An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis

The cytosol fraction of rat testicular homogenates contained a specific androgen binding protein (t-ABP), indistinguishable from, the androgen binding protean in. the epididymis(e-ABP) in terms of its chromatographic behaviour by gel filtration, sedimentation rate, mobility on polyacrylamide electrophoresis and steroid specificity(5α-diaydrotestosterone(DHT) > testosterone > estradiol-17β > parogesterone and estradiol-17α).The stability of t-ABP as well was similar to that of e-ABP. The aBP-DHT complexes were stable between pH 6.5–10, exhibiting the highest degree of binding between pH 8–9. The binding activity persisted after heating at 50°C for 30 min., whereas heating at 60°C for 30 min. completely destroyed the binding. DHT did not significantly increase the stability towards pH and temperature denaturation. Binding activity was not reduced by 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS), whereas similar treatment with 1 nM N-ethyl-maleimide(NEM) or 1 mM Ellman's reagent reduced it by some 10–50 per cent. Exposure to 10 mM β-mercaptoethanol(βME) reduced the binding by 60–70 per cent. These studies strongly indicate that t-ABP and e-ABP are identical proteins.Hemicastration for 4 weeks eliminated the ABP from the epididymal cytosol fraction on the castrated side, indicating that this protein is produced by the testis.     

Divalent cation binding to reduced and octanoyl acyl-carrier protein

Mn(II) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average Kd/site approximately 80 microM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e.g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average Kd/site approximately 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 1H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.     

Hymenolepis diminuta: Partial characterization of membrane-bound nucleotidase activities (ATPase and 5′-nucleotidase) in the isolated brush border membrane

Adenosine triphosphatase (ATPase; EC 3.6.1.3) and 5′-nucleotidase (5′-NTase; EC 3.1.3.5) activities of the isolated brush border membrane of Hymenolepis diminuta have been studied. The pH optimum for ATPase activity is 7.4, and divalent cations are necessary for maximum activity; no Na⁺-K⁺ activated ATPase is present in the isolated brush border membrane. ATPase activity is inhibited by molybdate and phosphorylated monosaccharides, but not by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), or fluoride. The pH optimum for 5′-NTase activity is 9.6–10.2, and divalent cations are necessary for maximum activity. 5′-NTase activity is inhibited by molybdate at pH 9.6 and 7.4, and activated by NEM and pCMB and pH 9.6 and 7.4, respectively; fluoride has no effect on 5′-NTase activity. Solubilization of the brush border membrane fraction in 1% sodium dodecyl sulfate has no inhibitory action on either enzyme activity.     

Induction and characterization of two types of ATPase on rat liver peroxisomes.

Clofibrate increased oligomycin-resistant ATPase activity in peroxisomes more than 17-fold (5.15 +/- 0.71 milliunits/mg protein) in rat liver. The activity was dependent on divalent cations (Mg2+ > Ca2+) with an optimum pH of 7.5. This activity was partially inhibited by N-ethylmaleimide (NEM), 4,4'-dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), silicotungstic acid (STA), and high concentrations of N,N'-dicyclohexylcarbodiimide (DCCD). Proteinase K digestion of intact peroxisomes severely reduced the NEM-sensitive activity, but little affected the NEM-resistant activity. NEM-sensitive and -resistant ATPases showed Km values for ATP of 780 and 73 microM, respectively. The NEM-sensitive activity was inhibited completely by DIDS, 7-chloro-4- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tributyltin chloride (TBT), and quercetin, and partially by DCCD and STA, whereas the NEM-resistant activity was totally insensitive to these chemicals except for STA. These activities had unique requirements for divalent cations, anions, and substrates, respectively. They were partially separated by gel filtration chromatography and had molecular masses of 520 kDa (NEM-sensitive enzyme) and 450 kDa (NEM-resistant enzyme), respectively.     

