Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.     

Kinetic studies on rat liver and beef heart mitochondrial adenosine triphosphatase: the effects of the chromium complexes of adenosine triposphate and adenosine diphosphate on the kinetic properties.

The kinetics of isolated rat liver and beef heart mitochondrial adenosine triphosphatase (ATPase) were studied by using the chromium ATP and ADP complexes as substrate analogs. It was found that both chromium ATP (CrATP) and chromium ADP (CrADP) are competitive inhibitors of ATP hydrolysis. The presence or absence of ATPase-activating anions, e.g., bisulfite, had little effect on the type or potency of the inhibition by these chromium complexes. Both CrADP and CrATP were noncompetitive inhibitors of the hydrolysis of ITP with both the heart and liver-derived enzymes. It was also found that CrADP was a consistently more effective inhibitor than the ATP complex with the beef heart enzyme. These results are consistent with the existence of two types of nucleotide binding sites on mitochondrial ATPases: One site is regulatory and is rather specific for adenosine polyphosphates, while the other site is relatively nonspecific and serves as the hydrolytic site.     

Kinetic and thermodynamic characterization of DNA duplex–hairpin interconversion for two DNA decamers: d(CAACGGGTTG) and d(CAACCCGTTG)

The duplex–hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kinetically by using uv-melting and nmr relaxation methods. Separately, each decamer shows slow exchange between hairpin and duplex conformations. The hairpin conformations have melting points of 47 and 50°C, respectively, and exhibit similar thermodynamic stabilities. The enthalpies of duplex formation, measured by nmr, were found to be very similar (ΔH_DH = 26 ± 3 kcal/mole) for both decanters at low salt concentrations (< 50 mM NaCl). However, as the salt concentration was increased the behavior of ΔH_DH, and kinetics is significantly different for each decamer. The d(CAACGGGTTG) decamer forms a duplex containing two central G·G mismatches at high salt and DNA concentration. Based upon the measurement of high interconversion activation energies and a decrease in hairpin formation rate with increasing salt, the interconversion between hairpin and duplex was concluded to proceed by complete strand dissociation. In contrast, the d(CAAC-CCGTTG) decamer was determined to form a duplex with two centrally located C·C mismatches at pH values less than 6.2, consistent with the formation of a hemiprotonated C⁺·C mismatch. At pH values greater than 6.4, the hairpin–duplex equilibrium is almost completely shifted toward the hairpin conformation at DNA concentrations of 0.5–7.0 mM and salt concentrations of 10–100 mM. The interconversion of duplex and hairpin conformations was ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary decamer. Although the interconversion proceeds by complete strand separation as suggested by high duplex-hairpin interconversion activation enthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the ΔH_DH, dependence on sail indicate that an intermediate internally bulged duplex (no C⁺·C formation) is stabilized by increasing ionic strength. These data support an interconversion mechanism where an intermediate internally bulged duplex may be the rate limiting step before strand separation. © 1995 John Wiley & Sons, Inc.     

Thermal Denaturation of Bacterial and Bovine Dihydrofolate Reductases and their Complexes with NADPH,Trimethoprim and Methotrexate

