Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Association-dissociation of the flavoprotein hog kidney D-amino acid oxidase. Determination of the monomer-dimer equilibrium constant and the energetics of subunit association.

The enzyme concentration dependence of spectrophotometric titrations of hog kidney D-amino acid oxidase [EC 1.4.3.3] with p-aminobenzoate was studied. The monomer-dimer equilibrium constant of the oxidized holoenzyme at 25 degrees C was estimated to be 7 X 10(5)M-1 at pH 7.5 and 4X 10(6)M-1 at pH 8.3. The energetics of subunit association are discussed.     

Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer

Tryptase was shown to be stabilized as an enzymatically active tetramer by association with heparin and dissociated to inactive monomers in the absence of heparin at 37 degrees C in physiologic buffer and in plasma. There was a 50% loss of tryptase activity at 37 degrees C by 6-8 min in both physiologic buffer and plasma. When heparin glycosaminoglycan was present, tryptase retained nearly full activity for 2 h in buffer and in plasma. Tryptase activity also decayed under standard assay conditions in the presence of synthetic ester and peptide substrates unless bound to heparin. That tryptase is bound to heparin at the pH and physiologic NaCl concentrations employed was shown by chromatography of tryptase on heparin-agarose, gel filtration, and velocity sedimentation. Elution of tryptase from heparin-agarose occurred at 0.8 M NaCl. Maximal stabilization of tryptase by heparin occurred at a weight ratio to tryptase that was equal to or greater than unity. Kcat/Km ratios for tryptase-heparin at 0.15 M NaCl and 37 degrees C were 0.9 X 10(6) s-1 M-1 for tosyl-L-Gly-Pro-Lys-p-nitroanilide and 1.7 X 10(6) s-1 M-1 for p-tosyl-L-arginine methyl ester and are among the highest reported for tryptic enzymes. The mechanism of heparin-dependent stabilization of tryptase was not due to indirect ion binding properties of heparin and was analyzed by Superose 12 high performance liquid chromatography. Active enzyme eluted with an apparent Mr of 132,000 +/- 10,000 (n = 3, +/- S.D.), whereas tryptase inactivated by incubation without heparin eluted with an apparent Mr of 34,000. The tetrameric structure of diisopropyl fluorophosphate-inhibited tryptase was also preserved after incubation with heparin at 37 degrees C but was reduced to monomeric subunits after incubation without heparin. That no appreciable degradation of tryptase occurs under conditions that cause dissociation of subunits was directly shown by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Two different subunits of 34,000 and 33,000 Mr (after reduction) present in the intact enzyme (calculated to be 134,000 Mr) were also detected unchanged after inactivation of tryptase by dissociation of its subunits. Thus, the selective localization and association of heparin and tryptase in the human mast cell secretory granule most likely plays a major role in the regulation of tryptase after secretion.     

Selective cytotoxic effect of an immunotoxin on human erythroid tumor cells]

The possibility of efficient directed elimination of human erythroblastoid cells by the conjugate of IgM-monoclonal antibody HAE9 directed against the erythroblast antigen and the A-chain of a plant toxin ricin has been demonstrated. The conjugate contained 2 molecules of A-chain per one antibody molecule. The efficiencies of the cytotoxic effect of native ricin and the conjugate were compared according to the number of binding sites on the surface of K562 cells as well as to the internalization rate of these molecules. As was shown, that the number of binding sites for the antibody approaches 2.7.10(4) molecules/cell, K a being equal to 1.7.10(8) M-1 while for ricin these indices constitute 2.4.10(5) and 4.6.10(8) M-1. Almost 100% of antibodies and 36% of ricin are internalized within 10 min at 37 degrees C. At a concentration 10(-11) of native ricin and 10(-10) of immunotoxin the 50% inhibition of growth of K562 cells carrying the erythroblast antigen on their surface is observed.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme substituted cytochrome b5

The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association kinetics and binding constants of nucleoside triphosphates with G-actin.

