A specific and saturable spermine-binding component present in bovine testis membranes

To determine if polyamine (PA)-binding components were present in testis membranes, a membrane fraction derived from homogenates of immature bovine testes was utilized in a radioligand assay. [14C]Spermine ([14C] Sp) bound to a spermine-binding component (SpBC) in a saturable manner and radioligand-binding data were analyzed and fit to a one-site model. Estimates of the binding constant (Kd = 7.2 +/- 1.2 X 10(-6) M) and of the concentration of sites (11.6 +/- 1.6 X 10(-6) M) were determined assuming a one-to-one stoichiometry. Metal cations (Ca+2, Mg+2, Mn+2 and Na+) as chloride salts had no effect on binding of [14C]Sp to SpBC when tested at concentrations up to 10 mM. Not all polyamines tested were effective competitors for [14C]Sp binding, which had an ID50 of 21.0 +/- 2.0 microM. The inhibitory potencies for putrescine and spermidine relative to spermine were 3.1 +/- 0.43 X 10(-4) and 0.1 +/- 0.01 (nmol/nmol) respectively, illustrating the specificity of SpBC binding. In addition, acetylation of polyamines, which is generally associated with degradation and interconversion of polyamines, resulted in a reduction of potency of 25.2 +/- 2.0-fold of spermine and 31.1 +/- 10.6-fold of spermidine in the SpBC radioligand assay. Binding of SpBC was decreased by tryptic digestion of the membrane fraction, suggesting that protein may be a part of the SpBC. Neuraminidase treatment had no effect on [14C]Sp binding but phospholipase-C digestion increased [14C]Sp binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

Atrial natriuretic peptide receptors and activation of guanylate cyclase in rat cardiac sarcolemma

Two classes of atrial natriuretic peptide (ANP) receptors are present in purified sarcolemmal membrane fractions isolated from rat ventricle. Scatchard analysis using [125I]-ANP reveals high affinity (Kd approximately 10(-11) M) and low affinity (Kd approximately 10(-9) M) binding sites. Basal guanylate cyclase activities associated with these membrane fractions range from 3.2 +/- 1.3 pmol/min/mg protein in the presence of Mg2+ to 129 +/- 17 pmol/min/mg protein in the presence of Mn2+. Millimolar concentrations of adenosine triphosphate (ATP) potentiates Mg2+- but not Mn2+-supported activity. Binding of ANP to the low affinity site but not the high affinity site results in a maximum 2-fold activation of Mn2+- and up to 6-fold activation of Mg2+/ATP supported guanylate cyclase activities.     

Glycerolipid biosynthesis in rat adipose tissue. 11. Effects of polyamines on Mg2+-dependent phosphatidate phosphohydrolase

The effect of polyamines (spermine, spermidine and putrescine) on the Mg2+-dependent phosphatidate phosphohydrolase was investigated. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. In the presence of various polyamines there was activation of the Mg2+-dependent phosphatidate phosphohydrolase activity. Under this condition, the Km of enzyme towards phosphatidase decreased from 1.6 x 10(-4) to 9.8 x 10(-5) M and the Mg2+ requirement decreased from 5 to 0.5 mM. These polyvalent cations did not replace Mg2+, but potentiate the phosphohydrolase activity in the presence of Mg2+. The activation of Mg2+-dependent phosphatidate phosphohydrolase activity by polyamines was observed in the presence of 3-sn-phosphatidylcholine, suggesting that these modulators of phosphatidate phosphohydrolase activity may be acting through different mechanisms. These studies demonstrate that polyamines may be important regulators of Mg2+-dependent phosphatidate phosphohydrolase activity in adipose tissue.     

Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver.

To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.     

Effect of polyamines on Mg(2+)-dependent ATP-hydrolase activity in plasmatic membrane of myometrium cells

Influence of aliphatic polyamines of spermine and spermidine on the enzymatic activity of the ouabain-sensitive Na+,K(+)-ATPase and the ouabain-resistant basal Mg(2+)-ATPase (specific activity--10.6 +/- 0.9 and 18.1 +/- 1.2 microM P(i)/hour on 1 mg of protein accordingly, n = 7) has been studied in the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that the polyamine spermine in concentration of 1 and 10 mM activated the Na+,K(+)-ATPase by 54 and 64% on the average relative to control value. Spermidine also stimulated the Na+,K(+)-ATPase activity, however it did it less efficiently than spermine: by 8 and 20% on the average at concentration of 1 and 10 mM, accordingly. Similarly, polyamines had affect on the basal Mg(2+)-ATPase: spermine in concentration of 1 and 10 mM activated it by 26 and 39% relative to control value; spermidine in concentration of 1 and 10 mM activated it by 10 and 32% relative to control. The magnitudes of the apparent activation constant K(a) of spermine were 0.35 +/- 0.07 and 0.10 +/- 0.02 mM for Na+,K(+)-ATPase and basal Mg(2+)-ATPase, accordingly (M +/- m, n = 5). It is supposed, that the obtained experimental data can be useful in the further research of the membrane mechanisms underlying of the cationic exchange in the smooth muscles, in particular, when investigating the role of the plasmatic membrane in providing electromechanical coupling in them, and also in regulation of ionic homeostasis in the smooth muscle cells.     

