Significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides produced by submerged culture of Tuber melanosporum

The significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides was systematically studied in the submerged fermentation of Tuber melanosporum. The lowest weight-average molecular weight (M_w) (i.e., 115.3 × 10⁴ g/mol) of intracellular polysaccharides (IPS) was obtained when Mg²⁺ and K⁺ was added in the fermentation medium. The IPS with the lower M_w exhibited a higher inhibition ratio against S-180 tumor cells. The compact conformation of extracellular polysaccharides (EPS) was formed when only K⁺ was supplied in the fermentation medium. Interestingly, EPS with compact conformation exhibited a higher inhibition ratio (i.e., 59.2%) than EPS with branched polymer chain (i.e., 9.2%) against A549 tumor cells. The highest inhibition ratio for EPS with α-glycosidic linkages against the tumor cell line HepG2 reached 32.2% when Mg²⁺ or K⁺ was supplied in the fermentation medium. The addition of metal ion Mg²⁺, K⁺, and their combination to the fermentation medium is a vital factor affecting the structures of Tuber polysaccharides, which further determine their anti-tumor activities. The information obtained in this work will be useful for the efficient and directed production of polysaccharides with anti-tumor activities by the submerged fermentation of edible fungi mycelium.     

金属离子对地衣芽孢杆菌合成多聚γ-谷氨酸的影响

多聚γ 谷氨酸 [γ Ｐｏｌｙ(ｇｌｕｔａｍｉｃａｃｉｄ) ,γ ＰＧＡ]是由某些杆菌 (Ｂａｃｉｌｌｕｓ)合成的一种细胞外水溶性高分子氨基酸聚合物 ,是由Ｌ 谷氨酸、Ｄ 谷氨酸两种构型的单体通过γ 酰胺键聚合形成的[1 ] 。γ ＰＧＡ具有极佳的成膜性、成纤维性 ,阻氧性、可塑性、粘结性、保湿性和可生物降解等许多独特的理化和生物学特性[2 ,3] 。因此 ,γ ＰＧＡ可以被广泛用于医药制造 ,食品加工 ,蔬菜、水果、海产品防冻、保鲜 ,化妆品工业 ,烟草、皮革制造工业和植物种子保护等许多领域 ,是一种有极大开发价值和前景的多功能新型生物制…     

Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Pseudomonas aeruginosa acid phosphatase. Activation by divalent cations and inhibition by aluminium ion.

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.     

Ribozyme-catalyzed dipeptide synthesis in monovalent metal ions alone

Previously we have identified a highly active ribozyme (R180, cis ribozyme) that can catalyze dipeptide synthesis using N-biotinylcaproyl-aminoacyl-adenylate anhydride (Bio-aa-5'-AMP) as its substrate. In this work, we re-engineered the cis R180 ribozyme into a 158-nt trans ribozyme (TR158) and designed a new substrate (5'-Phe-linker-20-mer). First, the metal ion requirements were examined and compared between the two ribozymes. Both R180 and TR158 ribozymes were active in Mg2+ and Ca2+ but inert with Zn2+, Cu2+, Mn2+, and Co2+. It is intriguing that both ribozymes were highly active in Li+, Na+, or K+ alone but showed very low activity with NH4+. The two ribozymes showed similar linear concentration dependence on Li+ and K+, while they displayed different dependency behavior on Mg2+. Moreover, by using the trans system, the detailed kinetic studies and pH dependent experiments were performed in either 10 mM Mg2+ or 1.0 M Li+. Analysis of kcat and Km values obtained at different pHs (6.0 to 9.0) indicated that it is the catalytic activity of the ribozyme but not the substrate binding affinity that changes significantly with pH. The slopes of the linear parts of the pH-rate plots were close to 1.0 in both Mg2+- and Li+-mediated reactions, suggesting that one proton transfer is involved in the rate-limiting step of catalysis. Overall, our results suggest that Mg2+ and Li+ function similarly in the ribozyme-catalyzed dipeptide synthesis.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Phosphorylation from adenosine triphosphate of sodium- and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme.

