Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

A simple, one-step assay for methyltransferase activity: direct partitioning of radioactive product into scintillation fluid

A simple, one-step procedure is described for measurement of the enzymatic transfer of radioactive methyl groups from S-adenosyl-l-methionine to O-demethylpuromycin. Reactions are carried out in small, stoppered scintillation vials and terminated by the addition of 0.1 m sodium borate-5 m NaCl (pH 9) buffer and scintillation fluid. The vials are then shaken to extract radioactive pyromycin into the organic phase and directly counted.     

Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect

Data are reported for T(m), the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na(2)SO(4) and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T(m) caused by anion binding at 0.1 M sulfate is 20 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees increase in T(m) between 0.1 and 1.0 M Na(2)SO(4), caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T(m) between 0 and 1.0 M is 14 degrees at pH 2 but only 1 degree at pH 9. The 14 degree increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na(2)SO(4) and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.     

A new procedure for the purification of rat testis-specific histone TH2B involving affinity-related chromatography

A new procedure for the isolation of rat testis-specific histone TH2B has been devised. First, rat testis chromatin fragments were applied to a hydroxylapatite column in 0.5 m NaCl, 0.1 m potassium phosphate buffer, pH 6.7, and histones were selectively stripped off the bound DNA in groups (H1/TH1, H2A/H2B/TH2B, and H3/H4). The fraction containing H2A, H2B, and TH2B, but lacking H3, was reduced, desalted, and applied to a p-chloromercuribenzoyl-aminoethyl-Sepharose 4B-CL column in 8 m urea, 0.1 m Tris-HCl, pH 8.1. After washing with the same buffer to remove H2A and H2B, covalently bound TH2B was eluted out with 10 mm dithiothreitol in the same buffer. No contaminants were detectable in the purified TH2B either by polyacrylamide gel electrophoresis in 0.4% Triton X-100, 2.5 m urea, 0.9 n acetic acid, or by N-terminal analysis with dansyl chloride.     

A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum

A procedure is described for the assay of bovine testicular hyaluronidase in human blood following intravenous administration of the enzyme. Inhibition of hyaluronidase by the reported nonspecific serum inhibitor is minimal. However, the presence of human serum does alter the pH profile of hyaluronidase and enhances the activity of the enzyme at low pH values. Preliminary data indicates that the effects caused by serum on the pH optimum and activity of the enzyme are largely associated with the albumin fraction and are not due to the presence of endogenous serum hyaluronidase. The activation effect is not specific for any particular blood type and is independent of whether serum or citrated plasma is used. A similar effect to that of serum on hyaluronidase activity is produced by different buffer mixtures or increased NaCl concentration. It is recommended that bovine testicular hyaluronidase be measured at pH 4.0 in 0.1 m sodium citrate buffer containing 0.15 m NaCl as under these conditions the addition of human serum or citrated plasma does not alter the pH optimum of the enzyme. These recommendations necessitate certain modifications of the reducing N-acetylhexosamine assay method of Reissig et al. (J. L. Reissig, J. L. Strominger, and L. F. Leloir, 1955, J. Biol. Chem.217, 959–966).     

Countercurrent distribution and partition of Chlorella-sensitive and -resistant strain to the lytic agent in two-phase system. I

The influence of various buffering ions and NaCl concentrations on the distribution of Chlorella cells of two strains having differing surface and size were studied in an aqueous dextran-polyethylene glycol phase system using the one-step partition and CCD method.As models for these experiments we used an artificial mixture of Chlorella pyrenoidosa A 24 or Chlorella sp. 366 (strains sensitive to the infective lytic agent) and cells of Chlorella vulgaris B. resistant to this agent. The average size of Chlorella pyrenoidosa A 24 cells was 5.3 ± 0.53 μm and of Chlorella vulgaris B. 6.8 ± 0.55 μm.The condition of complete distribution of this algal mixture in 0.01 M sodium and potassium phosphate buffer is fulfilled when the concentration of NaCl is equal to or greater than a critical concentration of 15 mM of NaCl in the first, and 10 mM of NaCl in the second buffer.In buffers containing—COOH groups at pH 7.3 and low molarity a complete separation of mixture components was obtained. The addition of NaCl to maleate- and fumarate-NaOH buffers of pH 7.3 slightly worsened the separation effect which was visualized when decreasing the r_max difference value of both algal components on comparison with the separation obtained in a system without NaCl.At pH 6.0 in various buffers containing —COOH groups a complete separation of the algal components of the mixture was observed only when NaCl was absent. The addition of NaCl to some buffers of pH 6.0, e.g. maleate, malonate, and succinate, destroyed completely the successful separation and gave only a common peak of small r_max value.The fumarate buffer of pH 6.0, in the absence of NaCl, was an exception and gave only one common peak of small r_max value in contrast to its stereo-isomer maleic acid where two peak separation was obtained.     

