多变鱼腥藻Mn^2＋,Ca^2＋和Mg^2＋之间取代作用和放氧的研究

借助于电子顺磁共振波谱仪(EPR)探测了多变鱼腥藻中 Mn~(2 )含量的变化。较高浓度的 CaCl_2和 Ca(NO_3),处理引起藻体中结合态 Mn 的游离和藻的放氧活性的下降。当结合Mn 游离量达57％时,放氧活性降为零。MgCl_2处理的效果小于 CaCl_2。较高浓度的 MnCl_2像 EGTA 一样抑制藻的放氧。与对照比较,在 CaCl_2、MgCl_2和 MnCl_2处理下藻体的低温荧光发射光谱发生变化,波长686nm处的肩消失,F_(130)/F_(695)比值下降。本文对光合放氧复合物中离子间可能在其结合位点上发生竞争性取代作用进行了讨论。     

Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions.

The ability of different metal ions to promote transformation of Pseudomonas aeruginosa by deoxyribonucleic acid of the plasmid RP1 was examined. CaCl2, MgCl2, and MnCl2 were found to promote such transformation, although at different frequencies and with the optimum response at different concentrations. Only MgCl2 promoted transfection of P. aeruginosa by the linear deoxyribonucleic acid of phage F116. CaCl2 was demonstrated to allow adsorption and entry into the cell of F116 deoxyribonucleic acid such that it became resistant to exogenous deoxyribonuclease, but phage production occurred only when MgCl2 was provided. Inactivation of linear phage deoxyribonucleic acid taken up in the absence of MgCl2 was observed. The transfection frequencies at various concentrations of MgCl2 were compared, and the optimum response occurred at the concentration which promoted the highest frequency of transformation by RP1 deoxyribonucleic acid.     

Temperature induced transition of the pH-activity curve of heavy meromyosin adenosine triphosphatase and inosine triphosphatase.

The pH-activity curve of heavy meromyosin ATPase [EC 3.6.1.3] was measured at various temperatures. The pH-activity curve at higher temperatures showed a maximum at low pH and a minimum at pH 7 to 8 as has been already reported. At lower temperatures it was sigmoidal in shape, similar to a simple dissociation curve of pKa 6 to 7. The pH-activity curve at intermediate temperatures appeared to be inbetween the two extreme shapes. These changes in pH-activity curve with temperature were found to be common in the presence of divalent cations such as Mg2+, Mn2+, and Ca2+. The ATPase mechanism may be identical in the presence of any divalent cation, and the rate determining step revealing the steady state rate alters by changing the temperature. The transition temperatures estimated at pH 8 were 10 degrees, 8 degrees, and about 5 degrees in the presence of MnCl2, CaCl2, and MgCl2, respectively. The difference in the temperature coefficients above and below the transition temperature was most distinct in the presence of MnCl2, and vague in the presence of CaCl2. A similar change of pH-activity curve with temperature was found with heavy meromyosin ITPase in the presence of MgCl2.     

EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition.

In the absence of magnesium ions, the EcoRV restriction endonuclease binds all DNA sequences with equal affinity but cannot cleave DNA. In the presence of Mg2+, the EcoRV endonuclease cleaves DNA at one particular sequence, GATATC, at least a million times more readily than any other sequence. To elucidate the role of the metal ion, the reactions of the EcoRV restriction enzyme were studied in the presence of MnCl2 instead of MgCl2. The reaction at the EcoRV recognition site was slower with Mn2+. This was caused partly by reduced rates for phosphodiester hydrolysis but also by the translocation of the enzyme along the DNA after cleaving it in one strand. In contrast, alternative sites that differ from the recognition site by one base pair were cleaved faster in the presence of Mn2+ relative to Mg2+. When located at an alternative site on the DNA, the EcoRV enzyme bound Mn2+ ions readily but had a very low affinity for Mg2+. The EcoRV nuclease is thus restrained from cleaving DNA at alternate sites in the presence of Mg2+, but the restraint fails to operate with Mn2+. A discrimination factor, which measures the ratio of the activity of the EcoRV nuclease at its recognition site over that at an alternative site, had values of 3 x 10(5) in MgCl2 and 6 in MnCl2.     