Abstract

Scanning microcalorimetry was used for the study of thermal denaturation of E.coli and bovine liver dihydrofolate reductases (cDHFR and bDHFR, respectively) and their complexes with NADPH, trimethoprim (TMP) and methotrexate (MTX) at pH 6.8. It was shown that the denaturation temperature of bDHFR is 7.2°C less than that of cDHFR and that ionic strength is equally important for the thermostability and cooperativity of the denaturation process of the two proteins. Binding of antifolate compounds significantly stabilizes DHFR against heat denaturation. The stabilizing effect and the transition cooperativity depend on the nature of the inhibitor, the presence of NADPH and the origin of the enzyme. The dependence of calorimetric denaturation enthalpy (calculated per gram of protein) on denaturation temperature for DHFRs, their complexes with NADPH and binary/ternary complexes with TMP/MTX fits to the same straight line with the slope of 0.66 J/K g. This relatively high value indicates an essential role of hydrophobic contacts in the stabilization of DHFR structure. The change of denaturation temperatures in binary complexes with MTX/TMP (in comparison with the free enzymes) is as much as 14.2°C/8.5°C and 13.3°C/3.2°C for cDHFR and bDHFR, respectively. The same change in ternary complexes with MTX/TMP is much more pronounced and equals to 21.9°C/16.8°C and 29.0°C/16.4°C. The vast difference of binary and ternary complexes thermostability demonstrates the important role of cofactor in the stabilization of enzyme. Moving from binary to ternary systems causes a significant increase in denaturation temperatures, even when corresponding association constants do not change (cDHFR binary/ternary complexes with MTX) or increases very slightly (bDHFR binary/ternary complexes with TMP). In all other cases the increase of denaturation temperature     

Structural Stability of Streptococcal Serum Opacity Factor

Streptococcal serum opacity factor (SOF) is a protein that clouds the plasma of multiple mammalian species by disrupting high density lipoprotein (HDL) structure. Intravenous infusion of low dose SOF (4 µg) into mice reduces their plasma cholesterol concentrations?~?40% in 3 h. Here we investigated the effects of pH, ionic strength, temperature, and denaturation with guanidinium chloride (GdmCl) on SOF stability and its reaction vs HDL. SOF stability was tested by pre-incubation of SOF at various temperatures, pH’s, and GdmCl concentrations and measuring the SOF reaction rate at pH 7.4 and 37?°C. SOF retained activity at temperatures up to 58?°C, at pH 4 to 10, and in 8.5 M GdmCl after being returned to standard buffer conditions. The effects of GdmCl, pH, and ionic strength on the SOF reaction rates were also measured. SOF was inactive at GdmCl?≥?1 M; SOF was most active at pH 5, near its isoelectric point and at an ionic strength of 3 (in NaCl). These data reveal that SOF is a stable protein and suggest that its activity is determined, in part, by the effects of pH and ionic strength on its overall charge relative to that of its reaction target, HDL.     

Collagen fibril formation in vitro at nearly physiological temperatures

Fibril formation by collagen from piglet skin was studied at temperatures of 28–39°C. Collagen fibrils obtained in this temperature range differ in the degree of ordering. Electron microscopy shows that fibrils of minimal diameter are formed at physiological pH, ionic strength (PBS), and temperature. The greater diameter of fibrils formed at 34.5°C is due to enhanced collagen hydration. Fibril diameter at 38.5°C is increased because of cooperative unfolding of the triple helix and weaker binding between collagen molecules. The optimal temperature for fibrillogenesis appears to be 36.5°C, and such fibrils are most similar to those observed in vivo.     

Multiple denaturational transitions in fibrillar collagen

The heat denaturation of pepsinized bovine nonfibrillar and fibrillar collagen was studied by differential scanning calorimetry. For fibrillar preparations that had been rapidly precipitated with stirring at low ionic strength, then resuspended at physiological ionic strength, multiple denaturational transitions were observed. At heating rates of 10°C/min, melting endotherms occurred at about 44, 50, 53, and 57°C. Fibrillar collagen that was slowly gelled without stirring at physiological ionic strength exhibited a similar series of endotherms, but the lower melting transitions were less conspicuous. In contrast, nonfibrillar bovine collagen in acidic solution showed only a single denaturational transition at 40°C. Nonfibrillar solutions at pH 7, to which inhibitors of fibrillogenesis were added, showed a major endotherm as high as 46°C. These results suggest that reconstituted fibrillar collagen contains a heterogeneous fibril population, possibly including molecules in a nonfibrillar state. It was proposed that the multiple melting endotherms of such preparations were due to sequential melting of molecular and fibril classes, each with a distinct melting temperature. The fibrillar classes may represent three or more types of banded and nonbanded species that differ from each other in packing order, collagen concentration, and possibly also in fibril width and level of cross-linking.     

