Isolation of low molecular weight cytoplasmic regulators from the rat liver inducing the electrophoretic transport of K+ ions and phosphate across the inner mitochondrial membrane

A water-soluble thermostable factor from rat liver cytoplasm whose activity decreases during starvation, causes the uncoupling of oxidative phosphorylation and stimulates pyruvate oxidation in rat liver mitochondria. The activity of this factor is insensitive to pronase treatment. Gel filtration and ion-exchange chromatography resulted in three low molecular weight water-soluble fractions which bear a negative charge at alkaline values of pH and induce electrophoretic transport of K+ and phosphate across the inner mitochondrial membrane. The effect of this factor on K+ transport is manifested at pH less than or equal to 7.0, that on phosphate transport-at pH 6.5-7.6.     

Effect of an abundant human skin melanosomal 66 kDa protein (MP 66) on m urine tyrosinase: Its physiological implications on melanogenesis

A single polypeptide protein (MP 66) of molecular weight 66 kDa purified to homogeneity from melanosomes of normal human skin epidermal melanocytes, was partially characterized. The isoelectric point of MP 66 is in the range of 7.3 to 7.6. This protein, which was shown to inhibit partially purified human skin tyrosinase activity at pH 6.8, also inhibits murine tyrosinase at pH 6.8. However, at pH 5.0, it stimulates murine tyrosinase activity. The physiological implications of these results are discussed.     

The denaturation-renaturation of chicken-muscle triosephosphate isomerase in guanidinium chloride

1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.     

Purification and properties of an endonuclease from the mitochondrion of Saccharomyces cerevisiae

An endonuclease, which is found only in the mitochondrion of the yeast Saccharomyces cerevisiae, has been purified. The protein has a sedimentation coefficient of 6.3 S, equivalent to a molecular weight of 105,000. The enzyme is active at pH 7.6, when it degrades single-stranded DNA about 10-times faster than double-stranded DNA, but at pH 5.4 only double-stranded DNA is degraded. In both cases the enzyme acts endonucleolytically, breaking a single phosphodiester bond at a random location within the DNA substrate. Mn2+ or Mg2+ are required for activity; Ca2+ and Zn2+ are ineffective cofactors. Enzyme activity at pH 7.6 is severely inhibited by low concentrations of NaCl or KCl, while activity at pH 5.4 is unaffected by salt. Ethidium bromide inhibits both the DNase activity at pH 5.4 and the activity with single-stranded DNA at pH 7.6, but has no effect on the DNase activity with double-stranded DNA at pH 7.6.     

Sedimentation equilibrium studies of human chymotrypsin II and bovine -chymotrypsin

Sedimentation equilibrium experiments indicate that neither human chymotrypsin II nor bovine δ-chymotrypsin molecules undergo association in the pH range 3–5 where dimerization occurs with α-chymotrypsin. The weight-average molecular weights of human chymotrypsin II and δ-chymotrypsin in a pH 4.4 0.1 ionic strength buffer are 26,200 and 26,400, respectively, using the measured partial specific volumes of 0.722 and 0.727 ml/g at 25 °C. Number-average molecular weight calculations also support the presence of monomeric species at this pH. In the pH range 6–7.6 where sedimentation velocity studies have shown that δ-chymotrypsin associates at concentrations above 3 mg/ml, no association was observed for either the human chymotrypsin II or bovine δ-chymotrypsin in the sedimentation equilibrium experiments where protein concentrations were below 1.2 mg/ml. These studies provide additional evidence that human chymotrypsin II is similar to bovine δ-chymotrypsin.     

Carboxylesterases (EC 3.1.1). The molecular sizes of chicken and pig liver carboxylesterases.

The molecular size of pig liver carboxylesterase has been investigated under a variety of conditions of pH and ionic strength. From equilibrium and velocity sedimentation at pH 4.0 and pH 7.5, and from chromatography on Sephadex G-200,we conclude that the monomeric molecular weight is similar to 65,000 daltons and that the enzyme associates to form trimers. Association equilibrium constants for the monomer-trimer system were estimated to be 0.02 1-2 g-2 at pH 4 (concentration-dependent molecular weight data) and 2 times 10-5 1-2g-2 at pH 7.5 (frontal gel chromatographic results). These studies were aided by comparisons of the properties of the pig liver enzyme with those of chicken liver carboxylesterase, which is shown to exhibit the velocity and equilibrium sedimentation characteristics of a homogeneous protein with molecular weight similar to 65,000. Studies of pig and chicken liver carboxylesterases in 6 M guanidinium chloride, 0.1 M in beta-mercaptoethanol, support the proposition that the monomeric species of these enzymes have molecular weights of similar to 65,000. On polyacrylamide gel electrophoresis in SDS, there is no evidence for a major species of molecular weight less than similar to 65,000 for the pig enzyme, but ca. 50 percent of the chicken esterase is dissociated into two species of molecular weight similar to 30,000.     

