Physiological Conditions Of Ionic Strength Research Articles

Two-dimensional crystallization of DNA gyrase B subunit on specifically designed lipid monolayers

The B subunit of DNA gyrase formed two-dimensional crystals when bound to a specifically recognized phospholipid spread into a monolayer at the air/water interface. The especially designed lipids consisted of novobiocin coupled through the 3' or 2″ hydroxyl group and a hydrophilous linker of a given length to dioleoylphosphatidic acid. Two-dimensional crystals of the gyrase B subunit are formed under physiological conditions of pH and ionic strength, with no precipitant added to the solution. Crystal diffraction extended to a 2.7 nm resolution in negative stain, with unit cell parameters a = 6.1 nm, b = 7.6 nm and γ = 64°.

Wet lipid-protein membranes imaged at submolecular resolution by atomic force microscopy

We have employed an AFM to determine the structural properties of supported planar membranes and membrane-bound proteins in an aqueous environment. Images of an asymmetric Langmuir Blodgett film of a charged phospholipid show long range positional as well as orientational order; individual headgroups are resolved. In order to study biofunctional membranes we have employed a recently introduced technique that allows the controlled formation of planar lipid-protein membranes on solid supports from a vesicle suspension. Combining this technique with the AFM permits the nondestructive imaging of these models of cell membranes at molecular resolution under physiological conditions of ionic strength and temperature.

Elastase activities of human bladder cancer cell lines derived from high grade invasive tumours

Elastase activities in intact human bladder cancer cell lines, established from three patients, were measured using a fluorogenic substrate highly specific for elastase, under conditions of physiological pH and ionic strength. This method allowed separation of cell-associated from secreted enzyme activity. As secreted elastase accounted for only 8% of the total, we concluded that the elastases were present at the cell surface. Inhibition studies using extracts of cell-surface elastases showed them to be serine proteinases which were also inhibited by α 1-antitrypsin. Partially purified fractions showing the highest specific activity towards the fluorogenic substrate hydrolysed insoluble elastin thus confirming the presence of elastases. This is the first time that elastase activity has been demonstrated in human bladder cancer cells and may represent a mechanism involved in tumour invasion.

Contractile activation and the effects of 2,3-butanedione monoxime (BDM) in skinned cardiac preparations from normal and dystrophic mice (129/ReJ).

Small cardiac myofibrillar preparations were obtained from the right ventricle of normal (129/ReJ) and dystrophic (129/ReJ dy/dy) mice and were chemically skinned in a relaxing solution by exposure to Triton X-100 (3% v/v). (2) The isometric force produced in these skinned cardiac preparations at different sarcomere lengths was measured in solutions of different [Ca2+] and ionic strength. The effect of the negative inotropic drug 2,3-butanedione monoxime (BDM), which is known to act at the myofibrillar level was also investigated. (3) The murine cardiac preparation from normal animals was found to develop 50% maximal force at a pCa (= -log10[Ca2+]) of 5.59 +/- 0.08 and 5.94 +/- 0.03 (mean +/- SD) under physiological (ionic equivalents concentration, I = 154 mM; pH 7.10; [Mg2+] 1 mM) and low ionic strength (I = 94 mM; pH 7.10; [Mg2+] 1 mM) conditions respectively. The isometric force curves were significantly shallower at low ionic strength (Hill coefficient, 1.8 +/- 0.1) than at physiological ionic strength (Hill coefficient, 2.6 +/- 0.3) and the sarcomere length effect on the force-pCa relation was markedly reduced at lower ionic strength. (4) Increasing BDM concentrations in solutions up to 100 mM reduced the maximum Ca2+-activated force of cardiac preparations from normal mice to less than 6% of the control values in a dose dependent fashion. BDM also rendered the cardiac preparations less sensitive to Ca2+ by a factor of up to 1.5 in a process which showed saturation at BDM concentrations higher than 15 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of the sedimentation properties of the U-snRNPs of mouse embryoid bodies (OTT6050) by sucrose gradient centrifugation under physiological ionic conditions and electrophoretic analysis of the RNA in various fractions.