The absorbance spectrum of the brown holo-membrane and the comparison of pI values of bacteriorhodopsin solubilized from purple membrane and from brown holo-membrane

Brown holo-membrane was prepared by the addition of all-trans-retinal to brown apo-membrane which was isolated from Halobacterium halobium grown in the presence of nicotine. The effects of pH and NaCI concentration on the absorbance spectrum of the brown holo-membrane were investigated in comparison with those of the purple membrane. The λ_max of the dark-adapted brown holo-membrane shifted from 560 to 600 nm by lowering pH. The pK value which was determined as the mid-point pH for the spectral red-shift was 5.8 in the absence of NaCl. It was lowered to 4.5 and 3.4 in 0.1 and 1 M NaCl solutions, respectively. The pK value for the brown holo-membrane was larger than the corresponding value for the purple membrane in the NaCl solution. Bacteriorhodopsins present in the purple membrane and in the brown holo-membrane were solubilized in the nonionic detergent, lauryl ester of sucrose. For both solubilized bacteriorhodopsins, the pK value of spectral red-shift was about 3.1 in water, and the pI value, determined by chromatofocusing, was about 4.6 at 22°C.     

Agar gel chromatography of rat liver ribosomal ribonucleic acids.

Ribosomal RNA (28s rRNA) of rat liver is selectively retained in 2 or 4% squashed agar gels equilibrated at 24–25°C with a sodium dodecyl sulfate-Tris-EDTA buffer containing 0.7 m NaCl. The absorbed polynucleotide could be recovered by elution with 0.1 m NaCl in the same buffer. Agar-gel electrophoresis and nucleotide composition indicate that the separation is close to quantitative. Column beds of 100–200 ml were used in the range of 3–6 mg of RNA. The procedure is simple, rapid and reproducible and gives excellent separation of 28 and 18s rat liver rRNA.     

Effects of salts and pH on the rate of erythrocyte diphosphoglycerate mutase

Erythrocyte diphosphoglycerate mutase is inhibited by several inorganic salts, the extent of the effect being characteristic of the anionic component, i.e., at ionic strength of about 0.1, SO₄^2? > Cl^? > CH₃COO^?. Using a partially purified enzyme preparation from human red blood cells, kinetic constants were determined in the presence of 0.1 m KCl to simulate the ionic environment of the cell. At pH 7.5, the addition of salt caused a 10-fold increase in the K_m of 1,3-diphosphoglycerate and a 46-fold increase in the K_i of 2,3-diphosphoglycerate. There was no effect of salt on the K_m of 3-phosphoglycerate or on the maximal velocity of the reaction. In the presence of 0.1 m KCl, the _i of inorganic phosphate increased from 0.3 mm to 0.6 mm. The K_m of 1,3-diphosphoglycerate was pH dependent, the values obtained being 3.6 μm at pH 6.75, 3.1 μm at pH 7.24, and 6.7 μm at pH 7.75. The K_i values for 2,3-diphosphoglycerate under the same conditions were: 12 μm at pH 6.75, 20μm at pH 7.24, and 53 μm at pH 7.75. The relative maximal velocity of the reaction has been evaluated over the same pH range. The maximal activity of the enzyme measured at 25 °C and pH 7.5 was 2 units/min/ml of packed red cells. From these studies, it is concluded that the effective enzymatic rate increases fourfold when the pH increases from 6.75 to 7.75.     

The dissociation of Helix pomatia alpha-hemocyanin. Microheterogeneity at pH 5.7 in 0.4 m NaCl.