Deamidation of glutaminyl residues: dependence on pH, temperature, and ionic strength

We have shown the dependence of the deamidation half-times of the peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly upon pH, temperature, and ionic strength. Increase in temperature or ionic strength, variation of pH to pH′s higher or lower than pH 6, and the use of phosphate buffer rather than Tris buffer at high pH all decrease the half-time of dcamidation. Temperature increase of 20°C or pH change of 2 pH units decreases the half-time about fivefold, while increase of one ionic strength unit decreases the half-time about twofold. In pH 7.4, I = 0.2, 37.0°C phosphate buffer, the deamidation half-times are 663 ± 74 and 389 ± 56 days respectively for the two peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly.These experiments should serve as a warning to peptide and protein experimenters that even the more stable glutaminyl residues are unstable with respect to deamidation in certain solvent conditions. These experiments also provide, along with previously reported experiments on asparaginyl peptides (7), some quantitative data to help with the extrapolation of in vitro deamidation experiments to in vivo deamidation conditions.     

Does collagen microunfolding stimulate fibril formation?

The data on the effect of temperature on the kinetics of collagen fibril formation at physiological pH values and ionic strength in the temperature range 26–39°C have been analyzed. The temperature of 35°C optimal for collagen fibril formation has been defined as the point of inflection for half-maximal turbidity and collagen molecule microunfolding values, which corresponds to the temperature of the first transition on the heat absorption curve. The temperature range (32–35°C) in which collagen microunfolding stimulates fibril formation has been determined.     

Effect of Temperature,pH, Ionic Strength,and Sodium Nitrate on Activity of LiPs: Implications for Bioremediation

The present work is aimed to show the effects of environmental parameters such as temperature, pH, ionic strength, and sodium nitrate on enzyme activity of a LiP Isoenzymes Mixture (LIM) obtained from an immobilized culture of Phanerochaete chrysosporium. LIM enzyme stability was also evaluated. The results are discussed in detail and a comparison with literature data is carried out. LIM showed high activity at pH 3.0 in the temperature range 30 to 40°C, it is able to catalyze oxidation reactions at acid pH (2.5<pH<6) and over a wide range of temperatures (25 to 60°C). Ionic strength below 0.2?M had no effect on enzyme activity at pH 4.7 and 39°C. An evaluation of the time decay constant of LIM activity under a specific combination of parameters was also conducted. Finally, an LIM activity and durability map shows the optimal working conditions that might be suitable for its practical application in waste bioremediation processes.     

The temperature-dependent self-association of beta-lactoglobulin C in glycine buffers

The self-association of β-lactoglobulin C at low pH (ca. 2.5) in glycine buffers has been studied at four temperatures, 10, 16, 20, and 25 °C, by low- and high-speed sedimentation equilibrium experiments. One buffer had an ionic strength of 0.1 and the other an ionic strength of 0.2. With either buffer the concentration dependence of the apparent weight average molecular weight, M_wa, was characteristic of a nonideal self-association. Like its genetic variants, β-lactoglobulin A and B, the self-association of β-lactoglobulin C increased with decreasing temperature; however, at the same temperature the association was always stronger in the buffer having the higher ionic strength. Several models were used to test the self-association, and a monomer-dimer self-association seemed to describe the self-association best with either buffer. Values of the association equilibrium constant, K₂, and the second virial coefficient, BM₁, are reported at each temperature for both series of experiments. Values of the thermodynamic functions, ΔG °, ΔH °, and ΔS °, are also reported for these experiments.     

Electron spin resonance study of the role of nitrosyl-heme in the activation of guanylate cyclase by nitrosoguanidine and related agonists.

