Chaotropic Series Research Articles

Characterization of Glial Cell K-Cl Cotransport

Background: The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells. Methods: We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl^--dependent bumetanide-insensitive Rb⁺ (a K⁺ congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb⁺ influx was measured at a fixed external Cl concentrations, [Cl^-]_e, as a function of varying external Rb concentrations, [Rb⁺]_e, and at a fixed [Rb⁺]_e as a function of varying [Cl^-]_e, and with equimolar Cl replacement by anions of the chaotropic series. Results: RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K_m for KCC-mediated Rb⁺ influx was 15 mM [Rb⁺]_e, and V_max 12.5 nmol Rb⁺ * mg protein^-1 * minute^-1. The calculated apparent K_m for external Cl^- was 13 mM and V_max 14.4 nmol Rb⁺ * mg protein^-1 * minute^-1. The anion selectivity sequence of the furosemide-sensitive Rb⁺ influx was Cl^->>Br-=NO₃^->I^-=SCN^->>Sfm^- (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N-ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb⁺ influx. A ñ50% NEM-induced loss of intracellular K⁺ was prevented by furosemide. Conclusion: We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.

Thermal aggregation of globulin from an indigenous Chinese legume, Phaseolus angularis (red bean)

The thermal aggregation behavior of red bean ( Phaseolus angularis) globulin (RBG) was studied at ≈1% (w/v) protein concentration in 0.01 M phosphate buffer, pH 7.4. The percentage of protein precipitated was affected by heating temperature, heating time and salt concentration. The influences of several salts of the chaotropic series and protein structure-modifying agents on thermal coagulation of RBG were also investigated. The effects of chaotropic salts did not follow the lyotropic series of anions. Sodium dodecyl sulfate caused a more pronounced reduction in heat-induced aggregation of RBG than did dithiothreitol, while N-ethylmaleimide did not affect aggregation until after a long heating period. Differential scanning calorimetric (DSC) data showed that heat aggregation of RBG was preceded by thermal denaturation. SDS-PAGE showed that heating led to the disappearance of some protein bands, and the basic polypeptide of 11S globulin (legumin) was not found in the buffer-soluble aggregates. Heating caused increases of surface hydrophobicity, again suggesting protein unfolding prior to aggregate formation. The buffer-insoluble aggregates did not show any DSC response, indicating extensive denaturation, and had a lower surface hydrophobicity and higher disulfide content than the buffer-soluble aggregates. The data suggest that electrostatic and hydrophobic interactions may play an important role in thermal aggregation of RBG, with disulfide bonds playing a limited role.

Glutamine Catabolism by Heart Muscle: Regulation of Phosphate-Activated Glutaminase by ATP, Citrate, and Chloride

Production of glutamate from glutamine by rat heart mitochondria was stimulated by citrate and ATP in a dose-dependent manner. Citrate required phosphate for manifestation of its stimulatory action, whereas ATP enhanced glutaminase activity with and without phosphate. At low concentrations (1-50 mM) ATP was more potent than citrate, whereas the opposite was true at high (50-150 mM) levels of these anions. Both citrate and ATP decreased the concentration of phosphate required for half-maximal stimulation of glutamate production (EC50) and reduced the value of the Hill coefficient. Phosphate lowered the EC50 for ATP. Chloride and other anions of chaotropic series inhibited glutamine catabolism, most likely by causing depolymerization of the enzyme. Rupture of mitochondrial membranes by freeze-thawing decreased the responsiveness of glutaminase to phosphate, ATP, and especially to citrate but it did not alter the inhibition of the enzyme by chaotropic anions. It is concluded that phosphate, ATP, and citrate act in concert to modulate glutaminase activity in heart in vivo. Chloride, which exhibits a small inhibitory effect at a concentration normally present in cardiac muscle, may assume a greater regulatory role in situations accompanied by a rise in the internal level of this anion.