Fractionation of nuclear extracts of mouse embryoid bodies (EBs; OTT6050) on sucrose gradients (5-20%) under conditions of physiological ionic strength (150 mM NaCl, 1mM MgCl2) and an analysis of the RNA in various fractions by gel electrophoresis revealed that the major U small nuclear ribonucleoproteins (U-snRNPs; U1a, U1b, U2, U4, U5 and U6-snRNPs) sediment over a wide region of the gradient, although they sediment preponderantly in the light region of the gradient. This result suggests that, under these experimental conditions, some of the populations of snRNPs exist as free particles separated from large nuclear RNP particles, while some of the populations associate with them in EBs. Furthermore, all species of these major U-snRNPs appear to associate with the larger nuclear RNP particles of EBs, since all these species sediment in the heavier fractions (approximate greater than or equal to 60S) of the 15-40%/50% gradients. The relative abundance of the various species of major U-snRNPs can also be observed to vary among the fractions of the gradients. A similar analysis of the post-mitochondrial cytoplasmic fraction showed that some leakage of the major U-snRNPs, but not the selective leakage of any particular species of U-snRNP, from the nuclear fraction, occurred during aqueous fractionation of the cells. Some species of RNA, larger than the snRNAs U1a/b and U2 respectively were also detected in the cytoplasmic fraction.

Specificity in formation of triple-stranded nucleic acid helical complexes: studies with agarose-linked polyribonucleotide affinity columns.

The binding of a variety of deoxyribo and ribo homo- and copolynucleotide complementary duplexes to agarose-linked homopolynucleotide affinity columns has been studied. The results provide information concerning the specificity of recognition of complementary base pairs of nucleic acids through a mechanism that involves triple-helix formation under physiological conditions of ionic strength, pH, and temperature. The method employed made it possible, for the first time, to survey the full range of base triplets conceivable from the canonical nucleic acid bases and, in addition, hypoxanthine and thereby to differentiate between those triplets which can and cannot form. Certain previously observed features of the stereochemistry of double-helical targets for third-strand binding are confirmed, and some unrecognized features are elaborated. These include a general requirement for clusters of purine residues in one strand, protonation of third-strand C residues, the ability of natural third-strand residues to distinguish between A.T/U and G.C base pairs, and a capacity of third-strand (unnatural) I residues to recognize all base pairs within such clusters. Thus, the basis for a third-strand binding code is demonstrated.

Potassium transport by pancreatic and parotid zymogen granule membranes.

Zymogen granules that were stable at physiological conditions of pH, ionic strength, and temperature were isolated from the rat pancreas and parotid. The cation permeability of these granules was evaluated to characterize the mechanism of secretagogue-stimulated fluid secretion by acinar cells. Granule swelling and lysis provide a measure of the rate of cation transport, since the use of ionophore combinations such as tripropyltin and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) will render cation conductance the rate-limiting step for salt influx. This technique supplies evidence for the existence of K+ conductance in the granule membrane. The pancreatic and parotid granules have a K+-selective conductance that is not inhibited by the K+ channel blockers barium, tetraethylammonium, quinidine, cesium, or 4-aminopyridine. Furthermore, the intragranular pH of pancreatic zymogen granules was measured to be approximately 6.5 and was identified as a factor that modulates the K+ conductance. Although the pancreatic and parotid granules were qualitatively identical, quantitatively the relative K+ transport rate constant was over twofold higher for the parotid than for the pancreatic granules. The zymogen granule K+ conductance may have an important role in active K+ secretion by exocrine glands, which is prominent in the parotid after stimulation with beta-adrenergic agents.

Crystallizations of human serum amyloid P component (SAP)

Human serum amyloid P component (SAP) crystallizes readily by batch methods at its isoelectric pH of 5.5 in the presence of calcium ions and small quantities of PEG 6000. Gel filtration under similar conditions shows the protein to be pentameric while under more physiological conditions of pH and ionic strength it is known to exist as stable decamers. The protein exhibits a calcium dependent affinity for the β-D-galactopyranoside pyruvate acetal moiety of agarose. The isolated sugar has been successfully employed as a crystallization additive to modify growth rate and crystal habit.

Selectivity of the anthracyclines for negatively charged model membranes: role of the amino group.

The equilibrium-binding affinities of six adriamycin analogues and four daunomycin derivatives for negatively charged dimyristoyl phosphatidylcholine/dimyristoyl phosphatidic acid (DMPC/DMPA) small unilamellar vesicles are compared with values for electroneutral DMPC liposomes. Binding of the daunomycin series to negatively charged dimyristoyl phosphatidyl glycerol (DMPG) vesicles was also examined. Under physiological conditions of pH and ionic strength, substitution of the amino group of adriamycin or daunomycin resulted in a reduced affinity for negatively charged bilayers, even if the substituent enhanced the degree of ionization of the amine. Decreasing the ionic strength increases the binding affinity for acidic membranes but decreases the drug affinity for neutral membranes. We propose that the electrostatic bond of the phosphate-amino group that has been shown to exist between anthracyclines and phosphatidic acid is sterically destabilized by substitution of the amino group. The results are consistent with a mode of anthracycline binding to negatively charged membranes which is driven by hydrophobic and electrostatic considerations but is destabilized by steric bulk at the amino group. The data also provide insight into the design of new anthracyclines with high membrane affinities and reduced uptake; such directed interaction with plasma membranes may enhance antineoplastic potential while reducing cardiac toxicity.

Characterization of anti-Z-RNA polyclonal antibodies: epitope properties and recognition of Z-DNA

Chemically brominated poly[r(C-G)] [Br-poly[r(C-G)]] containing 32% br8G and 26% br5C was recently shown to contain a 1:1 mixture of A- and Z-form unmodified nucleotides under physiological conditions of temperature, pH, and ionic strength [Hardin, C. C., Zarling, D. A., Puglisi, J. D., Trulson, M. O., Davis, P. W., & Tinoco, I., Jr. (1987) Biochemistry 26, 5191-5199]. Proton NMR results show that more extensive bromination of poly[r(C-G)] (49% br8G, 43% br5C) produces polynucleotides containing greater than 80% unmodified Z-form nucleotides. Using these polynucleotides as antigens, polyclonal antibodies were elicited in rabbits and mice specific for the Z-form of RNA. IgG fractions were purified from rabbit anti-Br-poly[r(C-G)] sera and characterized by immunoprecipitation, nitrocellulose filter binding, and ELISA. Two different anti-Z-RNA IgG specificities were observed. Decreased levels of brominated nucleotides in the immunogen correlated with an increased extent of specific cross-reactivity with Z-DNA. Inoculation of rabbits with polynucleotide immunogens containing 49% br8G and 43% of br5C produced specific anti-Z-RNA IgGs that do not recognize Z-DNA determinants. This suggests that the 2'-OH group is part of the anti-Z-RNA IgG determinant. In contrast, Br-poly[r(C-G)] immunogens containing 32% br8G and 26% br5C produced IgGs that specifically recognize both Z-RNA and Z-DNA. These results show that the bromine atoms are not required for recognition of the Z conformation by the antibodies. The affinity of these anti-Z-RNA IgGs for Z-RNA is about 10-fold higher than for Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

PH-Dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

Physiological ionic strength conditions prevented low pH-mediated destabilization of the adenovirion. A conformational change of the virion was induced at low pH as demonstrated by endoproteolytic cleavage of virions with dispase at pH 5.0. Hidden cleavage sites of the hexons were exposed and upon enzymatic digestion, virions still were intact as physical entities. Enzymatic cleavage of the hexon protein increased its hydrophobicity.

PH-Dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

Physiological ionic strength conditions prevented low pH-mediated destabilization of the adenovirion. A conformational change of the virion was induced at low pH as demonstrated by endoproteolytic cleavage of virions with dispase at pH 5.0. Hidden cleavage sites of the hexons were exposed and upon enzymatic digestion, virions still were intact as physical entities. Enzymatic cleavage of the hexon protein increased its hydrophobicity.

Glutathione as a scavenger of free hemin: A mechanism of preventing red cell membrane damage

The interaction of glutathione and hemin was studied at physiological ionic strength and pH conditions. Formation of a glutathione-hemin complex was assessed from the appearance of spectral changes in the visible region. In the presence of excess cyanide and histidine and upon oxidation of the sulfhydryl group, no complex was formed. From these results it was concluded that the thiol group of glutathione serves as a ligand for the heme iron. A binding constant of 3.1(±0.1) × 10 4M −1 was calculated by use of a Hill plot. The hemolytic effect of hemin on red cell was much reduced in the presence of glutathione. Since hemolysis results from association of hemin with membrane components its binding in the presence and absence of glutathione was studied. It was found that the affinity of hemin for the cytoskeletal membrane proteins as well as for the membrane lipid core was decreased in the presence of glutathione. It was concluded that glutathione competes with the membranes for hemin and by doing so can defend the membrane from injury by hemin.

Cranin: a laminin-binding protein of cell membranes.