Glycinebetaine stabilizes the association of extrinsic proteins with the photosynthetic oxygen-evolving complex.

The photosynthetic oxygen-evolving activity of the photosystem 2 complex, prepared from spinach, was labile when the complex was exposed to high-salt conditions under which the extrinsic proteins were dissociated from the complex. Glycinebetaine prevented the dissociation of the 18-kDa and the 23-kDa extrinsic proteins from the photosystem 2 complex in the presence of 1 M NaCl. It also prevented the dissociation of the 33-kDa extrinsic protein from the complex in the presence of 1 M MgCl2 or 1 M CaCl2. The oxygen-evolving activity of the photosystem 2 complex was stabilized by glycinebetaine when the complex was subjected to treatment with NaCl and MgCl2.     

Effect of divalent metal ions and glycerol on the GTPase activity of H-ras proteins

The product of the protooncogenic ras gene (p21N ras) exhibits a weak GTPase activity. A significant increase in the GTPase activity associated with p21N ras protein was obtained by using glycerol in the assay mixture. Of the several metal ions tested, only Mg++ and Mn++ are effective divalent cations that support the GTPase activity of p21N ras protein. p21N ras protein exhibits higher GTPase activity and yields higher [3H] GDP binding in the presence of MnCl2 than with MgCl2. Optimal GTPase and [3H] GDP binding are obtained at micromolar concentrations of MgCl2 or MnCl2. Concentrations in the millimolar range of either MgCl2 or MnCl2 are inhibitory to the GTPase activity, whereas [3H] GDP binding was not affected.     

Interaction and conformational changes of chromatin with divalent ions.

We have investigated the interaction of divalent ions with chromatin towards a closer understanding of the role of metal ions in the cell nucleus. The first row transition metal ion chlorides MnCl2, CoCl2, NiCl2 and CuCl2 lead to precipitation of chicken erythrocyte chromatin at a significantly lower concentration than the alkali earth metal chlorides MgCl2, CaCl2 and BaCl2. A similar distinction can be made for the compaction of chromatin to the "30 nm" solenoid higher order structure which occurs at lower MeCl2 concentration in the first group but at the same MeCl2 concentration within each group. In other experiments in which mixed solutions of NaCl and of MgCl2 were examined, it is shown that increasing NaCl concentration leads to increasing solubility in the presence of MgCl2. Best compaction of chromatin was obtained at 40 mM NaCl and 0.8 mM MgCl2 at a value A260 approximately 0.8. Similar experiments were undertaken with mixtures of NaCl and MnCl2.     

Studies on arginyl-tRNA synthetase from Escherichia coli B. Dual role of metals in enzyme catalysis.

Studies carried out in arginyl-tRNA synthetase from Escherichia coli indicate that metals may have two functional roles in the catalytic mechanism. Complete metal activation is observed when MgCl2, MnCl2, CoCl2, or FeCl2 is present at a concentration (5.0 mM) in excess of the total ATP concentration (2.0 mM). When CaCl2 is substituted for MgCl2, activity is not observed unless a small amount (0.1 mM) of MgCl2, MnCl2, CoCl2, FeCl2, or ZnCl2 (unable to produce activity alone at 5.0 mM) is added. A model, based on kinetic data, is proposed in which the enzyme possesses a site for free metal, which, when filled, lowers the Km for all three substrates (arginine, tRNAArg, and metal-ATP) and increases the Vmax of the reaction.     