The relative effectiveness of the ligands Mg2+, Na+, and ATP in preparing sodium plus potassium ion transport adenosine triphosphatase for phosphorylation was studied by means of a rapid mixing apparatus. Addition of 2 mM MgC12, 120 mM NaC1, and 5 muM [gamma-32P]ATP simultaneously to the free enzyme gave an initial phosphorylation rate of about 0.3 mu mol-mg-1-min-1 at 25 degrees and pH7.4. Addition of Mg2+ to the enzyme beforehand, separately or in combination with Na+ or ATP, had little effect on the initial rate. Addition of Na+ only to the enzyme beforehand increased this rate 1.5- to 3-fold. Early addition of ATP 130 ms before Na+ plus Mg2+ increased the rate 6- to 7-fold. Early addition of Na+ plus ATP was most effective; it increased the rate about 10-fold. The data indicate that Na+ and ATP bind in a random order and that each ligand potentiates the effect of the other. The rate of dissociation of ATP from the enzyme was estimated by a chase of unlabeled ATP of variable duration. This rate was slowest in the presence of Mg2+ (k = 540 min-1), most rapid in the presence of Na+ (k = 2000 min-1), and intermediate (k = 1100 min-1) in the absence of metal ions. The effect of Na+ concentration on the rate of phosphorylation was estimated when Na+ with Mg2+ was added to the enzyme-ATP complex. The rate followed Michaelis-Menten kinetics with a maximum of 2.9 mu mol-mg-1 and a Km of 8 mM. The effect of Na+ concentration was also estimated on the increment in the rate of phosphorylation produced by the presence of Na+ with the enzyme-ATP complex beforehand. The increment followed the same kinetics with a maximum of 3.75 mu mol-mg-1-min-1 and a Km of 5.4 mM. In both cases estimation of the Hill coefficient failed to show cooperativity between binding sites for Na+. In contrast, the dependence of ouabain-sensitive ATPase activity on Na+ concentration in the absence of K+ indicated two sites for Na+ with apparent Km values of 0.16 and 8.1 mM, respectively.     

Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity.     

Enzymic characteristics of ecto-adenosine triphosphatase in rat epididymal intact spermatozoa.

Ecto-ATPase in rat cauda-epididymal intact spermatozoa has a high degree of substrate specificity for the hydrolysis of ATP and dATP rather than of ADP, AMP, GTP, dGTP, CTP, dCTP, TTP and UTP. The enzyme is activated by bivalent metal ions in the order Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+. The apparent Km values of the enzyme for Mg2+, Mn2+, Co2+ and Ca2+ are approx. 80, 100, 100 and 150 microM respectively. Addition of Ca2+ (0.1 or 1 mM) gives no further stimulation of the Mg2+-activated ecto-ATPase activity. The apparent Km value of the enzyme for ATP is 95 microM. Pi (16 mM) inhibits the enzymic activity (by 25%), whereas Na+ (50 mM) or K+ (10 mM) alone or in combination, polyamines (spermine and spermidine; 1--12.5mM) and nucleic acids (yeast RNA and calf thymus DNA; 0.12 or 0.62 mg/ml) had no significant effect on the activity of the enzyme. Orthovanadate at a relatively low concentration (20 microM) strongly inhibits (approx. 50%) the ecto-ATPase activity. Vanadate inhibition can be reversed by noradrenaline (2.5 mM). The vanadate-sensitivity of the enzyme increases markedly during spermatozoal maturation in the epididymis. However, the activity of the spermatozoal ecto-ATPase decreases progressively during the epididymal transit of the testicular spermatozoa.     

The respiration of brain mitochondria and its regulation by monovalent cation transport.

The Na+ and K+ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose. Respiration was uncoupled by Na+ and less so by K+. Uncoupling was maximal in the presence of EDTA plus Pi and was decreased by Mg2+. Maximal uncoupler-stimulated respiration rates were inhibited by Na+ but largely unaffected by K+. The inhibition by Na+ was relatively insensitive to Mg2+. Membrane Na+ and K+ conductances as well as neutral exchanges (Na+/H+ and K+/H+ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations. Cation conductance, i.e. electrophoretic Na+ or K+ permeation, was increased by EDTA (Na+ greater than K+) and decreased by Mg2+. Magnesium preferentially suppressed Na+ conductance so as to reverse the cation selectivity (K+ greater than Na+). Neutral cation/H+ exchange rates (Na+ greater than K+) were not influenced by chelator or Mg2+. The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K+ conductor valinomycin. The metabolic influences of Na+ and K+ can be explained in terms of coupled flow of these ions with protons and their effect upon the H+ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Magnesium as NMDA receptor blocker in the traditional Chinese medicine Danshen

Aqueous extracts of the traditional Chinese medicine Danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae), blocked N-methyl-D-aspartate (NMDA) evoked currents in cerebrocortical neurons in vitro. The block of the NMDA-evoked currents was voltage dependent and showed the negative slope conductance reminiscent of the effect of Mg2+ ions. Atomic absorption spectrophotometry (AAS) revealed that aqueous Danshen extracts contained approximately 9mM magnesium. Fractionation of the extracts by high performance liquid chromatography followed by patch clamp recording and AAS indicated that magnesium ions were present in two distinct fractions. One fraction contained approximately 5 mM magnesium and blocked NMDA-induced currents indicating that it contained mostly free Mg2+ ions, while a second fraction did not possess NMDA antagonist activity despite the presence of approximately 4 mM magnesium suggesting that Mg2+ in this fraction was mostly chelated. Following removal of the free Mg2+ by ion exchange chromatography, the previously observed block of the NMDA-induced currents was abolished. These data demonstrate that Danshen contains both free and chelated Mg2+. Free Mg2+ ions account for the NMDA antagonist activity of Danshen in vitro.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Inhibition of smooth muscle 5'-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7-8, was found to depress the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg2+. Such an inhibitory effect of imidazole on the smooth muscle 5'-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusion of 5-10 mM Mg2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5'-nucleotidase in the absence of Mg2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5'-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5'-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5'-nucleotidase and alters the metal-enzyme interactions.     