The effects of various serine protease inhibitors on estrogen receptor steroid binding

Two serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP), were utilized to investigate the possible involvement of serine hydroxyl groups on 17 beta-estradiol binding to the rat estrogen receptor (ER). Single point saturation analysis and Scatchard analysis demonstrated that both 5 mM PMSF and 5 mM DFP were able to inhibit steroid binding to the ER after incubation at 37 degrees C, but neither were able to inhibit steroid binding of the nonactivated ER (0-4 degrees C). The reducing agent dithiothreitol (DTT) was used to differentiate between the interaction of PMSF with serine groups or with sulfhydryl groups of the receptor. When incubated in the presence of 5 mM PMSF, various concentrations of DTT up to 25 mM were not able to overcome the inhibition of this agent, indicating that there was no interaction of PMSF with sulfhydryl groups. Thus, these findings indicate that serine hydroxyl groups are involved in steroid binding of the rat ER.     

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-beta-(4-dimethylaminophenyl)-17a-hydroxy-17 beta-(3- hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one]). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [3H]ZK98299 with affinity approximately equal to that of [3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0 degrees C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [3H]ZK98299 and [3H]triamcinolone acetonide binding. While [3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide (NEM), [3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 degrees C) and receptor transforming (23 degrees C) conditions, and exhibited a faster rate of dissociation at 23 degrees C when compared with [3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

On the interaction of chromomycin A3 with calf thymus DNA in the presence of metal cations at different pH values

The interactions of the antitumor antibiotics, chromomycin A₃, with a variety of metal cations in the pH range of 3.0–8.5 were systematically studied by CD, absorption, and ¹H-nmr spectroscopies. Results were compared with those obtained in the presence of increasing amounts of calf thymus DNA. The negatively charged chromomycin A₃, pK_a 6.3, forms aggregates that become ordered and smaller in size, in the presence of variety of metal cations. Spectrophotometric titrations have shown that binding of the neutral drug to DNA at pH 4.5 does not require divalent cations, although the strength of the binding is greatly enhanced in their presence. At higher pH values (> 7.0) and low DNA/drug ratio ( > 20), the metal cations are necessary to induce the binding between chromomycin A₃ and DNA. At higher DNA/drug ratios (> 100: 1), an appreciable proportion of the drug is bound even in the absence of divalent cations. Its binding affinity to the DNA is enhanced in the presence of these cations and at low pH values. Therefore, we conclude that chromomycin A₃ binds in two related modes, in the presence and in the absence of divalent cations. The spectral data accumulated indicate the metal cation is involved in the binding of the drug to the DNA by forming a drug–metal–DNA ternary complex.     

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn²⁺, Mg²⁺, and Mn²⁺ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn²⁺ had the best effects among the three metal cations and the effect was about 20 times of that of Zn²⁺ or Mg²⁺ and its maximal protease activity was 2.3 × 10⁵ U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively.     

The PhoQ histidine kinases of Salmonella and Pseudomonas spp. are structurally and functionally different: evidence that pH and antimicrobial peptide sensing contribute to mammalian pathogenesis

The PhoQ sensor kinase is essential for Salmonella typhimurium virulence for animals, and a homologue exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST-PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST-PhoQ has a periplasmic Per-ARNT-Sim domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA-PhoQ). Here, we demonstrate that PA-PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ-null mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST-PhoQ and PA-PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA-PhoQ binding metal in a specific ligand-binding pocket. PA-PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA-PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.     

Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.     

Characterization of the receptor for epidermal growth factor-urogastrone in human placenta membranes

The binding of mouse epidermal growth factor-urogastrone (EGF-URO) to membranes from term human placenta is peptide-specific, saturable (about 20 pmol of EGF-URO bound maximally/mg of protein), reversible, and of high affinity (KD about 400 pM). Optimal binding is observed at pH 7.6. At low pH (3.5 to 5.0). EGF-URO can be reversibly dissociated from the receptor; however, exposure to pH < 3 irreversibly inactivates the receptor. The binding, which does not exhibit ligand cooperativity, exhibits an association rate constant of 6.1 x 10(-4) s-1 and a dissociation rate constant of 6.1 x 10(-4) s-1. The dissociation constant determined from the rate constants, 240 pM, is in reasonable agreement with the constant estimated by equilibrium methods. Both monovalent and divalent cations augment EGF-URO binding 2- to 3-fold. Although in general, divalent cations enhance binding at lower concentrations (optimum, 5 mM) than do monovalent cations (optimum, approximately 80 mM), there is no cation-specific effect. Neither guanine nor adenine nucleotides affect EGF-URO binding. Whereas the proteolytic enzymes (trypsin, chymotrypsin, papain, and pepsin) inactivate the receptor, neuraminidase and phospholipases A2, C, and D augment EGF-URO binding. Neuraminidase increases the number of available sites without affecting ligand affinity. Wheat germ agglutinin, concanavalin A, and phytohemagglutinin all compete for the binding of EGF-URO. The data complement previous observations of EGF-URO binding obtained in intact cells and provide a basis for the solubilization, characterization, and isolation of this receptor from a rich tissue source.     