The dissociation of the complex between 1:N6-ethenoadenosine, 5'-triphosphate (xiATP) and G-actin was initiated by dilution to concentrations between 1 micronM and 5 nM and monitored by the fluorescence change of xiATP. The results were quantitatively explained by a two-step mechanism: a reversible dissociation of the actin-nucleotide complex followed by a fast irreversible inactivation of nucleotide-free G-actin. Under normal conditions (0.8 mM CaCl2, pH 8.2,21 degrees C), the rate-limiting step was the dissociation of the nucleotide-G-actin complex. The half-time of the dissociation of xiATP from G-actin was 290 s as compared to only 13 s for the following denaturation step of nucleotide-free actin. 1 mM EDTA highly accelerated the dissociation step and, regardless of its concentration, the complex dissociated quantitatively within 1 min. Addition of Ca2+ within 20 s after EDTA addition induced a re-association of xiATP with nucleotide-free but still native G-actin. This reversal was kinetically resolved by means of a multimixing stopped-flow apparatus. The association rate constant was 6 X 10(6) M-1s-1. From the association and dissociation rate constant, a value of 2.5 X (10(9) M-1 was calculated for the binding constant of xiATP to G-actin. The binding constant of ATP (1.4 X 10(10) M-1) was derived from the relative binding constant of xiATP and ATP as determined by fluorescence titration of xiATP-G-actin with ATP. These binding constants are 10(3)-10(4) times higher than values reported earlier on the basis of more indirect data.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20 degrees C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 microM CaCl2, metal-deprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a second-order kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5 X 10(7) M-1 X s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7,500 M-1 X s-1 and 5-6 s-1, respectively. For PA-2, the rate constants for Ca2+ are 7 X 10(6) M-1 X s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3,500 M-1 X s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10 degrees C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.     

Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.     

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.     

Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.     

Codon-induced association of the isolated anticodon loop of tRNAPhe

The binding of the codon UUC to the isolated anticodon loop of tRNAPhe (yeast) has been studied as a model of codon recognition by a simple adaptor. Fluorescence titrations demonstrate that UUC binds to the isolated anticodon loop with an equilibrium constant of 1.4 X 10(3) M-1 (at 7.2 degrees C). Equilibrium sedimentation curves reveal that UUC binding induces association of anticodon loops beyond the dimer stage. A set of complete sedimentation curves obtained for various reactant concentrations was analyzed according to a model with an infinite number of subsequent association steps for UUC-anticodon loop complexes and with equal affinity for each step. The coupling of association and sedimentation was considered quantitatively, and the information resulting from conservation of mass was used by integration. According to this procedure, the experimental data can be described by an isodesmic association constant of 8 X 10(3) M-1 with satisfactory accuracy. Temperature-jump relaxation detected by fluorescence measurements provides independent evidence for codon-induced association of the anticodon loop. The data are consistent with the following mechanism: UUC preferentially binds to one of two loop conformations with a rate constant of 4.5 X 10(6) M-1 s-1; the UUC-anticodon loop complex undergoes association with a rate constant of 6.5 X 10(6) M-1 s-1. The reactions observed for the isolated anticodon loop are surprisingly similar to those observed previously for the complete tRNA, suggesting that simple hairpin loops are appropriate adaptors for a translation process at an early stage of evolution; the codon-induced association of the hairpin loop should be very useful to facilitate the transfer of cognate amino acids during translation.     

Binding and cytotoxicity of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and erythrocytes

The binding of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and human erythrocytes was studied in detail. Scatchard plots of binding of 125I-lectins to these cells gave biphasic lines except for HeLa cells at 0 degree C. The association constants of lectins for the three cell types at 37 degrees C were lower than those at 0 degree C. The numbers of total binding sites were estimated to be 7 to 16 X 10(7) per HeLa cell, 3 to 4 X 10(7) per Sarcoma 180 ascites tumor cell and 0.4 to 1 X 10(6) per erythrocyte. A fraction, 16 to 27% of the total amount of cell-bound lectin at 37 degrees C, appeared to be bound irreversibly as judged by non-removal on washing with 0.1 M lactose, whereas no lectin was irreversibly bound at 0 degree C. In the case of erythrocytes, no lectin became irreversibly bound even at 37 degrees C. The toxicity of lectins on HeLa cells and Sarcoma 180 ascites tumor cells was investigated. The toxicity of ricin D was 50 times for Sarcoma 180 ascites tumor cells and 140 times for HeLa cells as much as that for castor bean hemagglutinin. As to the sensitivities of both cell types to these lectins, it became apparent that Sarcoma 180 ascites tumor cells were more susceptible than HeLa cells.     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions

The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.