The careful equilibrium light-scattering study by Engelborghs and Lontie (1973, J. Mol. Biol., 77, 577–587) of the dissociation into half molecules of the α-component of Helix pomatia hemocyanin demonstrated that, although complete dissociation was achieved in the presence of 1 m NaCl in 0. 1 m sodium acetate buffer at pH 5.7, incomplete dissociation occurred in 0.4 m NaCl in the same buffer in such a way that it could not be explained by simple application of the law of mass action. On the basis of their light-scattering data in acetate buffer containing 0.4 m NaCl, these authors indicated that the protein sample behaved like a mixture of thermodynamic species characterized by a multiplicity of dissociation constants. In this respect their conclusions were similar to those of Siezen and van Driel (1973, Biochim. Biophys. Acta, 295, 131–139) from their study of the influence of pH on association-dissociation processes at alkaline pH values. One simple model which actually fits the data of Engelborghs and Lontie reasonably well assumes the presence of some incompetent whole molecules, unable to dissociate into halves in 0.4 m NaCl: With a formation constant of 0.18 – 0.23 liters/g for whole molecules from half molecules, 30–35% of the material would be in the form of incompetent whole molecules. On the basis of this model, the major point of reinterpretation is that the sedimentation velocity diagram contains a fast Schlieren peak representing incompetent whole molecules and a slow peak representing a reaction mixture, rather than half molecules. This picture explains why the ratio of peak areas is independent of concentration, why material separated by ultracentrifugation appears not to reequilibrate when resubjected to ultracentrifugation, and why, on the other hand, mixing experiments have indicated rapid reequilibration of species.     

Acidophilic haloarchaeal strains are isolated from various solar salts

Haloarchaeal strains require high concentrations of NaCl for their growth, with optimum concentrations of 10–30%. They display a wide variety of morphology and physiology including pH range for growth. Many strains grow at neutral to slightly alkaline pH, and some only at alkaline pH. However, no strain has been reported to grow only in acidic pH conditions within the family Halobacteriaceae. In this study, we isolated many halophiles capable of growth in a 20% NaCl medium adjusted to pH 4.5 from 28 commercially available salts. They showed growth at pH 4.0 to 6.5, depending slightly on the magnesium content. The most acidophilic strain MH1-52-1 isolated from an imported solar salt (pH of saturated solution was 9.0) was non-pigmented and extremely halophilic. It was only capable of growing at pH 4.2–4.8 with an optimum at pH 4.4 in a medium with 0.1% magnesium chloride, and at pH 4.0–6.0 (optimum at pH 4.0) in a medium with 5.0% magnesium. The 16S rRNA and DNA-dependent RNA polymerase subunit B' gene sequences demonstrated clearly that the strain MH1-52-1 represents a new genus in the family Halobacteriaceae.     

Monomers of the NhaA Na+/H+ antiporter of Escherichia coli are fully functional yet dimers are beneficial under extreme stress conditions at alkaline pH in the presence of Na+ or Li+

NhaA, the Na(+)/H(+) antiporter of Escherichia coli, exists in the native membrane as a homodimer of which two monomers have been suggested to be attached by a beta-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the beta-hairpin, NhaA/Delta(Pro(45)-Asn(58)), revealed that in contrast to the dimeric mobility of native NhaA, the mutant has the mobility of a monomer in a blue native gel. Intermolecular cross-linking that monitors dimers showed that the mutant exists only as monomers in the native membrane, proteoliposomes, and when purified in beta-dodecyl maltoside micelles. Furthermore, pull-down experiments revealed that, whereas as expected for a dimer, hemagglutinin-tagged wild-type NhaA co-purified with His-tagged NhaA on a Ni(2+)-NTA affinity column, a similar version of the mutant did not. Remarkably, under routine stress conditions (0.1 m LiCl, pH 7 or 0.6 m NaCl, pH 8.3), the monomeric form of NhaA is fully functional. It conferred salt resistance to NhaA- and NhaB-deleted cells, and whether in isolated membrane vesicles or reconstituted into proteoliposomes exhibited Na(+)/H(+) antiporter activity and pH regulation very similar to wild-type dimers. Remarkably, under extreme stress conditions (0.1 m LiCl or 0.7 m NaCl at pH 8.5), the dimeric native NhaA was much more efficient than the monomeric mutant in conferring extreme stress resistance.     