Characterization of 20S component in tubulin from mammalian brain.

Tubulin from porcine brain, purified by at least two cycles of assembly and disassembly, was characerized at different pH values by sedimentation velocity analysis and turbidimetric measurements. At pH 6.4 the depolymerized material was composed of two major species sedimenting with so20,w values of 6 and 36 and a minor one of 20S. By raising the pH, the amount of the 20S component increased and that of the 36S decreased, whereas that of the 6S component was unaltered. At pH 7.6 the mixture contained 20S and 6S components but hardly any 36S. The 20S species can be separated from the 6S ones by gel filtration on agarose A-15m at pH 7.6. On electron microscopic examination this preparation contains far fewer double rings compared to the material at pH 6.4, but single rings could often be seen. Sodium dodecyl sulfate gel electrophoresis of the 20S and 36S components showed that they consist almost entirely of tubulin and some higher and lower molecular weight fractions. Turbidity measurements showed that the minimal protein concentration necessary for polymerization increases with increasing pH. The turbidity plateau reached at a given pH can be raised by decreasing the pH. From these results it is suggested that the 20S component is an intermediate of the 36S species. The results further indicate the existence of a pH-dependent equilibrium between the 20S species and the 36Soligomers.     

Purification and kinetic properties of chalcone-flavanone isomerase from soya bean.

The chalcone-flavanone isomerase from soya bean seed has been purified 8300-fold. A molecular weight of 15,600 plus or minus 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form. The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.     

Properties of pig liver transketolase]

Some properties of homogeneous transketolase from pig liver were studied. It was shown that the pH optimum of the transketolase reaction lies within the range of 7.8--8.2. The isoelectric point is at pH 7.6--7.8. The molecular weight of transketolase is 138,000 +/- 3,000 as determined by the sedimentation equilibrium method and about 152,000 according to the data from gel filtration through Sephadex G-200. The enzyme molecule is a tetramer of the alpha 2 beta 2 type. The molecular weights of the alpha- and beta- subunits determined by polyacrylamide gel in the presence of sodium dodecyl sulfate are 52,000--56,000 and 27,000--29,000, respectively. Transketolase contains about two moles of TPP per mole of protein and does not require metal ions for its catalytic activity.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites.

The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.     

Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12. Concentration-dependent dissociation to dimers in the presence of L-threonine.

The concentration-dependent association-dissociation equilibrium of the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 has been investigated at pH 7.6 in the presence of 10 mM L-threonine and 0.1 M KCl by equilibrium gel permeation monitored by a single-photon counting spectrophotometer. The results obtained are consistent with the existence of a dimer-tetramer equilibrium with the association constant of 2.6 X 10(7) M-1 (deltaG0 = -9.9 kcal/mol of dimer). The limiting partition cross-sections estimated by a three-parameter least squares minimization procedure indicate that the molecular radii of the dimer and tetramer are 53.8 A and 70 A, respectively. Both the dimeric and tetrameric forms of the enzyme possess dehydrogenase activity. Treatment of the enzyme with the chaotropic salts, potassium thiocyanate or potassium trichloroacetate, generates a monomeric form that is devoid of dehydrogenase activity. The catalytically inactive monomeric form of the enzyme has a molecular radius between 43 and 45.5 A and a molecular weight of approximately 80,000 as determined by small zone gel chromatography and sedimentation equilibrium studies.     

Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5+/-2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)(6). At pH 5.8, (RepA)(6) associates to form a dimer with a molecular mass of 366.2+/-4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)(6) at pH 5.8 and 7.6. The dissociation constants, K(d), for the equilibrium binding of (dA)(30) to the (RepA)(6) dimer at pH 5.8 and to (RepA)(6) at pH 7.6 were determined at 10 degrees C in the presence of 0.5 mM ATPgammaS, 10 mM MgCl(2) and 60 mM NaCl as K(d5.8)=0.94+/-0.13 microM at pH 5.8 and K(d7. 6)=25.4+/-6.4 microM at pH 7.6. The stoichiometries, n, for the two complexes (dA)(30)/(RepA)(6) dimer and (dA)(30)/(RepA)(6) at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)(30) molecule was bound per (RepA)(6) dimer, while at pH 7.6 one (dA)(30) molecule was bound to one (RepA)(6). Binding curves were compatible with a single ssDNA binding site present on the (RepA)(6) dimer and on (RepA)(6), respectively, with no indication of cooperativity. (RepA)(6) tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)(6) dimer was observed both in the absence and presence of (dA)(30).     

Cobalt hemoglobin: pH dependence of Adair constants and the Bohr effects.