Proteolytic activities in cortex of apical parts of <Emphasis Type="Italic">Vicia faba</Emphasis> ssp. <Emphasis Type="Italic">minor</Emphasis> seedling roots during kinetin-induced programmed cell death

Programmed cell death (PCD) is a crucial process in plant development. In this paper, proteolytically related aspects of kinetin-induced PCD in cortex cells of Vicia faba ssp. minor seedlings were examined using morphological, fluorometric, spectrophotometric, and fluorescence microscopic analyses. Cell viability estimation after 46 μM kinetin treatment of seedling roots showed that the number of dying cortex cells increased with treatment duration, reaching maximum after 72 h. Weight of the apical root segments increased with time and was about 2.5-fold greater after 96 h, while the protein content remained unchanged, compared to the control. The total and cysteine-dependent proteolytic activities fluctuated during 1–96-h treatment, which was not accompanied by the changes in the protein amount, indicating that the absolute protein amounts decreased during kinetin-induced PCD. N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), and Z-Leu-Leu-Nva-H (MG115), the respective cysteine, serine, and proteasome inhibitors, suppressed kinetin-induced PCD. PMSF significantly decreased serine-dependent proteolytic activities without changing the amount of proteins, unlike NEM and MG115. More pronounced effect of PMSF over NEM indicated that in the root apical segments, the most important proteolytic activity during kinetin-induced PCD was that of serine proteases, while that of cysteine proteases may be important for protein degradation in the last phase of the process. Both NEM and PMSF inhibited apoptotic-like structure formation during kinetin-induced PCD. The level of caspase-3-like activity of β1 proteasome subunit increased after kinetin treatment. Addition of proteasome inhibitor MG-115 reduced the number of dying cells, suggesting that proteasomes might play an important role during kinetin-induced PCD.     

Biological Significance of Divalent Metal Ion Binding to 14-3-3 Proteins In Relationship to Nitrate Reductase Inactivation

In this report we address two questions regarding the regulationof phosphorylated nitrate reductase (pNR; EC 1.6.6.1 [EC] ) by 14-3-3proteins. The first concerns the requirement for millimolarconcentrations of a divalent cation in order to form the inactivepNR: 14-3-3 complex at pH 7.5. The second concerns the reducedrequirement for divalent cations at pH 6.5. In answering thesequestions we highlight a possible general mechanism involvedin the regulation of 14-3-3 binding to target proteins. We showthat divalent cations (e.g. Ca²⁺, Mg²⁺ and Mn²⁺) bind directlyto 14-3-3s, and as a result cause a conformational change, manifestedas an increase in surface hydrophobicity. A similar change isalso obtained by decreasing the pH from pH 7.5 to pH 6.5, inthe absence of divalent cations, and we propose that protonationof amino acid residues brings about a similar effect to metalion binding. A possible regulatory mechanism, where the 14-3-3protein has to be "primed" prior to binding a target protein,is discussed. ¹Co-operative investigations of the U.S. Department of Agriculture,Agricultural Research Service, and the North Carolina AgriculturalResearch Service, Raleigh, NC. This work was supported by agrant from the U.S. Department of Agriculture-National ResearchInitiative (Grant 93-37305-9231 to JLH and SCH). Mention ofa trademark or proprietary product does not constitute a guaranteeor warranty of the product by the USDA or the North CarolinaAgricultural Service. Nor does it imply its approval to theexclusion of other products that might also be suitable.     

