Stability of the hexagonal lattice structure formed by an R-form lipopolysaccharide of Klebsiella: decrease in the stability by electrodialysis and recovery by addition of the magnesium

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non-electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon-like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non-electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.     

Disintegration of Mg2+ -induced hexagonal assembly of an R-form lipopolysaccharide from Klebsiella pneumoniae by treatment with CaCl2

R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-: K1-), which was precipitated by the addition of 2 volumes of ethanol containing 10 mM MgCl2 for the purification process, ultrastructurally exhibited membrane pieces consisting of an ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm. When the R-form LPS was suspended in 50 mM tris (hydroxymethyl) aminomethane buffer (at pH 8.5) containing 1 mM or higher concentrations of CaCl2 and kept at 4 C for 10 hr, the ordered hexagonal lattice structure of the R-form LPS was disintegrated and changed to an irregular rough, mesh-like structure. By treatment with CaCl2, the content of Mg in the LPS was markedly decreased, and conversely, the content of Ca was increased to a level depending upon the concentration of CaCl2. Results indicate that the addition of CaCl2 to suspensions of the Mg-bound R-form LPS result in a tighter binding of Ca2+ to the R-form LPS and the release of Mg2+ from the R-form LPS, and as a consequence, destroys the Mg2+ -induced ordered hexagonal lattice structure of the R-form LPS.     

Relationship between Mg2(+)-induced hexagonal assembly of R-form lipopolysaccharides and chemical structure of their R-cores

The relationship between formation of the Mg2(+)-induced hexagonal lattice structure by R-form lipopolysaccharides (LPS) and chemical structure of their R-cores was investigated using different kinds of R-form LPS from a series of mutants of Salmonella minnesota or S. typhimurium. The optimal experimental condition for formation of the hexagonal lattice structure was to suspend LPS preparations, from which cationic material was removed by electrodialysis, in 50 mM tris (hydroxymethyl) aminomethane buffer at pH 8.5 containing 10 mM MgCl2. Under this experimental condition, Rb1 LPS formed the hexagonal lattice structure with the lattice constant of 14.0 +/- 0.2 nm. Ra LPS, which possesses the full length of R-core, also formed the hexagonal lattice structure but its lattice constant was larger (18.1 +/- 0.2 nm) than that of Rb1 LPS (the lattice structure by Ra LPS was looser than that by Rb1 LPS). All the other R-form LPS preparations tested, RcP+, PcP-, Rd1P-, and Re LPS, whose R-cores are shorter than that of Rb1 LPS, did not form the hexagonal lattice structure, but formed membranous structures showing various shapes which consisted of multiple bilayer structures. Failure to form the hexagonal lattice structure was the common feature of these kinds of R-form LPS irrespective of temperature at which the LPS suspensions in 10 mM MgCl2-50 mM Tris buffer were incubated. From the results of the present study it was concluded that capability of R-form LPS to form the hexagonal lattice structure has a close correlation with the chemical structure of their R-cores.     

In vitro hexagonal assembly of R-form lipopolysaccharides: effect of pH on the Mg+2-mediated hexagonal assembly

The R-form lipopolysaccharide (LPS) from Escherichia coli K-12, from which cationic material had been removed by electrodialysis and the pH of which had fallen to 3.6, formed a rough hexagonal lattice structure with the lattice constant of about 19 nm. The rough hexagonal structure was maintained in buffers at pH 5 or lower but disintegrated into the ribbon-like structures in buffers at pH 6 or higher. However, in the presence of 10 mM Mg2+, the hexagonal lattice structure was not disintegrated even at alkaline pH levels but conversely it became more dense. At pH 8.3 to 8.9, the hexagonal lattice structure with the shortest lattice constant (15 nm) was formed. The same optimal pH levels were obtained for formation of the dense hexagonal lattice structure (lattice constant, 14 to 15 nm) by the electrodialyzed LPS from Klebsiella pneumoniae strain LEN-111 (O3-:K1-). The ability of Mg2+ to induce formation of the dense hexagonal lattice structure of the K-12 LPS depends upon the presence of buffers showing the optimal pH levels, since a very high concentration of Mg2+ such as 500 mM was required for the lattice formation in distilled water. The amount of the magnesium bound to the K-12 LPS did not significantly differ throughout the pH range of 3 to 9. Therefore, the optimal pH range is another essential factor for formation of the dense hexagonal lattice structure of the LPS in addition to binding of the magnesium to the LPS.     

Stability of the hexagonal lattice structure formed by an R-form lipopolysaccharide of Klebsiella: study of long-range stability

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The stability of this hexagonal lattice structure in long-term incubation at 4 C was investigated. The hexagonal lattice structure was stable for at least 220 days when the LPS was suspended in distilled water, but it had been disintegrated into a rough mesh-like structure when the LPS was suspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.5, at 4 C for 60 days. Half of the Mg bound to the LPS was released when the LPS was suspended in Tris buffer for 60 days, whereas Mg was not released when it was suspended in distilled water even for 220 days. By contrast, it was stable for at least 220 days in Tris buffer containing 5 mM MgCl2. The LPS suspended in Tris buffer for 60 days, at which time the structure had been disintegrated, could be restored to the original hexagonal lattice structure within 24 hr by addition of 5 mM MgCl2. From these results it is concluded that the hexagonal lattice structure of the LPS retains long-range stability if Mg bound to the LPS is not released from the LPS.     

In vitro hexagonal assembly of lipopolysaccharide of Escherichia coli K-12

We examined Escherichia coli K-12 lipopolysaccharide (LPS), which is known to be an R-form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K-12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2 +/- 0.3 nm and that of the calcium salt form 18.5 +/- 0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.     

Hexagonal assembly of the magnesium salt of an R-form lipopolysaccharide from Klebsiella pneumoniae: its lowered stability compared with original non-electrodialyzed preparation

The magnesium salt of R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-) that was prepared after the removal of cationic materials by electrodialysis formed essentially the same ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm as the original non-electrodialyzed preparation of the R-form LPS. When the magnesium salt was suspended in 50 mM glycine buffer or Tris buffer at pH 1.4 to 9.5 and kept at 4 C for 24 hr, its content of Mg was markedly decreased, and its hexagonal lattice structure was changed to a swollen hexagonal lattice structure with extended lattice constants at pH 1.4 and to a loose mesh-like structure at pH 3.0 or higher. In the original non-electrodialyzed preparation of the R-form LPS, the release of Mg and disintegration of the hexagonal lattice structure did not occur by suspending in buffers at pH 1.4 to 8.5 at 4 C for 24 hr, but occurred only at pH 9.0 or higher. The results suggest that organic cations that can be removed by electrodialysis play some part in tight binding to Mg2+ and in stabilizing the ordered hexagonal assembly of the R-form LPS.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella sp.

We extracted an R-form lipopolysaccharide (LPS) by the phenol-water method from Klebsiella sp. strain LEN-111 (O3-:KI-) and followed the changes in ultrastructure of the LPS during the extraction procedure. When the LPS was obtained from the water phase of an extract by addition of 2 volumes of 10 mM MgCI2-ethanol, it consisted of membrane pieces with a hexagonal lattice structure with a lattice constant of 14 to 15 nm. The lattice structure of the LPS was disrupted into short rods with sodium dodecyl sulfate, but the same hexagonal lattice structure was again formed by precipitation with 2 volumes of 10 mM MgCI2-ethanol. The LPS preparation after two cycles of treatment by the phenol-water method, which contained no detectable amounts of proteins, kept an unaltered ability to form the hexagonal lattice structure. Extensive treatment with pronase and extraction with chloroform did not impair the ability of the LPS preparation to form the lattice structure. When the other salts, NaCI, CaCI2 or Zn(CH3COO)2, were used for precipitation of the LPS with ethanol in place of MgCI2, the LPS did not form the hexagonal lattice structure. However, if the LPS precipitated with NaCI-ethanol was converted to the magnesium salt form after it was electrodialyzed, it formed the same hexagonal lattice structure as the LPS precipitated with MgCI2-ethanol. From these results, it was concluded that the R-form LPS has the ability of in vitro self-assembly into a hexagonal lattice structure in the presence of Mg2+ without the help of other components such as proteins and free lipids from outer membrane.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: effect of various divalent cations on the lattice formation

The R-form lipopolysaccharide from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was previously shown to form a hexagonal lattice structure with the lattice constant of 14 to 15 nm when suspended in 50 mM tris(hydroxymethyl)aminomethane buffer at pH 8.5 containing 10 mM Mg2+. Under this experimental condition, effects of other divalent metal cations on the hexagonal assembly of the electrodialyzed LPS were compared with that of Mg2+. The Zn2+, Hg2+, Cu2+, and Ni2+ could produce essentially the same hexagonal lattice structure with the lattice constant of 14.5 to 15.0 nm as that formed with Mg2+. The Cd2+, Co2+, and Fe2+ produced the hexagonal lattice structure with the lattice constant of 15.5 to 16.0 nm, and Ba2+, Sr2+, and Ca2+ produced that with the lattice constant of 18 to 19 nm. In addition, the hexagonal lattice structures formed with the latter three cations were less orderly than those formed with the other cations. When the higher concentrations of Ba2+, Sr2+, and Ca2+ were used, the lattice constants were not shortened. The length of lattice constants of the hexagonal lattice structures formed with the divalent cations did not relate to the quantity of the cations bound to the LPS. Among the divalent cations tested, Hg2+ was bound to the LPS in the smallest amount (its atomic ratio to P, 0.07), and Zn2+ and Fe2+ were bound in very large amounts (their atomic ratios to P, 2.94 and 8.28, respectively).     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: comparative study of preparations extracted by the phenol-water and the phenol-chloroform-petroleum ether methods

An R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) by the phenol-chloroform-petroleum ether method was compared with that extracted by the phenol-water method in the ability to form a hexagonal assembly. The LPS which was extracted by the phenol-water method and dialyzed against tap water to remove phenol showed ribbon-like structures, and it formed a hexagonal lattice structure with a lattice constant of 14.5 +/- 0.3 nm when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The LPS which was extracted by the phenol-chloroform-petroleum ether method and lyophilized consisted of ribbon-like structures and their fragments and it often formed small pieces of a hexagonal lattice, although the LPS before lyophilization did not form such a lattice. When the LPS extracted by the phenol-chloroform-petroleum ether method was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, it formed essentially the same hexagonal lattice structure as that formed by the LPS extracted by the phenol-water method. From these results it is concluded that the ability of the LPS to form a hexagonal lattice structure does not depend upon the method of its extraction from bacterial cells.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: relationship between lattice formation and uniform salt forms

Various uniform salt forms of an R-form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN-111 (O3-:K1-) were prepared and their ultrastructure was examined. The LPS, which was extracted by the phenol-water method, freed from contamination with RNA by treatment with RNase, and precipitated by addition of two volumes of 10 mM MgCl2-ethanol, was used as the original preparation for uniform salt forms. The original LPS preparation formed a hexagonal lattice structure with a lattice constant of 14.9 +/- 0.2 nm. The LPS after electrodialysis retained the ability to form a hexagonal lattice structure, although its lattice constant was large (18.7 +/- 0.5 nm) and the lattice structure of the electrodialyzed LPS was labile at pH 8.0 in contrast to that of the original LPS preparation. The magnesium salt form of the LPS formed essentially the same ordered hexagonal lattice structure (lattice constant of 15.0 +/- 0.2 nm) as that of the original LPS preparation. The calcium and ammonium salt forms formed a hexagonal lattice structure, but the lattice constants of the calcium and ammonium salt forms were larger (18.6 +/- 0.6 nm and 19.3 +/- 0.4 nm, respectively) than that of the magnesium salt form. The sodium and potassium salt forms consisted of freely branching ribbon-like structures with an average width of 13 nm and an average thickness of 9 nm. The triethylamine salt form consisted principally of short rods (10 nm X 9-13 nm).     

Disparate efficacy of tobramycin on Ca(2+)-, Mg(2+)-, and HEPES-treated Pseudomonas aeruginosa biofilms.

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2.2H2O or MgCl2.4H2O. MEP treated with HEPES or < 5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca(2+)-treated MEP precipitated but Mg(2+)-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having < 1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES +/- 5.0 mM CaCl2.2H2O or MgCl2.4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, +/- 125 micrograms/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg(2+)-, and Ca(2+)-treated biofilms were similar and gradual from T = 0-6 h. The viability of the HEPES- and Mg(2+)-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 micrograms/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

Effect of pH and different substrates on the electrokinetic properties of (Na+, K+)-ATPase vesicles

Some biophysical properties of a (Na+, K+)-ATPase preparation from guinea-pig kidney have been analysed. The recently developed technique of laser Doppler spectroscopy was applied to measure particle mobility under electrophoretic conditions. The following results were obtained: 1. magnesium ions at pH 7.3 decrease the mobility of the ATPase containing vesicles by binding to negatively charged surface groups. At pH 3.3 the competitive binding of protons causes a shift of the mobility vs. [Mg2+] curve to higher values of [Mg2+], 2. binding of ATP at pH 7.3 (Kd = 0.9 X 10(-4) M for (mM 1 NaCl, 0.2 KCl, 0.1 MgCl2, 0.1 Tris) was measured as an increase in particle mobility depending also on [Mg2+]. At pH 3.3 also unspecific ATP-binding occurred, 3. ITP and GTP had the same Kd value as ATP; ADP a slightly lower one (Kd = 1.2 X 10(-4) M). Tris-H3PO4 (Kd = 2.6 X 10(-4) M) was also able to increase particle mobility, but only at higher concentrations and not to the same extent as ATP; AMP induced only very small changes, 4. from the mobility-pH curve an isoelectric point of 4.1 is derived (buffer: 1 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl2, 0.1 mM Tris). In the presence of 0.9 mM ATP the isoelectric point is shifted to 3.2. As the electrophoretic mobility is directly proportional to the net charge of the vesicles, the results may be interpreted as changes in surface charge density, originating from both a conformational change of the ATPase polypeptide and a decrease in vesicle size.     

Formation of crystalline arrays of chlorophyll a/b - light-harvesting protein by membrane reconstitution.

The structure of the major protein constituent of photosynthetic membranes in higher plants, the chlorophyll a/b-light harvesting complex (LHC), was studied by x-ray diffraction and electron microscopy. The LHC was purified from Triton X-100 solubilized thylakoid membranes of the pea, and contained 6 mol of chlorophylls a and b per mole of a polypeptide of 27,000 molecular weight. X-ray diffraction showed that in the presence of 10 mM MgCl2, purified LHC forms planar aggregates that stack with a period of 51 A. Within each layer, LHC molecules pack with a center-to-center distance of 85 A but without long-range order. However, when LHC is incorporated into single-walled vesicles of plant lecithin, the addition of NaCl above 10 mM, or MgCl2 above 2 mM, led to the formation of plaques of hexagonal lattices, with a lattice constant of 125 A. The large domain size and high degree of order in the plane of the membrane are evident from the sharp lattice lines observed to 7 A resolution on the equator of the x-ray pattern. Freeze-fracture electron micrographs demonstrated an aligned stacking of the lattices in adjacent membranes, resulting in crystallinity in the third dimension over short distances. Micrographs of negatively stained membranes revealed a hexagonal lattice of the same lattice constant, formed by surface-exposed parts of the LHC molecules which are probably responsible for the ordered stacking of lattices. In both the LHC aggregates and in the reconstituted membrane lattices the diffracted x-ray intensities at 10-A spacing on the equator indicate that the LHC molecule contains paralled alpha-helices or beta-sheets that are oriented perpendicular to the planar arrays.     

Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.     

Stabilization of glucocorticoid receptors in vitro using divalent cations plus cation chelators

The specific glucocorticoid receptor binding of rat liver cytosol was very unstable in vitro at 25 and 4 degrees C. However, 5 mM CaCl2 added with 5 mM EDTA to cytosol prior to incubation markedly stabilized unbound glucocorticoid receptors at both temperatures. Optimal effectiveness was achieved using equimolar (5 mM) amounts of CaCl2 and EDTA. On the other hand, 5 mM CaCl2 (added alone) further destabilized the unbound glucocorticoid receptor, while 5 mM EDTA (added alone) had no effect at 25 degrees C. EGTA (in lieu of EDTA) added with CaCl2 stabilized hepatic receptor binding at 25 degrees C. On the other hand, citrate added with calcium was ineffective in stabilizing the hepatic glucocorticoid receptor. MgCl2 effectively replaced CaCl2 as a stabilizing agent at 25 degrees C if added with 5 mM EDTA. When added alone, MgCl2 slightly destabilized the unbound receptor. Sucrose density gradient analysis (in low salt) revealed that CaCl2 plus EDTA enhanced the steroid-receptor complex sedimentation coefficient from 7 S to about 10 S. Unlike molybdate, CaCl2 plus EDTA had no apparent effect on steroid-receptor complex thermal transformation into a nuclear binding form, while MgCl2 plus EDTA partially reduced transformation. These results suggest a novel means to chemically stabilize unbound hepatic glucocorticoid receptors in vitro which may be of particular importance for receptor purification studies.     

Effects of calcium and magnesium ions on the interaction of corticosterone with rat brain cytosol receptor(s).

The apparent maximum corticosterone binding (B max) with rat brain cytosol and the apparent dissociation constant of this steroid-receptor binding (Kd) estimated with a Scatchard plot was 2.9 X 10(-13) moles/mg cytosol protein and 4.0 X 10(-9) M, respectively. When increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, a specific [3H] corticosterone binding increased 4-fold by CaCl2 at concentrations of 1.0-2.0 mM and 1.5-fold by MgCl2 at concentrations of 0.5-5.0 mM. The addition of MnCl2 and KCl did not affect this binding. Binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EGTA and complete inhibition was observed at concentrations equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.     

Interaction of Lys-61 labeled actin with myosin subfragment-1 and the regulatory proteins

Actin modified at Lys-61 with fluorescein 5-isothiocyanate (FITC) recovers the ability to polymerize following the binding of phalloidin. The resulting polymer (FITC-P-actin) activates the S1-Mg2+-ATPase activity to the same extent as non-labeled F-actin. However, in the absence of phalloidin, FITC-actin (0.5 mg/ml) neither polymerized nor activated the S1-Mg2+-ATPase activity effectively even when it was preincubated with S1 for 3 h in 0.1 mM ATP, 0.1 mM CaCl2, and 1 mM Tris/HCl (pH 8.0), in contrast to the previous report [Miller, L., Phillips, M., & Reisler, E. (1988) Eur. J. Biochem. 174, 23-29]. The modification of Lys-61 did not impair the ability to bind tropomyosin or tropomyosin-troponin. On the other hand, the fluorescence polarization of FITC-P-actin increased when tropomyosin or troponin-tropomyosin was added. Moreover, the modification of Lys-61 affected the regulation of the actin activation of the S1-Mg2+-ATPase activity by the tropomyosin and troponin complex. In 30 mM KCl, 2.5 mM ATP, and 5 mM MgCl2, tropomyosin alone has been shown to inhibit the actin-activated S1-Mg2+-ATPase. This inhibition did not occur with FITC-P-actin even though tropomyosin was tightly bound. When troponin-tropomyosin was added, the FITC-P-actin activation of S1-Mg2+-ATPase activity was regulated in response to micromolar Ca2+ concentrations. On the other hand, in 30 mM KCl, 2.5 mM ATP, and 2 mM MgCl2, tropomyosin alone did not inhibit the actin-activated S1-Mg2+-ATPase activity with either non-labeled F-actin or FITC-actin.(ABSTRACT TRUNCATED AT 250 WORDS)