Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.     

Role of bivalent and monovalent cations in the functioning of myosin ATPase

The effects of bivalent (Mg2+, Ca2+, Sr2+) and monovalent (K+, Na+, NH4+) cations on the ATPase activity of subfragment 1 of myosin (SI) with a decreased Mg2+ content (EDTA-SI) were studied. Mg2+ activate the EDTA-SI ATPase, but only in the absence of other activating cations. K+, NH4+, a2+ and Sr2+ have a much stronger activating effect on EDTA-SI ATPase than on Mg-SI (SI enriched with Mg2+) ATPase. Monovalent cations inhibit Mg2+-ATPase and Ca2+-ATPase of EDTA-SI, while K+ and NH4+ activate Sr2+-ATPase of EDTA-SI. Based on experimental results and literary data, a hypothesis on the participation of the cations in the functioning of myosin ATPase was postulated. This hypothesis entails the existence of two closely interconnected cation-binding sites in the vicinity of the myosin active center (one for bivalent and one for monovalent cations); the ATPase activity of myosin is at any moment dependent on the nature of cations present in these two sites. An attempt to explain the role of the cations in the accomplishment of the ATPase reaction by myosin was made.     

Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.     

Activity and properties of fructose bisphosphatase of turbellarian Phagocata sibirica

Activity and properties of fructose bisphosphatase (FBPase) was studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. There was studied dependence of the FBPase reaction rate on the substrate concentration. For realization of the enzyme activity, the high affinity to substrate and presence of bivalent cations (Mg2+ or Mn2+) are necessary. The was studied the effect of various effectors as well as of monovalent (Na+, K+, Li+, and NH4+) and bivalent (Zn2+ and Cu2+) cations.     

Binding of bivalent cations by hyaluronate in aqueous solution

The interaction between sodium hyaluronate and bivalent cations was investigated by conductometry, viscosimetry, circular dichroism and nuclear magnetic resonance spectroscopy. It is shown that the hyaluronate chains (Mn approximately 4.0 x 10(5)-1.7 x 10(6)g/mol) bind various bivalent cations (Ca2+, Mg2+, Mn2+, Fe2+, Cu2+, Zn2+, Cd2+ and Pb2+) at pH 6 in aqueous solutions. Hyaluronate deriving from Streptococcus equi was studied in comparison with dextran from Leuconostoc mesenteroides which was shown to develop no specific interactions with the bivalent cations. The molar relation between the bivalent cations and the disaccharide units of the resulting complex was determined with the result that one bivalent cation is bound by approximately five disaccharide units. Heavy metal ions (Cd2+, Pb2+) seem to bind stronger to the hyaluronate chain than their lighter counterparts (Ca2+, Mg2+). Circular dichroism spectra of the hyaluronate exhibit a cation-induced change in the n-pi* transition, indicating that the acetamide group of the aminoglucane unit is involved during the complexation. NMR spectra of hyaluronic acid in presence of paramagnetic manganese cations show strong interactions between the acetamide as well as the carboxylate groups and the cations. Based on these data, a structure of the binding complex is proposed which involves two disaccharide units.     

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.     

Raman spectroscopic study of the effects of Ca2+, Mg2+, Zn2+, and Cd2+ ions on calf thymus DNA: binding sites and conformational changes

The interaction of Mg2+, Ca2+, Zn2+, and Cd2+ with calf thymus DNA has been investigated by Raman spectroscopy. These spectra reveal that all of these ions, and particularly Zn2+, bind to phosphate groups of DNA, causing a slight structural change in the polynucleotide at very small metal: DNA (P) concentration ratio (ca. 1:30). This results in increased base-stacking interactions, with negligible change of the B conformation of DNA. Contrary to Zn2+ and Cd2+, which interact extensively with the nucleic bases (particularly at the N7 position of guanine), the alkaline-earth metal ions are bound almost exclusively to the phosphate groups. The affinity of both the Zn2+ and Cd2+ ions for G.C base pairs is comparable, but the Cd2+ ions interact more extensively with A.T pairs than Zn2+ ions. Interstrand cross-linking through the N3 atom of cytosine is suggested in the presence of Zn2+, but not Cd2+.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

Effect of the membrane potential on the Mg2+,ATP-dependent transport of Ca2+ across smooth muscle sarcolemma