Appearance of magnesium guanylate cyclase activity in rat liver with sodium azide activation.

Native soluble and particulate guanylate cyclase from several rat tissues preferred Mn2+ to Mg2+ as the sole cation cofactor. Wtih 4mM cation, activities with Mg2+ were less than 25% of the activities with Mn2+. The 1 mM NaN3 markedly increased the activity of soluble and particulate preparations from rat liver. Wtih NaN3 activation guanylate cyclase activities wite similar with Mn2+ and Mg2+. Co2+ was partially effective as a cofactor in the presence of NaN3, while Ca2+ was a poor cation with or without NaN3. Activities with Ba, Cu2+, or Zn2+ were not detectable without or with 1 mM NaN3. With soluble liver enzyme both manganese and magnesium activities were dependent upon excess Mn2+ or Mg2+ at a fixed MnGTP or MgGTP concentration of 0.4 mm; apparent Km values for excess Mn2+ and Mg2+ were 0.3 and 0.24 mM, respectively. After NaN3 activation, the activity was less dependent upon free Mn2+ and retained its dependence for free Mg2+, at 0.4 mM MgGTP the apparent Km for excess Mg2+ was 0.3 mM. The activity of soluble liver guanylate cyclase assayed with Mn2+ or Mg2+ was increased with Ca2+. After NaN3 activiation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+. After NaN activation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+ or Mg2+. The stimulatory effect of NaN2 on Mn2+-and Mg2+-dependent guanylate cyclase activity from liver or cerebral cortex supernatant fractions required the presence of the sodium azide-activator factor. With partially purified soluble liver guanylate cyclase and azide-activator factor, the concentration (1 mjM) of NaN3 that gave half-maximal activation with Mn2+ or Mg2+ was imilar. Thus, under some conditions guanylate cyclase can effectively use Mg2+ as a sole cation cofactor.     

Properties of Na+, K+-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.     

Activation of partial reactions of the Ca2+-ATPase from human red cells by Mg2+ and ATP.

(1) At ATP concentrations up to 30 micrometer addition of 0.5 mM MgCl2 in the reaction mixture increases both the rate of formation and the steady-state level of the phosphoenzyme of the Ca2+-ATPase from human red cell membranes. Under these conditions Mg2+ has no effect on the rate of dephosphorylation, which remains slow. (2) In the presence of Mg2+ the rate of dephosphorylation is increased 5 to 10 times by high (1 mM) concentrations of ATP. (3) Provided Mg2+ has reacted with the phosphoenzyme, acceleration of dephosphorylation by ATP takes place in the absence of Mg2+. This suggests that the role of Mg2+ on dephosphorylation is to convert the phosphoenzyme into a form that, after combination with ATP, reacts rapidly with water. (4) The results are consistent with the idea that combination of ATP at a non-catalytic site is needed for rapid dephosphorylation of the Ca2+-ATPase.     

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.     

Na+/K(+)-ATPase: modes of inhibition by Mg2+.

Adding 15 mM free Mg2+ decreased Vmax of the Na+/K(+)-ATPase reaction. Mg2+ also decreased the K0.5 for K+ activation, as a mixed inhibitor, but the increased inhibition at higher K+ concentrations diminished as the Na+ concentration was raised. Inhibition was greater with Rb+ but less with Li+ when these cations substituted for K+ at pH 7.5, while at pH 8.5 inhibition was generally less and essentially the same with all three cations: implying an association between inhibition and ion occlusion. On the other hand, Mg2+ increased the K0.5 for Na(+)-activation of the Na+/K(+)-ATPase and Na(+)-ATPase reactions, as a mixed inhibitor. Changing incubation pH or temperature, or adding dimethylsulfoxide affected inhibition by Mg2+ and K0.5 for Na+ diversely. Presteady-state kinetic studies on enzyme phosphorylation, however, showed competition between Mg2+ and Na+. In the K(+)-phosphatase reaction catalyzed by this enzyme Mg2+ was a (near) competitor toward K+. Adding Na+ with K+ inhibited phosphatase activity, but under these conditions 15 mM Mg2+ stimulated rather than inhibited; still higher Mg2+ concentrations then inhibited with K+ plus Na+. Similar stimulation and inhibition occurred when Mn2+ was substituted for Mg2+, although the concentrations required were an order of magnitude less. In all these experiments no ionic substitutions were made to maintain ionic strength, since alternative cations, such as choline, produced various specific effects themselves. Kinetic analyses, in terms of product inhibition by Mg2+, require Mg2+ release at multiple steps. The data are accommodated by a scheme for the Na+/K(+)-ATPase with three alternative points for release: before MgATP binding, before K+ release and before Na+ binding. The latter alternatives necessitate two Mg2+ ions bound simultaneously to the enzyme, presumably to divalent cation-sites associated with the phosphate and the nucleotide domains of the active site.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.