One major component of lens plasma membrane is a glycoprotein that SDS-polyacrylamide gel electrophoresis shows to possess an apparent molecular weight of 26,000. When this protein is solubilized in low ionic strength buffers containing SDS, and heated to 100° for 1 to 3 min prior to electrophoresis, conversion into high molecular weight aggregate results. The heat lability of this protein is greatly enhanced if it solubilized and heated in buffers containing 0.1 M NaCl. At this ionic strength, incubation for 3 h at 38° results in conversion of 20% of the protein into high melecular weight aggregates. Most other membrane proteins isolated from lens membrane are insensitive to heat treatment. It is concluded that temperature and ionic strength must be recorded and controlled carefully when using SDS-polyacrylamide gel electrophoresis to study this membrane protein.     

Studies on the assembly of a spherical plant virus. I. States of aggregation of the isolated protein

The aggregates formed at equilibrium by purified protein from cowpea chlorotic mottle virus have been characterized on the basis of their sedimentation behaviour and appearance in the electron microscope. Between pH 3.5 and 7.5, at ionic strengths greater than 0.2, most of the protein is found in aggregates sedimenting at either 3 S or 50 S. The 50 S aggregate is identified as the reassembled capsid of cowpea ehlorotic mottle virus. Decreasing the ionic strength favours the formation of multi-shelled particles. Below pH 5.5 single- and multishelled particles predominate, while above this pH most of the protein sediments at 3 S.Varying the temperature from 5 °C to 20 °C has little effect on the equilibrium proportions of aggregates although some real differences can be detected. Ionic strength is not as important a variable as pH in determining which protein forms are present (but increasing ionic strength does result in a steady decrease in the proportion of protein in the multi-layer aggregates). The dependence of the equilibrium upon protein concentration shows that capsid formation is a quasi-crystallization: beyond a certain total protein concentration the concentration of 3 S aggregate remains at this “critical” concentration and all further protein goes into 50 S capsid. In addition to shells and variations upon shells, tubes and hexagonal nets of protein subunits have occasionally been seen with the electron microscope.     

A specific stain for α-glucosidases in isoelectric focusing gels

The separation of the four diastereomers of β,γ-bidentate Cr · ATP using reverse-phase HPLC techniques is described. This technique provides complete resolution of the diastereomers within 10 min and relies on the use of methanesulfonic acid (in the ionized form) as an ion-pairing agent. To identify the screw sense of these resolved isomers, the CD spectra of each isomer were done, and substrate and inhibition specificities were examined using hexokinase. The results were then correlated with the isomeric assignments made by D. Dunaway-Mariano and W. W. Cleland (1980, Biochemistry19, 1506–1515). Further studies included the monitoring of isomer interconversion at pH 6.2 to an equilibrium concentration of all four, and specific rotation measurements of the pure isomers at pH 2.5, 23°C, and 546 nm.     

Purification and properties of microbody malate dehydrogenase from Spinacia oleracea leaf tissue

The microbody isoenzyme of malate dehydrogenase (EC 1.1.1.37) from leaves of Spinacia oleracea was purified to a specific activity of 3000 units/mg protein and examined for a number of physical, kinetic, and immunological properties. The purified enzyme has a molecular weight of approximately 70,000 and an isoelectric point of 5.65. Thermal inactivation first order rate constants were 0.068 (35 °C), 0.354 (45 °C), and 2.11 (55 °C) for irreversible denaturation. Apparent millimolar Michaelis constants are 0.34 (NAD, pH 8.5) 0.16 (NADH, pH 7.5), 3.33 (malate, pH 8.5), 0.07 (OAA, pH 6.0), 0.06 (OAA, pH 7.5), and 0.50 (OAA, pH 9.0). The enzyme is stablized by 20% glycerol and can be stored for several months at 4 °C without detectable loss of activity. The purified enzyme is sensitive to the ionic strength of the assay medium exhibiting a pH optimum of 5.65 at high ionic strength and 7.00 at low ionic strength. Rabbit antiserum prepared against the purified microbody MDH shows a single precipitin band on immunodiffusion analysis. Immunological studies indicate that rabbit antiserum prepared against the purified microbody enzyme cross reacts approximately 10% with the mitochondrial isoenzyme of MDH. No cross reaction was shown with the soluble isoenzyme. In general, the data presented in this report tend to support the notion of organelle specific isoenzymes of malate dehydrogenase in higher plant tissues and uniqueness of the microbody form of malate dehydrogenase in particular.     