Complement C4bC2 complex formation: an investigation by surface plasmon resonance

Complex formation between the human complement proteins C4b and C2 was investigated by surface plasmon resonance. C4b was immobilised and C2 was used in the fluid phase to measure interaction at different ionic strengths (30-830 mM NaCl) and in the absence and presence of MgCl2. Maximum binding was observed at 30 mM NaCl, and was negligible above 300 mM NaCl. Binding was not greatly influenced by variation in Mg(2+) in the range of 2.5-15 mM. C4bC2 affinity (Kd) was determined by steady-state analysis to be 7.2x10(-8) M in physiological conditions (10 mM Hepes, 2.5 mM MgCl2, 0.75 mM CaCl2 and 140 mM NaCl, pH 7.4). For C4(H2O)C2 complex formation, a Kd of 4.0x10(-8) M was calculated. As far as detected by the applied method, complex formation does not involve conformational changes of one of the binding partners. Consistent with previous reports, C4bC2 binding takes place as a multiple-site binding event in the presence of Mg2+. C4bC2 complex formation in 10 mM Hepes, 2.5 mM EDTA and 140 mM NaCl (pH 7.4) was also observed and the interaction showed characteristics of a single-site binding event. Kd was 1.5x10(-8) M. Complement factor B (FB) was also tested for its binding to immobilised C4b. Weak interaction was observed at FB concentrations in the physiological range (500-1000 nM). Kd was 1.2x10(-6) M, indicating possible cross-reactivity between classical and alternative pathways of the activation of the complement system.     

Structural analysis of DNA interactions with biogenic polyamines and cobalt(III)hexamine studied by Fourier transform infrared and capillary electrophoresis

Biogenic polyamines, such as putrescine, spermidine, and spermine are small organic polycations involved in numerous diverse biological processes. These compounds play an important role in nucleic acid function due to their binding to DNA and RNA. It has been shown that biogenic polyamines cause DNA condensation and aggregation similar to that of inorganic cobalt(III)hexamine cation, which has the ability to induce DNA conformational changes. However, the nature of the polyamine.DNA binding at the molecular level is not clearly established and is the subject of much controversy. In the present study the effects of spermine, spermidine, putrescine, and cobalt(III)hexamine on the solution structure of calf-thymus DNA were investigated using affinity capillary electrophoresis, Fourier transform infrared, and circular dichroism spectroscopic methods. At low polycation concentrations, putrescine binds preferentially through the minor and major grooves of double strand DNA, whereas spermine, spermidine, and cobalt(III)hexamine bind to the major groove. At high polycation concentrations, putrescine interaction with the bases is weak, whereas strong base binding occurred for spermidine in the major and minor grooves of DNA duplex. However, major groove binding is preferred by spermine and cobalt(III)hexamine cations. Electrostatic attractions between polycation and the backbone phosphate group were also observed. No major alterations of B-DNA were observed for biogenic polyamines, whereas cobalt(III)hexamine induced a partial B --> A transition. DNA condensation was also observed for cobalt(III)hexamine cation, whereas organic polyamines induced duplex stabilization. The binding constants calculated for biogenic polyamines are K(Spm) = 2.3 x 10(5) M(-1), K(Spd) = 1.4 x 10(5) M(-1), and K(Put) = 1.02 x 10(5) M(-1). Two binding constants have been found for cobalt(III)hexamine with K(1) = 1.8 x 10(5) M(-1) and K(2) = 9.2 x 10(4) M(-1). The Hill coefficients indicate a positive cooperativity binding for biogenic polyamines and a negative cooperativity for cobalt(III)hexamine.     

A study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease I and to deoxyribonucleic acid by electron paramagnetic resonance.

EPR studies of Mn2+ binding to bovine pancreatic deoxyribonuclease I show that the enzyme can bind three Mn2+ ions at pH 7.5 and 2 degrees. Two sites bind Mn2+ strongly, with a Kd of 10(-4)M, and the third binds Mn2+ weakly, with a Kd of 10(-3)M. Ca2+ competes with the two strong sites, whereas Mg2+ competes only with one of them, indicating that both sites are not equivalent. Mn2+ binding to DNA has been confirmed by EPR measurements. Two types of sites, with different affinities for Mn2+ binding, were found on DNA molecules, one with a Kd of 1.2 times 10(-4)M and the other with a Kd of 10(-3)M. Mg2+ ions can displace Mn2+ from the high affinity sites, but not from the low affinity sites. These results suggest the Mn2+ binds not only to the phosphate groups, but also to the electron donor groups of the base rings.     

The ribosomal E site at low Mg2+: coordinate inactivation of ribosomal functions at Mg2+ concentrations below 10 mM and its prevention by polyamines

Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM. The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J. Biol. Chem. 261, 9133 (1986] are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.     

Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.     