Role of divalent metals in the activation and regulation of insulin receptor tyrosine kinase

Multiple equilibrium equations were solved to separate the individual effects of ionic divalent metals, free nucleotides and their chelated species on insulin receptor tyrosine kinase (IRTK). Basal IRTK is activated by divalent metal cations when present in excess of that required for substrate formation, indicating the presence of a divalent cation-dependent regulatory site on the kinase. The activatory order for basal activity was Mn2+ greater than Co2+ greater than Mg2+ and Ca2+ = 0. The insulin-dependent activation of IRTK was minimal in the absence of excess free divalent metal, even when the concentration of MnATP or MgATP substrate present exceeded the apparent Km of the kinase. The activatory order for insulin-dependent activation of IRTK changed to Mg2+ greater than Mn2+ and Co2+ = 0. The titration of the MnCl2 saturation response at several concentrations of MgCl2 revealed that the insulin-dependent response of IRTK increases as a function of increasing MgCl2, while basal activity was unaffected. This enhancement of the responsiveness to insulin in the presence of both cations was not due to differing affinities of the kinase for substrate, as evidenced by nearly identical apparent Km values for MnATP and MgATP. The Mg2+-dependent increase in the response of the kinase to insulin may be due to Mg2+ inducing a stronger coupling between receptor and kinase than that observed with Mn2+ alone. The plotting of the effect of several concentrations of free divalent metals on substrate saturation curves revealed that an increase in either of the reactants increased the affinity of the insulin-activated kinase for the other respective reactant. Accordingly, free divalent metal and metal-ATP substrate interact with IRTK in a mutually inclusive manner. CaCl2 saturation curves in the presence of constant MnCl2 and increasing MgCl2 showed that the affinity of IRTK for Ca2+ decreases and the affinity for CaATP increased with increasing Mg2+. Our data suggests that IRTK contains three sites for interaction with divalent metal cations: a MeATP (active) site, a regulatory site, and a metal-dependent site acting to couple the receptor with the kinase.     

Purification and properties of an endonuclease endogenous to rat-liver nuclei

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a pI value of 5.7 and migrated as a single band to a molecular weight position of 46,000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl2 and/or by 5-15 mM MgCl2 in the presence of 2 mM CaCl2 (an optimum pH, 7.0), but was lowered by CaCl2 alone and inhibited strongly by ZnCl2 or MnCl2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5'-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.     

Spontaneous release of transmitter from the growth cones of Xenopus neurons in vitro: the influence of Ca2+ and Mg2+ ions

To determine whether spontaneous release of transmitter from the growth cones of neurons exhibits properties similar to the spontaneous release which occurs from the neurons at the neuromuscular junction, release of transmitter from the growth cones of Xenopus neurons in culture was monitored in salines containing varying calcium and magnesium concentrations. Release was monitored by use of an outside-out piece of muscle membrane attached to a patch clamp electrode. Spontaneous release of transmitter from the growth cones in standard saline (2 mM CaCl2, 1 mM MgCl2) produces clusters of single-channel openings in the muscle membrane. Clusters are seen to consist of two types: a series of high-frequency channel openings, called "bursts," and clusters of low-frequency channel openings called "singles." The bursts were identified and examined for their possible relationship to MEPP-producing release, and the singles were identified and examined for their possible relationship to "leak" release of the neuromuscular junction. When the external saline contains high calcium (10 mM CaCl2, 1 mM MgCl2) or high magnesium (2 mM CaCl2, 9 mM MgCl2), the frequencies of both "bursts" and "singles" was greatly reduced. This reduction in release persists if the neurons are grown in the high-calcium or high-magnesium solutions. When the saline is a low-calcium solution (0 mM CaCl2, 3 mM MgCl2) the growth cones release transmitter at rates similar to those from standard saline. These results indicate that although the spontaneous release from the growth cone shares one characteristic with the leak release, neither the burst nor the singles release from the growth cones share exact relationship with either the MEPP producing release or the leak release. This suggests that further development of the mechanisms for spontaneous release of neurotransmitter occurs after nerve-muscle contact.     

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.     

Inorganic compounds have dual effect on recombinant protein production: influence of anions and cations on serine alkaline protease production

AIMS: Investigation of concerted effects of cations, i.e. Mg2+ and Mn2+, in combination with their anions, i.e. sulphate, chloride and acetate (Ac), on the physiology of Bacillus licheniformis carrying pHV1431::subC to improve the fermentation medium for serine alkaline protease (SAP) production, whereupon, determination of the acid that can be used in pH control. METHODS AND RESULTS: The cell concentrations increased with the increase in MnSO4 and Mn(Ac)2 concentrations, and the highest values were obtained at Co(MnSO4) = 0.20 mmol l-1 and Co(Mn(CH3COO)2) = 4.0 mmol l-1, as 2.3 and 2.2 g l-1, respectively. However, Co(MnCl2) did not influence biomass concentration. SAP production was inhibited with MnCl2 after Co(MnCl2) = 0.60 mmol l-1, but with MnSO4 SAP production was inhibited drastically. Whereas, at high concentrations of Mn(Ac)2 SAP production increased and the highest activity was obtained as ASAP = 1285 U ml-1 at t = 65 h. With the Mg compounds, cell concentrations increased with the increase in the concentrations of MgSO4, MgCl2 and Mg(Ac)2; and the anions did not show any influence on the cell growth. Similar to the results of Mn compounds, the glucose consumption rate increased with the increase in MgSO4 and MgCl2 concentrations; contrariwise, decreased with the increase in Mg(Ac)2 concentrations, due to the use of acetate as the second carbon source. Co(MgSO4) = 0.40 mmol l-1, Co(MgCl2) = 1.60 mmol l-1 and Co(Mg(Ac)2) = 0.40 mmol l-1 were the optimum concentrations separately, and the highest SAP activity was obtained with Mg(Ac)2 as ASAP = 1338 U ml-1 at t = 47 h. Consequently, ion acetate and its acid HAc appear, respectively, as the superior anion for the essential cations and the control agent for pH control in the bioreactor. Finally, optimum initial concentrations and the concerted effects of Mg(Ac)2 and Mn(Ac)2 were investigated, and the optimum concentrations were found respectively as 0.40 and 0.80 mmol l-1, while the maximum activity was obtained as ASAP = 1010 U ml-1 at a shortened cultivation time of t = 39 h. CONCLUSIONS: Mn(Ac)2 and Mg(Ac)2 together enhanced the cell formation and SAP synthesis rates, moreover, SAP synthesis started at an earlier cultivation time. SIGNIFICANCE AND IMPACT OF THE STUDY: Each inorganic compound with its cation and anion has dual effect on the metabolism. Mg2+ and Mn2+ at their specific concentrations influence the regulation of the pathways that might cause better coupling of supply and demand for the amino acids on the basis of the amino acid composition of the enzyme molecule.     

The binding of cations to the surfaces of cells from early chick blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl2 and MnCl2 at 3 X 10(-2) M while SrCl2 and BaCl2 were not able to reverse the cells' charge at concentrations up to 6 X 10(-2) M. CaCl3 reversed the cells' EPM at concentrations as low as 5 X 10(-3) M. Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoplasmic proteins of Streptococcus mutans (serotype c) and their interaction with fluoride.

The protein profile of the cytoplasmic proteins of Streptococcus mutans GS-5 was determined by two-dimensional gel electrophoresis. Use of this recently developed, high-resolution analytical tool showed in excess of 140 cytoplasmic proteins. The profile consisted of mostly acidic components with pI values between 3.70 and 5.30 and relative molecular weights mainly in the 13,000 to 90,000 range. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were resolved into 40 to 45 components. The binding of fluoride by the proteins reached a maximum value in 15 min, and it was linear with exogenous F- doses of up to 60 to 80 ppm per mg of protein (60 to 80 micrograms/g). The proteins bound 22 to 138 times more F- from assay mixtures containing 1 mM CaCl2 than from assay mixtures containing such ions as HgCl2, ZnCl2, CuCl2, MgCl2, MnCl2, or SnCl2. When NaF, SnF2, NH4F, CsF, (CH3)4NF, and Na2PO3F were used as sources of F- (adjusted to 10 ppm of F- in all cases), the proteins bound 2.1, 1.8, 1.6, 1.4, and 0.3 ppm of F- per mg of protein, respectively. Initial fractionation of the plasma proteins by preparative column isoelectric focusing indicated that proteins with pI values of 4.1 to 4.5 as well as those with pI values of 5.0 to 5.3 bound twice as much F- as did the proteins outside these pI values.