Isolation and Partial Characterization of Heat-denatured Products of Soybean 11S Globulin and Their Analysis by Electrophoresis

Heat denaturation of soybean 11S globulin was examined at 70° and 100°C in phosphate buffer (pH 7.6), at 0.01 and 0.5 ionic strength. Gel electrophoresis (Davis system) indicated that heat-denatured soybean 11S globulin contained two major components (buffer-soluble form). But they were not identified at 70°C-0.5 ionic strength. Gel filtration followed by SDS-gel electrophoresis showed that the major components were composed of a monomer and at least three of kinds of oligomers containing only an acidic subunit. Gel filtration of the precipitate formed at 100°C at 0.5 ionic strength gave two peaks. SDS-gel electrophoresis indicated that the first peak contained aggregates of highly polymerized subunits, and the second peak contained a monomer of basic subunit and seven kinds of oligomers with various proportions of basic subunits to an acidic subunit.     

The application of surface tension measurements to the study of conformational transitions in aqueous solutions of poly-L-lysine

The change in surface tension of solutions of poly-L -lysine in water has been studied as a function of temperature at various pH values. The changes at various temperatures have been correlated with changes in the circular dichroic spectra reflecting conformational change. In addition to the major transition at 50°C attributed to the conversion of the α-helical → β conformation, two other transitions have been observed at 30°C and 80°C. A minimum in the surface tension value was observed at pH 10, near the pK value for poly-L -lysine. It was concluded that at this pH the concentration of hydrophobic groups at the surface was a maximum.     

Analysis of the calcium-dependent interaction of calmodulin with bovine serum albumin

An air-driven ultracentrifuge has been used to investigate the calcium-dependent association between calmodulin and bovine serum albumin. Procedures were described which allowed the interaction to be analyzed to yield the equilibrium constant. At low ionic strength (25 mm Tris-HCl, pH 7.5, pCa 6.68, 9°C) the equilibrium constant for the interaction was estimated to be 2.1 × 10⁴m⁻¹, while at high ionic strength (25 mm Tris-HCl, pH 7.5, 150 mm KCl, pCa 6.68, 9°C) the value was 4.5 × 10³m⁻¹. Under similar conditions, calmodulin was also found to interact with β-lactoglobulin A and gelatin, but no detectable association was observed with ovalbumin.     

Affinity of arginase for ornithine carbamoyltransferase in Saccharomyces cerevisiae.

The association between the two trimeric enzymes ornithine carbamoyltransferase and arginase, which is under the control of arginine and ornithine, is endothermic (ΔH° = + 14.6 kcal mol^?1). The process is clearly entropy-driven (ΔS° = + 94.7 cal mol^?1 deg.^?1) allowing a dissociation constant of 0.1 nm for the complex at optimal pH 8, 30 °C and an ionic strength of 0.025. The stability of the complex is moderately sensitive to pH and ionic strength. The dissociation constant of the complex was measured in a medium at cellular pH and salt concentration and found to be close to the constant expected for operative inhibition of ornithine Carbamoyltransferase in the yeast cell. The importance of the presence of arginine as an effector for the formation of the complex under the above conditions is clearly demonstrated.     

Dynamic Light Scattering Studies on Hydrodynamic Properties of Fibrinogen-Fibronectin Complex

Abstract

A high molecular weight ‘cryogel’ was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37°C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20°C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20°C in 0.05 M Tris- HCl(pH7.4) containing 0.5 M NaCl was estimated as 8.5× 10^?8 cm²s^?1. The complex did not dissociate over the temperature range from 20 to 37°C. The diffusion coefficients of the complex decreased significantly at 12°C and 40°C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40°C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15°C, but not at near- freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.