Adhesion of Pseudomonas fluorescens (ATCC 17552) to Nonpolarized and Polarized Thin Films of Gold

The adhesion of Pseudomonas fluorescens (ATCC 17552) to nonpolarized and negatively polarized thin films of gold was studied in situ by contrast microscopy using a thin-film electrochemical flow cell. The influence of the electrochemical potential was evaluated at two different ionic strengths (0.01 and 0.1 M NaCl; pH 7) under controlled flow. Adhesion to nonpolarized gold surfaces readily increased with the time of exposition at both ionic-strength values. At negative potentials (−0.2 and −0.5 V [Ag/AgCl-KCl saturated {sat.}]), on the other hand, bacterial adhesion was strongly inhibited. At 0.01 M NaCl, the inhibition was almost total at both negative potentials, whereas at 0.1 M NaCl the inhibition was proportional to the magnitude of the potential, being almost total at −0.5 V. The existence of reversible adhesion was investigated by carrying out experiments under stagnant conditions. Reversible adhesion was observed only at potential values very close to the potential of zero charge of the gold surface (0.0 V [Ag/AgCl-KCl sat.]) at a high ionic strength (0.1 M NaCl). Theoretical calculations of the Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy for the bacteria-gold interaction were in good agreement with experimental results at low ionic strength (0.01 M). At high ionic strength (0.1 M), deviations from DLVO behavior related to the participation of specific interactions were observed, when surfaces were polarized to negative potentials.     

Structure of the histone tetramer (H3-H4)2: 1. Peculiarities of optical absorption,fluorescence and light scattering in response to changes in ionic strength and pH of the medium

Using the methods of spectrophotometry, spectrofluorimetry, light scattering and gel filtration, it was shown that, at pH 5.6 and 7.4 and various ionic strengths, the histone tetramer (H3-H4)₂ may have several structural states with different packing of the polypeptide chains of histones H3 and H4. Two structural changes of the tetramer (H3-H4)₂ at pH 7.4 in the ranges 0.1–0.3 m and 0.7–0.9 m NaCl were observed. In the high ionic strength solution, the tetramer (H3-H4)₂ had a more compact structure at pH 7.4 than at pH 5.6. At pH 3.0 destruction of the histone tetramer (H3-H4)₂ and formation of non-specific aggregates took place.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.     

Use of Gradient Plates To Study Combined Effects of Temperature, pH, and NaCl Concentration on Growth of Monascus ruber van Tieghem, an Ascomycetes Fungus Isolated from Green Table Olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35°C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35°C, whereas at 35°C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

The effect of potassium sorbate, NaCl and pH on the growth of food spoilage fungi

In this study, the hurdle technology approach was used to prevent fungal growth of common spoilage fungi in naturally fermented black olives (Alternaria alternata, Aspergillus niger, Fusarium semitectum andPenicillium roqueforti). The factors studied included a combination of different concentrations of potassium sorbate (100 up to 1000 mg/L), a range of pH values (4.5, 5, 5.5, 6, and 6.5) and levels of NaCl (0, 3.5, 5, 7.5, and 10%).Alternaria alternata was the most sensitive fungus whereasP. roqueforti was the most resistant fungi against all hurdle factors. The combination of all hurdles completely inhibitedA. alternata andF. semitectum by lowest inhibitory factors, such as 100 mg/L potassium sorbate with 3.5% NaCl at pH 5. On the other hand, at pH 5, A.niger andP. roqueforti were totally prevented by a combination of 300 mg/L potassium sorbate with 10% NaCl and 400 mg/L potassium sorbate with 7.5% NaCl, respectively. Potassium sorbate and 5–10% NaCl interaction had significant stimulation effect onp. roqueforti andA. niger (p<0.05). This study indicates that potassium sorbate is a suitable preserving agent to inhibit growth of fungi in fermented products of pH near 4.5 regardless levels of NaCl. For products of slightly higher pH, the addition of potassium sorbate is suggested in combination with NaCl.     

Fluorescence analysis of G·C versus A·T binding of quinacrine to DNA

The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m, NaCl0.1 m, EDTA 10^?3m, pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.