The use of chaotropic salts for separation of ribonucleic acids and proteins from yeast nucleoproteins

The effect of chaotropic salts on the dissociation of ribonucleic acid from yeast nucleoprotein complex was studied. The effectiveness of various salts on the dissociation of ribonucleic acid followed the chaotropic series; i.e., Cl(3)CCOONa = NaClO(4) > NaBr > NaCl. Treatment of the nucleoprotein complex with 0.5M Cl(3)CCOONa or NaClO(4) resulted in RNA removal of about 80%, whereas NaCl and NaBr removed only about 10 and 25%, respectively. Based on the results presented, a simple and novel method for industrial-scale preparation of single-cell proteins with low levels of nucleic acid is proposed.

Mechanism of lysis of Escherichia coli by ethanol and other chaotropic agents.

Ethanol has been shown to inhibit the assembly of cross-linked peptidoglycan and to induce cell lysis in Escherichia coli. These effects of ethanol appear to result from the weakening of hydrophobic interactions by ethanol rather than from the intercalation of ethanol into membranes. Other chaotropic agents also inhibited cross-linking and induced lysis. The potency of chaotropic anions with regard to this effect followed the expected chaotropic series. Antichaotropic agents, which strengthened hydrophobic interactions, antagonized ethanol-induced lysis. The weakening of hydrophobic interactions by ethanol is proposed as a general mechanism by which ethanol and other chaotropic agents could affect membrane-associated enzyme activities.

Factors affecting terminal deoxynucleotidyl transferase activity in cacodylate buffer

For reproducible terminal deoxynucleotidyl transferase activity in cacodylate buffer, assay tubes need scrupulous cleaning, and for maximal activity with the commercial enzyme, zinc ion is required when dATP is polymerized. Potassium ion is also required for maximal activity, though ammonium ion may be used in some situations. The enzyme is readily inhibited by a variety of negative ions, and inhibition by the monovalent anions follows the chaotropic series. This result suggests that cacodylate ion belongs at the beginning of the chaotropic series and so may be generally useful in assaying enzymes with labile activity.

Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase.

As chaotropic salts are generally believed to affect water structure in a manner which increases lipophilicity of water, they may seem to be capable of substituting for detergents in the solubilization of particulate enzyme. Although solubilization either by detergents or by chaotropic salts has been demonstrated with several membrane proteins, the effects these agents have on the properties and activity of an enzyme may be quite different. This is illustrated by the effects on mammalian mitochondrial dihydroorotate dehydrogenase. Stability of the solubilized enzymic activity is dependnet on the presence of a detergent and maximum enzymic activity is observed at the critical micelle concentration of the detergent. Addition of low concentrations of various anions of the chaotropic series further enhances activity while higher concentrations of these anions, although increasing solubility of the enzyme, irreversibly inhibit catalysis.

Purification of staphylococcal alpha-toxin by adsorption chromatography on glass.

Staphylococcal alpha-toxin was purified from Staphylococcus aureus growth medium using adsorption chromatography on controlled pore glass beads. Elution of alpha-toxin from the unmodified glass surface of the beads with various anions generally followed the chaotropic series. Alpha-toxin, purified by glass bead chromatography, is composed of a single electrophoretic form, containing less than 2% of other forms.

Effect of free magnesium and salts on the inorganic pyrophosphatase purified from a slightly halophilic Vibrio alginolyticus

1. 1. Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) of a slightly halophilic marine Vibrio alginolyticus was purified 100-fold. This enzyme has an optimum at pH 8.7 and exhibits an absolute requirement for free Mg 2+, MgPP i 2− acts as a true substrate for this enzyme, but Mg 2PP i is also able to serve as a substrate. 2. 2. The enzyme is inactivated by preincubation with salts, which are dependent upon the species of monovalent anions; the order of their effectiveness follows a chaotropic series: ClO 4 −, SCN > Br − ⩾ NO 3 − > Cl −. The dissociation constant for the enzyme chloride complex was estimated to be 150 mM. The enzyme is effectively protected from inactivation only when both Mg 2+ and MgPP i 2− are coexistent, either one of which is ineffective separately. 3. 3. The enzyme is inhibited by salts independently of the species of monovalent cations and anions. NaCl exhibits a parabolic non competitive inhibition. The salt inhibition is prevented by increasing Mg 2+. 4. 4. It is suggested that the inactivation and inhibition of this enzyme by salt might be protected by the presence of high concentration of Mg 2+ under physiological conditions.