We report that a 120-kDa glycoprotein is the predominant laminin-binding protein detected within plasma membranes of rodent NG108-15 neural hybrid cells, embryonic chicken brain, and mouse 3T3 fibroblasts. This protein was detected when membrane extracts were separated by PAGE, transferred to nitrocellulose, and incubated with laminin at concentrations as low as 2.8 X 10(-11) M, under conditions of physiological ionic strength and pH and in the presence of calcium ions. It behaves as an integral membrane component, and its laminin-binding moiety is accessible to the external face of the cell surface. Moreover, it appears to bind to a site on laminin that is very sensitive to proteolysis. The properties of this protein, which we have termed "cranin," distinguish it from other known laminin receptors and make it a candidate to mediate some of the effects of laminin upon cells.

ChemInform Abstract: Aluminium Speciation Studies in Biological Fluids. A New Investigation of Aluminium Hydroxide Equilibria Under Physiological Conditions

Abstract(37 °C, 0.15 M aqueous NaCl).

Differential stability of the higher order structure of chromatin associated with genes having different transcriptional activity

We have investigated the stability of the higher order structure of chromatin associated to genes with display a different transcriptional activity in adult rat liver. Nuclei were digested with micrococcal nuclease and chromatin was fractionated by sedimentation in sucrose gradients. Specific DNA sequences were revealed by dot-blotting. In conditions of physiological ionic strength the distribution of the inactive γ-casein gene sequences is similar than the bulk of chromatin. In the same conditions the relative content of the albumin gene, highly expressed in adult rat liver, revealed an enhanced instability of the chromatin superstructure. The distribution of the potentially active but silent α-fetoprotein sequences in adult liver showed an intermediate unfolding of its chromatin superstructure. These distinct behaviour was not observed in non-physiological ionic strength conditions. Our results suggest that distinct folding of the local higher order structure of chromatin actually occurs in the region of active, potentially active and inactive genes.

Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects.

The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels.

Speciation studies in relation to magnesium bioavailability. Formation of Mg(II) complexes with glutamate, aspartate, glycinate, lactate, pyroglutamate, pyridoxine and citrate, and appraisal of their potential significance towards magnesium gastrointestinal absorption

Associations of Mg 2+ ions with organic ligands are frequently advertised as likely to enhance the bioavailability of magnesium from orally administered commercial preparations. However, no systematic study of the relevant equilibria has been produced so far to substantiate these assertions, and no superiority has yet been demonstrated for any magnesium salt on clinical grounds. After a review concerning different aspects of magnesium gastrointestinal absorption, in particular with respect to calcium interactions, this paper deals with the determination of formation constants for magnesium complexes with glutamate, aspartate, glycinate, lactate, pyroglutamate, pyridoxine and citrate, under physiological conditions of ionic strength (0.15 mol dm −3) and temperature (37 °C). Corresponding results are then used to assess the potential capacity of each of these ligands to mobilise Mg 2+ ions into membrane diffusible complexes. At usual therapeutic concentrations, pyroglutamate and pyridoxine do not coordinate magnesium in appreciable amounts, and glycinate and lactate do not form any neutral complex. In contrast, glutamate, aspartate and citrate do form neutral magnesium species; the extent of their expected effects with respect to magnesium uptake by enterocyte membranes is discussed on the basis of relevant computer simulations. Potential calcium interactions are also examined, which required the determination of formation constants for calcium complexes with these three ligands.

The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities.

A 50-kDa polypeptide that is rapidly phosphorylated on addition of [gamma-32P]ATP to isolated clathrin-coated vesicles is shown here to be identical to the 50-kDa component (AP50) of the clathrin assembly protein (AP), a complex that promotes the assembly of clathrin coat structures under physiological conditions of pH and ionic strength. Phosphorylation of the AP50 occurred readily at 0 degrees C, almost exclusively on a threonyl residue(s). This reaction is attributable to autophosphorylation, since the AP50 was able to covalently incorporate 32P from [gamma-32P]ATP after separation by either one- or two-dimensional sodium dodecyl sulfate gel electrophoresis. Kinetic studies in solution were consistent with an intramolecular phosphorylation event; in addition, a concentration-dependent increase in AP50 phosphorylation was observed that may reflect intermolecular AP-AP activation of autophosphorylation. The phosphorylated AP50 was resistant to several inorganic phosphatases tested but was a substrate for protein phosphatases 1 and 2A, suggesting that a physiological phosphorylation-dephosphorylation cycle may exist. The phosphorylation state of the AP50 did not affect the ability of the AP to promote in vitro clathrin coat assembly. These and other data suggest that unique structural domains of the assembly protein are responsible for assembly (the 100-kDa components) and autophosphorylation (the AP50) and that the latter may be active as a protein kinase in the intact cell.