Binding of beta-scorpion toxin: a physicochemical study

The binding to rat brain synaptosomes of a beta-scorpion toxin, i.e., toxin II of Centruroides suffusus suffusus (Css II), was studied as a function of pH, temperature, and concentration of some monovalent and divalent cations. At 10 degrees C and pH 6.0, the specific binding of 125I-labeled Css II corresponds to a single class of noninteracting high-affinity binding sites (KD = 0.18 nM) with a capacity (4.2 pmol/mg of protein) that is almost identical with that generally accepted for saxitoxin. The equilibrium dissociation constant of beta-scorpion toxin is pH independent, but the maximum binding capacity is reduced with increasing pH. Li+, guanidinium, Ca2+, Mg2+, and Mn2+ modified the apparent KD of the 125I-labeled Css II toxin. The equilibrium dissociation constant varies markedly with the temperature. The van't Hoff plot of the data is curvilinear, corresponding to a standard free-energy change associated with an entropy-driven process. The association rate constant also varies considerably with the temperature whereas the Arrhenius plot is linear between 1 and 30 degrees C. The energy of activation determined from these data is 17.6 kcal/mol. These results support the hypothesis that a cluster of nonpolar amino acid residues present on one face of the molecule is involved in the toxin-receptor interaction.     

Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters

Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.     

Agonist binding to multiple muscarinic receptors

The binding of agonists to muscarinic cholinergic receptors is well described by a binding model of multiple affinity states (superhigh, high, and low) in most central and peripheral tissues. Although previous studies of the influences by divalent cations, guanine nucleotides, and sulfhydryl reagents support the concept that these regulators act through closely related sites to alter the relative proportions of muscarinic agonist affinity states, it has become apparent that muscarinic receptor subtypes (as defined with the nonclassical antagonist pirenzepine) are differentially affected by the regulators. For example, in tissues that have few high-affinity [3H]pirenzepine-binding sites (heart, ileum, cerebellum), magnesium ions promote the formation of a high agonist affinity state, whereas exposure of these tissues to the sulfhydryl reagent N-ethylmaleimide (NEM) or guanine nucleotides promotes the formation of a low agonist affinity state. Conversely, tissues rich in high-affinity [3H]pirenzepine-binding sites (cerebral cortex, corpus striatum, hippocampus) show little, if any, change in agonist binding site affinity when magnesium ions or guanine nucleotides are present. Furthermore, NEM enhances the muscarinic binding site affinity for agonists in these tissues. Taken together, these results support the concept of muscarinic receptor heterogeneity, as proposed from previous physiological studies, and indicate that the aforementioned regulators (guanine nucleotides, magnesium ions, NEM) differentially alter the agonist-binding properties of these muscarinic receptor subtypes.     

Self-association of a high molecular weight subfragment-2 of myosin induced by divalent metal ions

The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form ordered, high molecular weight aggregates whereas short S-2 does not associate. The association process is co-operative and results from binding two to four divalent cations within the light meromyosin-heavy meromyosin (LMM-HMM) hinge region of long S-2. Optical diffraction of electron micrographs of the long S-2 aggregates revealed several periodicities including reflections near 143 A. High molecular weight HMM showed a similar divalent metal induced self-association. Chymotryptic digestion studies of rod filaments reveal that cleavage within the LMM-HMM hinge is also strongly dependent on the presence of divalent cations. At pH 8, in the absence of divalent cations, the S-2 region appears to be displaced away from the filament backbone resulting in rapid proteolysis in the hinge domain. At high cation concentrations (greater than 10 mM) proteolytic cleavage is suppressed. A similar depression of the (substantially lower) hinge cleavage rate was also observed at neutral pH following addition of these divalent metal ions. Results suggest that binding of Mg2+ within the hinge domain under physiological conditions may act to lock the cross-bridge onto the thick filament surface in its resting-state orientation.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

Thermal denaturation and gelation of rubisco: effects of pH and ions

In order to understand the mechanism of thermal gelation of rubisco, its native and heat denatured states were characterized by absorbance, fluorescence and circular dichroïsm spectroscopies as well as by differential scanning calorimetry in the presence of various salts. It appears that during the denaturation process, divalent anions are released while divalent cations are fixed by the protein, while it is disorganized and while the environment of its aromatic chromophores becomes more hydrophilic. The pH transition of gelation is shifted 1–2 pH units higher than the transition of denaturation temperature which occurs near the isoelectric point of the native molecule. This shift probably corresponds to the breaking of saline bridges within the protein molecule. Finally, a large effect of divalent cations on the phase diagram indicates that a particular denatured state is attained when these cations are in the denaturation medium.