption of the S-S-bridge between the A- and B-chains of thrombin.     

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.     

Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.

2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.     

Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.

Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.     

Direct measurement of the equilibrium between glutathione and dithiothreitol by high performance liquid chromatography

The equilibrium constant between reduced glutathione (GSH), oxidized glutathione (GSSG), reduced dithiothreitol (DTTSHSH), and oxidized dithiothreitol (DTTSS) has been directly measured by high performance liquid chromatography analysis of equilibrium mixtures. The equilibrium constant at 25 degrees C for the reaction GSSG + DTTSHSH in equilibrium 2GSH + DTTSS varies from approximately 200 M, below pH 8, to approximately 2800 M, above pH 11. The observed pH dependence is generally consistent with published values of acid dissociation constants of these thiols.     

Dependence of epidermal growth factor activation on its affinity and oligomerization]

Epidermal growth factor receptor (EGF-R) oligomerization has been followed on A-431 cells using covalent labeling by 125I-EGF and EGF-dependent autophosphorylation of receptor-kinase. High molecular weight complexes corresponding to monomeric, dimeric, and trimeric forms of EGF-R are detected. The process of oligomerization occurs effectively at 37 degrees C while at 4 degrees C no oligomer formation is detected. PMA or ATP treatment reduces the number of high-affinity EGF-binding sites but has no influence on dimer formation. Dimerisation of the EGF-R in the absence of the ligand has been established on formalin-fixed A-431 cells.     

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.     

The solubilization of the L and M antigens from sheep red cell membranes

The Lp, L1 and M antigens from sheep red cells were solubilized using the non-ionic detergent Triton X-100 in the presence of dithiothreitol. Recovery rates were improved when membranes were sonicated at 4 degrees C in the presence of the detergent; values in the range 16-25% (M) and 9-17% (Lp and L1) were achieved for recovery.     

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.     

Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.     

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.     

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A-431 cells

The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.     

Temperature-dependent variation in the synthesis of streptococcal group-specific carbohydrate. II. Biosynthetic studies in group A and variant strains.

The biosynthesis of the cell wall polysaccharide and peptidoglycan of group A and A-486-Var streptococci was studied with N-acetyl-[14C]glucosamine, UDP-N-acetyl-[14C]glucosamine, and [14C]glucose. The incorporation of N-acetyl-[14C]-glucosamine into the cell wall four times greater in the A-486-Var cells than in the group A cells. However, the percentage of the total label incorporated into the cell wall polysaccharide at 37 degrees C by the A-486-Var strain was 12%, compared with 66% for the group A cells. When the A-486-Var was grown at 22 degrees C, the proportion of the label incorporated into the cell wall polysaccharide increased to 41%. At 37 degrees C, N-acetyl-[14C]glucosamine was incorporated preferentially into the peptidoglycan of the A-486-Var; almost three times as much of the label was incorporated into the peptidoglycan at 37 degrees C as was incorporated at 22 degrees C. Studies with protoplast membranes of these organisms showed similar differences, with a fourfold greater uptake of UDP-N-acetyl-[14C]glucosamine by the A-486-Var membranes at both incubation temperatures. These studies suggest that a defect in the incorporation of N-acetylglucosamine into the side chain of the polysaccharide is present in the A-486-Var strain at a step following the synthesis of UDP-N-acetylglucosamine. This defect, which may involve the UDP-N-acetylglucosamine transferase, is temperature dependent in the A-486-Var strain.     

Evidence for a novel growth factor in Xenopus oocytes

Xenopus oocytes contain a novel growth factor, as determined by its effect on the anchorage-dependent and -independent growth of various rat cells and on a Xenopus cell line; it is destroyed by trypsin, acidification (pH 2.0), heating (100 degrees C, 3 min), 8 M urea but not by dithiothreitol. Gel filtration of this activity in nondissociating conditions suggests the existence of aggregates and the presence of a very high (approximately 2000 Kd) and a much lower (approximately 30 Kd) form.     

Effect of reduction of the interchain disulfide bridge of human thrombin on its enzymatic activity and structure

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.     

Radioimmunoassay of ricin A- and B-chains applied to samples of ricin A-chain prepared by chromatofocusing and by DEAE Bio-Gel A

A radioimmunoassay for ricin and ricin A- and B-chains was developed. Amounts as low as 100 pg of A-chain and 500 pg of B-chain could easily be quantitated. We showed, however, that the free chains were more reactive in the radioimmunoassay than the equivalent quantity of the individual chains when combined in intact ricin. The usefulness of the assay was demonstrated by determining the concentration of contaminating A- or B-chains in preparations of the separate polypeptides purified by DEAE Bio-Gel A chromatography and by chromatofocusing.