Stimulation of pyruvate dehydrogenase phosphatase activity by polyamines

Pyruvate dehydrogenase phosphatase requires Mg2+ or Mn2+, and its activity in the presence of Mg2+ is markedly stimulated by Ca2+. At saturating Mg2+ and Ca2+ concentrations, the polyamines spermine, spermidine and putrescine stimulated the activity of pyruvate dehydrogenase phosphatase 1.5- to 3-fold. Spermine was the most active of the polyamines. At a physiological concentration of Mg2+ (1 mM) and saturating Ca2+ concentration, the stimulation by 0.5 mM spermine was 4- to 5-fold, and at 0.3 mM Mg2+, the stimulation was 20- to 30-fold. In the absence of Mg2+ or Ca2+, spermine had no effect. These results suggest that a polybasic factor may be involved in the regulation of pyruvate dehydrogenase phosphatase activity.     

Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.     

Polyamines secreted by cancer cells possibly account for the impairment of the human erythrocyte sodium pump activity.

Using an original microcalorimetric method, we previously showed that in erythrocytes from cancer patients, the sodium pump activity was decreased and returned to normal in patient in remission. In addition we suggested that a plasma-borne factor probably secreted by cancer cells accounted for this impairment of the sodium transporter. In the present study we sought to identify this factor as well as its mechanism of action. First we determined the effect of culture media from undifferentiated and differentiated colon cancer cell lines (Caco-2 and HT29-D4) on the sodium pump activity of normal human erythrocytes. The inhibitory powers of culture media from undifferentiated cells were higher than those of differentiated cells (38.6 +/- 3.5% vs 6.9 +/- 4.6%, p<0.05 for Caco-2 and 45.8 +/- 6.2% vs 9.0 +/- 5.0%, <0.05 for HT29-D4). The use of alpha difluoro-methylomithine (2 mM) to inhibit ornithine decarboxylase, the rate-limiting enzyme for polyamine biosynthesis, dramatically reduced the sodium pump inhibition induced by the two undifferentiated cell lines (75% for Caco-2 and 89% for HT29-D4). Polyamines secreted by undifferentiated cells and then taken up by human erythrocytes thus appeared as inhibitors of sodium pump of these red blood cells. Putrescine, spermidine, and spermine (the main polyamines) exerted a similar inhibitory effect (33 +/- 2%). Tested in vitro on Na,KATPase, these polyamines (3 mM) were inhibitors (putrescine = 23 +/- 2%; spermidine= 48 +/- 3%; spermine= 55 +/- 2%) when assay condition for the ATPase reaction was suboptimal (Na+ = 10 mM; K+ = 1 mM). The inhibitory effect appeared to be related to their charge and their aliphatic chain length. The effect of spermidine and spermine on the ionic substrates and ATP-Mg showed that molecules decreased the affinity (Km) of the Na,K-ATPase for Na+ (11.24 +/- 0.49 mM for control vs 23.51 +/- 1.53 mM for spermine and 18.86 +/- 0.98 mM for spermidine), indicating that polyamines exerted their inhibitory effect in a competitive manner.     

Effect of spermine on activity of purified Ca2+, Mg2+-ATPase from plasma membranes of myometrium cells

Effect of endogenous polyamine spermine, a relaxant of smooth muscle, on the activity of myometrium cell plasma membrane Ca2+, Mg(2+)-ATPase was studied. It was observed a tendency to activation of enzyme at the spermine concentrations 0.1-0.5 mM, the increase of the polyamine concentrations up to 10 mM inhibited. ATPase by 80% (I50 = 5.5 +/- 0.3 mM). Spermine inhibited enzyme decreasing its turnover rate and affinity for Ca2+. The ATPase affinity for Mg2+ increased in the presence of spermine. It was revealed, that the inhibitory effect of spermine is changed by the stimulatory effect under the increase of Ca2+ concentration (up to 2.6 microM), that correlates with the relaxing effect of this polyamine on the smooth muscle.     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.     

Divalent ion-binding properties of the two avian beta-parvalbumins

Birds express three parvalbumins, one alpha isoform and two beta isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T-cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca2+- and Mg2+-binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepes-buffered saline, ATH binds Ca2+ with apparent microscopic binding constants of 2.4 +/- 0.2 x 10(8) and 1.0 +/- 0.1 x 10(8) M(-1). The corresponding values for CPV3-C72S are substantially lower, 4.5 +/- 0.5 x 10(7) and 2.4 +/- 0.2 x 10(7) M(-1), a 1.9-kcal/mol difference in binding free energy. Thus, the beta-parvalbumin lineage displays a spectrum of Ca2+-binding affinity, with ATH and the mammalian beta isoform at the high- and low-affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca2+ affinity, CPV3-C72S exhibits increased affinity for Mg2+, relative to ATH. Whereas the latter displays Mg2+-binding constants of 2.2 +/- 0.2 x 10(4) and 1.2 +/- 0.1 x 10(4) M(-1), CPV3-C72S yields values of 5.0 +/- 0.8 x 10(4) and 2.1 +/- 0.3 x 10(4) M(-1).     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.