The effect of the membrane potential (K(+)-valinomycin system) on the Mg2+, ATP-dependent transport of Ca2+ in inside-out vesicles of myometrium sarcolemma has been studied. The membrane potential was identified by using a cyanine potential-sensitive probe, diS-C3-(5). In the presence of valinomycin (5.10(-8) M) the inside-out directed K+ gradient (delta psi = -86 mV, with a negative charge inside) stimulated the initial rate of the energy-dependent accumulation of Ca2+ transfer whereas the oppositely directed K+ gradient (delta psi = +72 mV, with a positive charge inside) had no effect on this process. The K+ gradient was formed by isotonic substitution of K+ in intra- or extravesicular space for choline +. At the same time, in the absence of K+ gradient the Mg2+, ATP-dependent accumulation of Ca2+ in membrane vesicles did not depend on the chemical nature of the cations (K+ or choline+) used for isotonicity. The decrease of delta psi from 0 to -86 mV affects the initial rate of Ca2+ accumulation but not the maximal content of the accumulated cation. Preliminary dissipation of the membrane potential (delta psi = -86 mV) in Mg2(+)-free isotonic (with respect of K+ and choline+) media containing ATP and Ca2+ resulted in the inhibition of Mg2+, ATP-dependent Ca2+ transport induced by subsequent addition of Mg2+. These results indicate that the negative (intravesicular) electrical potential activates the Ca-pump of smooth muscle sarcolemma. This activation is based on the increase in the turnover number of the Ca2+ transporting system but not on its affinity for the transfer substrate. The use of the absolute reaction rates theory made it possible to establish that the Ca-pump effectuates the transport of a single positive charge in inside-out vesicles of smooth muscle plasma membranes, i.e., the energy-dependent transport of Ca2+ occurs either as a symport (with an anion (Cl-) or an antiport with a monovalent cation (K+) or a proton. It is assumed that the potential dependence of the Ca-pump in the smooth muscle plasma membrane plays a role in the realization of effects of mediators and physiologically active substances that are manifested as stimulation of the contractile response and depolarization of the sarcolemma. In is quite probable that the delta psi-dependent Ca-pump is also responsible for the maintenance of intracellular homeostasis of monovalent cations (K+, H+, Cl-) in smooth muscle tissues.     

Perturbation of hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations

Perturbation of the hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine...thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at O6 leads to destabilization of the adenine...thymine pair; divalent cations (Mg2+, Ca2+) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

Zn2+, not Ca2+, is the most effective cation for activation of dolichol kinase of mammalian brain

The cation specificity of dolichol kinase of mammalian brain and the potential involvement of a Ca2+-calmodulin system in regulation of this enzyme have been studied. Among 10 divalent cations examined, Zn2+ was found to be most effective for the activation of dolichol kinase of rat and calf brain and cultured C-6 glial cells. The activations with Ca2+, Co2+, and Mg2+ were 53%, 32%, and 18% of the full activation with Zn2+, respectively. No combinations of the cations could activate the enzyme as much as Zn2+ alone. A role for a Ca2+-calmodulin system in the regulation of brain dolichol kinase was not supported by our data. First, the concentration of free Ca2+ required for the maximum activation of dolichol kinase was two to three orders of magnitude greater than the concentration required by typical calmodulin-dependent enzymes. Second, neither the depletion of calmodulin from the microsomal fraction nor the addition of exogenous calmodulin caused an alteration in the activation of dolichol kinase by Ca2+ (or Zn2+). Third, antagonists of calmodulin failed to suppress the activation of the enzyme by Ca2+ (or Zn2+). The data raise the possibility that Zn2+ is involved in the regulation of dolichol kinase in brain.     

Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.     

Cell damage by cytolysin. Spontaneous recovery and reversible inhibition by divalent cations

Cytolysin-induced membrane damage (which requires low Ca2+) has been studied 1) in E by assay of hemolysis, 2) in Lettre cells by measurement of transmembrane potential, intracellular content of K+ and Na+, leakage of phosphoryl[3H]choline or 51Cr from [3H]choline-labeled or 51CrO4(2-)-labeled cells and leakage of lactate dehydrogenase, and 3) in phospholipid bilayers by measurement of electrical conductivity changes. In Lettre cells, damage is restricted and reversible: little lactate dehydrogenase leaks from cells that leak substantial amounts of Na+, K+, and phosphoryl[3H]choline; at low amounts of cytolysin, membrane potential and intracellular content of Na+ and K+ recover within minutes. In E and Lettre cells, membrane damage is inhibited by Zn2+, by high Ca2+, or by low pH. Inhibition is reversible: addition of EGTA to Zn2+-protected E or Lettre cells (incubated in the presence of cytolysin, low Ca2+ and Zn2+) initiates leakage; removal of Zn2+ (and cytolysin and Ca2+) by washing also initiates leakage; such leakage is again sensitive to Zn2+, high Ca2+, or H+. In phospholipid bilayers, channels induced by cytolysin (at low Ca2+) are partially closed by negative voltage; Ca2+, Zn2+, or H+ promote channel closure. Channels are re-opened (only partially in the case of Zn2+) by positive voltage. From all these results it is concluded that the action of cytolysin on membranes is similar to that of other pore-forming agents: damage does not necessarily lead to lysis of nucleated cells, and can be prevented by Ca2+, Zn2+, or H+.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Evaluation of a role for intracellular Na+, K+, Ca2+, and Mg2+ in hyperthermic cell killing