Precise oxygen equilibrium curves have been obtained for cobalt hemoglobin at pH values from 5.5 to 8.2. The Hill plots are symmetric having asymptotes with slopes of unity. At pH 7.0, cobalt hemoglobin has p0.5 = 116 toor (15.45 kPa), pm = 117 torr (15.58 kPa) and a Hill coefficient of n = 1.72. The values of n decrease slightly with either decrease or increase of pH; the protein is almost non-cooperative at pH greater than 8.2. The Adair constants have been calculated with a non-linear least-squares program. From deltalnpm/deltapH a maximum of 2.5 Bohr protons was calculated at physiological pH values. The majority of alkaline Bohr protons are released after binding of the first and the third oxygen with maxima at pH 7.6 and 7.3, respectively. The acid Bohr effect was also observed with the majority of the protons taken up following the first and third oxygen bound. Smaller alkaline Bohr effects were obtained by differential titration and at higher pH than that calculated from oxygen equilibria. The discrepancy can be largely attributed to the binding of salt components to cobalt hemoglobin.     

Purification and properties of phosphofructokinase (EC 2.7.1.11) of Saccharomyces carlsbergensis.

A procedure for the purification of phosphofructokinase from brewer's yeast (Saccharomyces carlsbergensis) is reported. Treatments with organic solvents and heat were avoided and chromatographic and filtration techniques in the presence of phenylmethane sulfonyl fluoride were mainly used. The purified enzyme is homogeneous in disc gel electrophoresis and according to sedimentation velocity and equilibrium measurements in the ultracentrifuge. The isoelectric point determined by focusing was 5.3. Absorption spectra, fluorescence spectra and circular dichroism spectrum are given. The molecular weight of the purified enzyme determined by gel filtration was 720 000, in agreement with that of the enzyme in the raw extract. This confirms the results of sedimentation velocity experiments which gave a value of SO20, W equals 19.4. Alkaline treatment leads to a dissociation of the native enzyme, yielding an inactive species with a molecular weight of 360 000. In 6M guanidine hydrochloride the enzyme dissociates into subunits with a mean molecular weight of 90 000 as obtained by ultracentrifugation analysis. This suggests a structure composed of 8 monomers. The specific activity of the enzyme was 116 U/mg under optimum conditions. The enzyme activity was proportional to the enzyme concentration in the range of 6 times 10- minus 12 M to 3 times 10- minus 7 M. The Michaelis constants and Hill coefficients for fructose 6-phosphate and AMP, the pH optima, and the stability properties of the enzyme are reported. Furthermore, the activation energy is given and it is shown that under saturating conditions, a straight Arrhenius plot obtains, whereas the plot is discontinuous at high ATP concentrations and at pH 7.6.     

Cooperative proton and calcium binding by sarcoplasmic reticulum ATPase.

The classic work on binding of calcium to CaATPase is analyzed by an objective non-linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K1K2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH less than 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of a small, less than statistical, amount of a ternary complex bearing one calcium and one proton.     

Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37 degrees C and pH 6.6-9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H(+) and undissociated acetic acid. At pH values below 7.6, the H(+) inhibited the fermentation following non-competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45-6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na(+)) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Purification and properties of phosphotransacetylase from the eucaryotic green alga Chlorogonium elongatum

Phosphotransacetylase (EC 2.3.1.8) was detected in cell-free crude extracts of starch-fermenting eucaryotic green algae. The enzyme was purified from autotrophically grown Chlorogonium elongatum. The purified enzyme fraction, after affinity chromatography, shows a single protein band upon acrylamide gel electrophoresis and has a molecular weight of 280 000. It consists of six subunits of identical molecular weight (44 000). The pH and temperature optima for the eucaryotic phosphotransacetylase are 7.6 and 28°C, respectively. The K_m values at 25°C (pH 7.6) for acetyl-CoA and phosphate are 0.078 mM and 5.440 mM, respectively, and in the reverse reaction (acetyl-CoA synthesis) for CoA and acetyl phosphate 0.093 mM and 0.310 mM, respectively. The maximum velocity of the forward reaction was 1627 nkat/mg protein and of the reverse reaction 8582 nkat/mg protein. The activity of the eucaryotic phosphotransacetylase strictly depends on the presence of univalent cations (ammonium, K_a = 9 mM; potassium, K_{a = 12.5 mM}). Inactivation studies with iodoacetamide and iodoacetic acid revealed the presence of an essential sulphhydryl group at the catalytic site. Arsenolytic and product inhibition studies indicate a rapid equilibrium random bi-bi reaction mechanism for the enzyme from C. elongatum. The control of the enzyme activity in the forward reaction by both pyruvate and NADH gives evidence for a physiological function of phosphotransacetylase in anaerobic energy metabolism of eucaryotic green algae rather than in aerobic acetate activation.