A dose of heat which renders 98% of a population of Chinese hamster ovary cells reproductively dead has no significant effect on their Na+, K+, or Mg2+ content by 28 h postheat. In contrast, the cellular Ca2+ content increases in a dose-dependent manner as observed at 22 h after heating for 15-35 min at 45 degrees C. However, the rates of both influx and efflux of Ca2+ were reduced by heating. Increasing the cellular Ca2+ content by incubating the cells in high extracellular Ca2+, either at the time of heating or for a period of 22 h following heat, does not potentiate the lethal effect of heat. Completely blocking the heat-induced increase in Ca2+ content by incubating the cells in medium containing a low Ca2+ concentration does not protect the cells. Therefore, we conclude that heat does not produce any significant changes in the Na+, K+, or Mg2+ content of cells and that the heat-induced increase in Ca2+ does not play an important role in hyperthermic cell killing.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.     

Cationic distribution in vessels, heart, skeletal muscle, and blood of normotensive (NWR) and spontaneously hypertensive rats (SHR)

Electrolytic analysis carried out with the atomic absorption spectrophotometer permitted to compare the cationic (Ca2+, Mg2+, Zn2+, K+ and Na+) tissue content of spontaneously hypertensive male rats (SHR) and normotensive male Wistar rats (NWR). In all SHR-tissues, Zn2+ is augmented, but mainly in the atria (left atrium), inferior vena cava, left ventricle, and skeletal muscle. The inferior cava vein and the right atrium have a similar, accentuated high bivalent (Ca2+, Mg2+, and Zn2+) cation content; Ca2+ and Mg2+ is present in a minor content in the right ventricle and type II "pale" skeletal muscle while only Mg2+ was also reduced in the left atrium, aorta, and the whole blood. A higher K+ concentration is seen in the right atrium, aorta, and type I "red" skeletal muscle. In the aortic wall and the whole blood a higher Na+- tissue content is found, confirming earlier observations. There is a discret water retention in the tissues from the left ventricle and skeletal muscle, simultaneously with a small depletion in the whole blood. We have concluded, that there is a specific cationic profile in SHR structural and functional different cardiovascular tissues, including skeletal muscle. The cationic tissue distribution is related to underlying genetic and adaptative factors and may be involved into specific drug effects.     

二价金属离子对嵌有线粒体H~+-ATP酶的脂酶体不同层次脂质流动性的影响

本文报道用荧光偏振及顺磁共振两种方法研究Mg~(2+)及其它二价金属离子对嵌有H~+-ATP酶的脂酶体不同层次脂质流动性的影响。 (1)顺磁标记探剂5-、12-、16-氮氧基硬脂酸测定结果表明Mg~(2+)和其它二价金属离子都能降低膜脂双分子层表层的流动性。降低流动性的顺序为Mg~(2+)=Ca~(2+)>Sr~(2+)>Cd~(2+)。较深层脂则无明显变化。 (2)荧光探剂7-、12-(9-蒽酰)硬脂酸及16-(9-蒽酰)棕榈酸的测定结果也表明Mg~(2+)和其它二价金属离子降低了膜脂表层的流动性,尤以Mn~(2+)、Ca~(2+)降低流动性最显著,流动性降低的顺序为;Mn~(2+) Ca>Sr~(2+) Mg~(2+) Cd~(2+)。除Mn~(2+)、Ca~(2+)还能影响膜脂深层的流动性外,其它与对照无明显差异。     

Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K n.m.r.

The interactions of the S100 protein (S100) with metal cations such as Ca2+, Mg2+, Zn2+ and K+ were studied by the metal n.m.r. spectroscopy. The line widths of 43Ca, 25Mg, 67Zn and 39K n.m.r. markedly increased by adding all S100s. A broad 43Ca n.m.r. band of Ca(2+)-S100a solution was not affected by Zn2+ and K+, while it was greatly decreased by adding Mg2+. The 43Ca n.m.r. spectra of Ca(2+)-S100a0 and -S100b solutions consisted of two slow-exchangeable signals which corresponded to Ca2+ bound to two environmentally different sites of the S100a0. These two 43Ca n.m.r. signals were not affected by Zn2+ and K+. The line width of broad 25Mg n.m.r. band of the Mg(2+)-S100 solution greatly decreased by adding Ca2+, while it did not change by adding Zn2+ and K+. Further, the addition of Ca2+, Mg2+ and K+ did not affect the line width of the 67Zn n.m.r. of the Zn(2+)-S100 solutions. These findings suggest that: (1) Mg2+ binds to all S100s, and at least one of the Mg2+ binding sites of S100 molecule is the same as the Ca2+ binding site; (2) Zn2+ binds to S100s, although the binding site(s) is/are different from Ca(2+)- or Mg(2+)-binding site(s), and the environment of Zn2+ nuclei will not change even though Ca2+ binds to S100s.     

A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves

A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump.