Volutin Granules in Zoogloea ramigera

Zoogloea ramigera, a gram-negative bacterium found in activated sludge, formed volutin granules when excess orthophosphate was added to a phosphate-starved culture. These volutin granules were stainable by hydrogen sulfide after lead acetate treatment and extractable by N-perchloric acid but were not adsorbed by activated charcoal. They appeared to consist of inorganic polyphosphate. Optimum granule formation in the arginine broth required 10 g of glucose, 3 mg of phosphate, and 1 to 20 mg of magnesium per liter of medium. At an Mg(2+) concentration of 1 mg/liter, very large granules appeared which often appeared to fill the cell. An excess of glucose, orthophosphate, or magnesium reduced granule formation. In the absence of sulfate, moderate granulation occurred in arginine broth before the addition of excess orthophosphate; granulation did not increase after the addition of phosphate.     

Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance

The structure of the divalent metal ion binding site of S-adenosylmethionine synthetase from Escherichia coli has been studied by using the vanadyl(IV) ion (VO2+) as probe. VO2+ binds at a single site per subunit in the presence or absence of substrates. Single turnover experiments measuring S-adenosylmethionine (AdoMet) formation from methionine and the ATP analogue 5'-adenylyl imidodiphosphate show that complexes containing VO2+ and either Mg2+ or Ca2+ as a second metal ion are catalytically active, while a complex containing VO2+ alone is inactive. Electron paramagnetic resonance spectra of the enzyme-VO2+ complex, as well as complexes also containing AdoMet or methionine, indicate the coordination of two water molecules and at least two protein ligands to the VO2+. In complexes with polyphosphate substrates or products (e.g., enzyme-VO2+-ATP-methionine, enzyme-VO2+-PPi-Mg2+), EPR spectral changes reveal ligand substitutions on the VO2+, and 8.5-G isotropic superhyperfine coupling to two 31P nuclei can be resolved. 17O superhyperfine coupling from [17O]pyrophosphate indicates coordination of two oxygen atoms of PPi to the VO2+ ion. Thus the polyphosphate compounds are bidentate ligands to the VO2+, demonstrating that the VO2+ binds at the active site and suggesting a catalytic role for the protein-bound metal ion.     

Evidence that the etiology of the syndrome containing type 2 diabetes mellitus results from abnormal magnesium metabolism

Evidence is reviewed supporting the presence of an inherited structural defect in the plasma membranes of somatic cells of humans who have type 2 diabetes mellitus and sodium-sensitive essential hypertension. This magnesium-binding defect (MgBD) consists of a decreased content of tightly bound Mg2+ ion in the cell membrane and limits the amount of Mg2+ that enters the cell, some of which combines with ATP4-, produced by the cell, to form MgATP2-, the currency of metabolic energy. Consequently, in both prediabetes and overt diabetes, the intracellular concentration of the interdependent Mg2+ and MgATP2- ions is significantly less than normal. These 2 ions are required as cofactors and (or) substrates for some 300 enzyme systems in human metabolism, many of which are involved with insulin. Thus the decreased activities of particular ones of these enzyme systems due to the decreased intracellular [Mg2+] and its dependent [MgATP2-] are responsible for (i) insulin resistance and (ii) decreased insulin secretion and (or) production, the 2 pathophysiological processes required for the occurrence of type 2 diabetes mellitus. These 2 processes can account for all of the morbid symptoms associated with this disease. Thus, the decreased intracellular concentration of the interdependent Mg2+ and MgATP2- ions constitutes the etiology of genetic predisposition to type 2 diabetes mellitus and can be corrected by 2 identified peptide Mg2+-binding promoters that are derived from the carboxyl terminal of the tachykinin substance P and occur in normal blood plasma. Decreased intracellular [Mg2+] and [MgATP2-] can also result from a dietary deficiency of magnesium or from an abnormal accumulation of saturated fatty acids in cell membranes, which inhibits the entrance of Mg2+ into the cell; thus it is also the etiology not only of diabetes caused by magnesium deficiency, but also of the "lipotoxic" type 2 diabetes mellitus. Although these pathologies cannot be corrected by the Mg2+-binding promoters, they can be corrected, respectively, by dietary magnesium supplementation or by exercise plus dietary caloric and lipid restriction. Theoretically, the disease syndrome containing type 2 diabetes mellitus may involve approximately 30% of the population.     

The elemental composition dynamics of large polyphosphate granules in Acinetobacter strain 210A

Electron microscopy and energy dispersive X-ray micro-analysis were used to examine the elemental composition of large polyphosphate granules in unfixed and unstained intact cells of Acinetobacter strain 210A. When grown in medium with butyrate, Acinetobacter strain 210A possessed 1 or 2 large granules with a diameter of 0.4 m besides a relatively large number of small granules. The large granules were composed of phosphorus, magnesium and potassium. A decrease in the Mg/Ca-ratio of the medium from 5.95 to 0.0073 resulted in a decline in the intracellular Mg/Ca-ratio from 15 to 0.56. At a high intracellular Mg/Ca-ratio, magnesium was the dominant counterion in the polyphosphate granule. Calcium became the major cation in the polyphosphate bodies at a low intracellular Mg/Ca-ratio. Omission of Ca²⁺ or modification of the K/Mg ratio in the medium did not significantly affect the cation composition of the polyphosphate granules. The dissociation constants for Mg- and Ca-polyphosphate were 9.3×10^-2 mol/l and 1.5×10^-1 mol/l, respectively.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

Aminoglycosides inhibit hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg2+ uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca2+ and Mg2+. The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg2+ uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, elicited transient increases in intracellular Ca2+ from basal levels of 102 +/- 13 nM to 713 +/- 125 nM, suggesting a receptor-mediated response. In order to determine Mg2+ transport, MDCT cells were Mg(2+)-depleted by culturing in Mg(2+)-free media for 16 h and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+ uptake, d([Mg2+]i)/dt, was 138 +/- 24 nM/s in control MDCT cells. Gentamicin (50 microM) did not affect basal Mg2+ uptake (105 +/- 29 nM/s), but inhibited PTH stimulated Mg2+ entry, decreasing it from 257 +/- 36 nM/s to 108 +/- 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 +/- 2.5 to 23 +/- 1 pmol/mg protein x 5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg2+ entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg2+ absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of these antibiotics.     

Respiration-dependent uptake and extrusion of Mg2+ by isolated heart mitochondria

It has been known for some time that isolated heart mitochondria can both take up and extrude Mg2+ by respiration-dependent, uncoupler-sensitive processes. A re-examination of these reactions reveals that the respiration-dependent uptake of Mg2+ can be quite rapid and efficient and is apparently preceded by a passive binding to the inner membrane. The rate of Mg2+ uptake can exceed 30 ng ion/min/mg protein at an efficiency of about 1 ng ion Mg2+ accumulated per ng atom O2 consumed. Passive binding and respiration-dependent accumulation of Mg2+ are strongly inhibited by K+ and other monovalent cations and the uptake reaction is further decreased by the presence of ATP or ADP. Under conditions approaching those faced by mitochondria in situ (state 3 respiration in a KCl medium) the rate of Mg2+ uptake, as estimated from 28Mg2+ distribution, is no more than 0.25 ng ion/min/mg. When heart mitochondria are suspended in a Mg2+-free medium, a slow, respiration-dependent Mg2+ efflux is seen. This reaction is quite insensitive to external K+ and otherwise shows an inhibitor profile markedly different from that of the Mg2+ accumulation reaction. Neither the uptake nor the loss of Mg2+ is inhibited by ruthenium red or diltiazem. These reactions therefore appear unrelated to those involved in the uptake and release of Ca2+. It is concluded that heart mitochondria have separate pathways available for Mg2+ uptake and release.     

Effect of potassium chloride on the uptake and storage of phosphate by Saccharomyces mellis

The inorganic and polyphosphate pools of Saccharomyces mellis, grown in a medium containing excess phosphate, remain associated with the cells when the cells are suspended in a saline medium. If the cells are incubated in a medium containing 2 m KCl, the cells are altered in some manner which permits most of the orthophosphate and approximately one-third of the polyphosphate to be subsequently eluted by osmotic shock. At lower salt concentrations, beta-mercaptoethanol enhances this salt effect but is inactive by itself in this respect. At equivalent ionic strengths, the sodium salt of ethylenediaminetetraacetic acid behaves exactly like KCl or any other monovalent ionic compound in altering the cell to susceptibility to osmotic shock. No special effect of this anion at either high or low concentration could be detected. Resting cells are refractory to being altered in this manner by salts if an energy source, such as glucose, is included in the reaction mixture. Cells which are depleted of phosphate reserves will immediately incorporate phosphate when suspended in a medium containing inorganic phosphate and an energy source. These cells exhibit the phenomenon of "überkompensation." In resting cells, the inclusion of KCl in the reaction mixture prevents the conversion of orthophosphate into polyphosphate and, also, gradually decreases the ability of the organism even to assimilate orthophosphate. This effect is reversible, however, since the cells will incorporate phosphate in a normal manner if the cells are transferred to a non-salinized medium, or if a nitrogen source is included in the salinized reaction mixture so that the cells are now in a medium adequate for growth.     

Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes

31P-NMR spectra have been recorded on erythrocytes stored at 4 degrees C in various preservation media. Storage was always associated with an upfield shift of the inorganic phosphate (Pi) resonance and a pronounced upfield shift of the ATP beta resonance, indicating decreased intracellular pH (pHi) and decreased intracellular free magnesium ([Mg2+]i). The decreased [Mg2+]i occurred in preservation media not containing citrate and even in media supplemented with Mg2+. It could not be attributed to the changes in pHi, Na+, K+, lactate, Pi or 2,3-diphosphoglycerate, that occur with storage. The decrease in [Mg2+]i was largely reversed when stored cells were incubated for 1 h at 37 degrees C in fresh plasma. Stored cells were found to contain significant amounts of inorganic pyrophosphate, up to about 200 mumol per liter cell water. Being a tight binder of Mg2+, pyrophosphate could account for some of the observed decrease in [Mg2+]i. Additional mechanisms may involve precipitation of some other Mg2+ complex during cold storage or enhancement of Mg2+ binding to membrane components.     

Effects of anions, pH and magnesium on calcium accumulation and release by sarcoplasmic reticulum vesicles

In the absence of oxalate, Ca2+ accumulation by isolated sarcoplasmic reticulum vesicles may show a transient behavior in which the vesicles accumulate during the first 2 min of incubation as much as twice the amount of Ca2+ which is retained after 5-7 min, when Ca2+ accumulation approaches a steady state. Before Ca2+ release begins, the Ca2+ accumulation can reach 200-250 nmol/mg protein. The spontaneous release of the "extra" Ca2+ initially accumulated appears to be triggered by the attainment of a sufficiently high concentration of free Ca2+ inside the vesicles. The amplitude of the transient phase of Ca2+ accumulation reaches a high value near pH 6.0 and is increased by free Mg2+. At optimal concentrations of H+ and Mg2+, the amount of Ca2+ accumulated during the transient is augmented by various anions, in the order maleate > or = propionate > or = succinate > chloride > sulfate > acetylglycine. The divalent anions have their maximum effects at 20-40 mM and the monovalent anions, at 40-200 mM. At 200 mM, all of the carboxylic anions tested significantly reduce the amount of Ca2+ retained in the steady state.     

Magnesium magnetic isotope effect: a key towards mechanochemistry of phosphorylating enzymes as molecular machines

A discovery of the huge magnesium isotope effect in enzymatic ATP synthesis provides a new insight into mechanochemistry of enzymes as the molecular machines. It has been found that the catalytic activity values of ATPase, creatine kinase and phosphoglycerate kinase are 2 to 4-fold higher once their active sites contain magnetic (25Mg) not spinless, non-magnetic (24Mg, 26Mg), magnesium cation isotopes. This clearly proves that the ATP synthesis is a spin-selective process involving Mg2+ as the electron accepting reagent. The formation of ATP takes place in an ion-radical pair resulted by two partners, ATP oxyradical and Mg+. The magnesium bivalent cation is a key player in this process, this ion transforms the protein molecule mechanics into a mere chemistry. This ion is a most critical detail of structure of the magnesium dependent phosphorylation enzymes as the mechanochemical molecular machines.     

Altered Mg2+ transport across liver plasma membrane from streptozotocin-treated rats

Type-I diabetes is associated with a decrease in magnesium content in various tissues, including liver. We have reported that hepatocytes from streptozotocin-injected rats have lost the ability to accumulate Mg2+ following hormonal stimulation. To assess whether the defect is inherent to the Mg2+ transport mechanism located in the hepatocyte cell membrane, plasma membrane vesicles were purified from diabetic livers. Diabetic plasma membranes do not retain intravesicular Mg2+ as tightly as vesicles purified from livers of age-matched non-diabetic rats. In addition, the amount of intravesicular Mg2+ these vesicles exchange for extravesicular Na+ or Ca2+ is 2-3-fold larger than in non-diabetic vesicles. The partition of Ca2+/Mg2+ and Na+/Mg2+ exchange mechanisms in the apical and basolateral domains of liver plasma membrane is maintained under diabetic conditions, although the Na+/Mg2+ exchanger in diabetic basolateral membranes has lost the ability to operate in reverse and favor an accumulation of extravesicular Mg2+ within the vesicles in exchange for entrapped Na+. These data indicate the occurrence of a major alteration in Mg2+ transport across the hepatocyte membrane, which can explain, at least in part, the decrease in liver magnesium content observed in diabetic animals and patients.     

Magnesium magnetic isotope effect: A key to the mechanochemistry of phosphorylating enzymes as molecular machines

The discovery of the powerful magnesium isotope effect on enzymatic ATP synthesis provides a new insight into the mechanochemistry of enzymes as molecular machines. The catalytic activities of ATPase, creatine kinase, and glycerophsphate kinase containing a Mg²⁺ ion with magnetic isotope nuclei (²⁵Mg) were found to be two to four times higher than those of the enzymes with spinless, nonmagnetic magnesium cation isotopes (²⁴Mg or ²⁶Mg). This demonstrates unambiguously that ATP synthesis is a spin-selective process involving Mg2+ as the electron-accepting reagent. ATP synthesis proceeds in an ion-radical pair consisting of an ADP oxyradical and Mg²⁺. In this process, the magnesium bivalent cation is the key agent that transforms the mechanics of a protein molecule into chemical processes. This ion is the crucial structural component of enzymes as mechanochemical molecular machines.     

升温对湖泊底泥藻细胞复苏及其生理特性的影响

为探讨藻细胞复苏过程中环境因子的作用及其细胞生理特性的变化,在连续升高温度条件下,比较了在不同N:P值的培养基中复苏藻细胞的丰度、藻群落组成动态、藻光合活性变化,同时检测了这一过程中藻细胞中Na+K+-ATPase和Ca2+Mg2+-ATPase活性的变化。结果表明:实验期间共检测到7门,62种藻,表明太湖的底泥可以作为"种源",为藻细胞的复苏提供"种子"。6℃时蓝藻就能够萌发复苏,16℃左右是最适宜藻细胞复苏的温度。在设定的温度范围内,底泥中复苏蓝藻的光合效率随着温度的升高一直增加,表明温度越高越有利于蓝藻从底泥中的萌发和复苏;但是复苏的绿藻和硅藻的光合活性一直处在被抑制状态。低N:P值培养基中复苏的藻细胞丰度远远大于其他2种培养基中复苏的藻细胞丰度,低N:P值能够显著性的激发藻细胞从底泥中的复苏。同时,低N:P比培养液中复苏藻细胞的Na+K+-ATPase和Ca2+Mg2+-ATPase活性都显著高于其他培养液中复苏藻细胞的ATPase活性;16℃时2种ATPase活性的骤然升高与最适宜藻细胞复苏的温度相吻合,而且这个温度提前于复苏藻细胞显著增加的温度(21℃)。此外,复苏藻细胞的比生长速率与Na+K+-ATPase和Ca2+Mg2+-ATPase活性都呈现显著性的线性相关(*P<0.05)。因而,藻细胞中Na+K+-ATPase和Ca2+Mg2+-ATPase活性的恢复和升高,对推动藻细胞从底泥迁移到水柱中的萌发和复苏过程具有重要的意义。     

Two internal pools of soluble polyphosphate in the cyanobacterium Synechocystis sp. strain PCC 6308: an in vivo 31P NMR spectroscopic study

Two intracellular pools of soluble polyphosphate were identified by in vivo ³¹P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate. The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. ³¹P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment. Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value. These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA. Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells. Received: 3 July 1997 / Accepted: 26 September 1997     

The activation of the nicotinic acetylcholine receptor by the transmitter

Experimental evidence has been published from isolated guinea pig muscle in vitro, and from direct ligand binding to receptors from T. californica, indicating that two agonist ions react with the nicotinic receptor by exchanging for one magnesium ion. It is the basis of the ion exchange receptor pair model, in which two acetylcholine ions exchange for one magnesium ion in contact with and between a pair of negatively charged receptor groups about 4 A apart. In the resting state the electrostatic attraction between the negatively charged receptor groups and the Mg2+ ion exerts a binding force. This binding force is opposed by the quantum mechanical repulsions of the electron clouds of the charged groups and ions in contact, together with the mutual repulsion of the pair of receptor oxyanions. When the Mg2+ ion is replaced by two acetylcholine ions the quaternary heads of the latter are positioned so that they form two mutually repelling ACh+ receptor group dipoles. As the Mg2+ ion leaves, its rehydration energy contributes to the sum of the electron cloud repulsions and the ACh+ receptor group dipole repulsions, causing the receptor groups to be forced apart activating the receptor macromolecule. The subsequent decrease in ACh+ concentration results in the reestablishment of the resting state. The coulombic electrostatic energy, the Born repulsion energy, the London attraction energy and the oxyanion ACh+ dipole repulsion energies have been calculated and shown to be consistent with the model. The displacement of the Mg2+ by two ACh+ ions makes several hundred kcals of energy available for receptor group separation and receptor activation.     

Sodium transport and phosphorus metabolism in sodium-loaded yeast: simultaneous observation with sodium-23 and phosphorus-31 NMR spectroscopy in vivo

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.     

45Ca2+ uptake and peptide hormones secretion in the presence of excess Mg2+ content

Studies have been performed on the relationship between PRL and GH production and the 45Ca2+ influx in high magnesium content in vitro. The obtained data show that an elevated magnesium concentration in Krebs-Ringer solution is capable of inhibiting some hormonal function of the pituitary gland. It has been found, that PRL and GH released into the media in normal KRB solution revealed nearly two times higher concentration than in the presence of high Mg2+. Instead the cellular iPRL and iGH did not show any significant differences in control and in treated cultures. The incorporation of 4.5-3H-leucine into the prolactin and growth hormone demonstrate a significant decrease in the presence of high Mg2+ indicating that the ion is able to inhibit the secretion of newly synthesized PRL an GH. High concentration of Mg2+ abolished either the stimulation effect of releasing hormones on calcium uptake.     

Polyphosphate-cation interaction in the amino acid-containing vacuole of Neurospora crassa

The vacuoles of Neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-P. Vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. Thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. Polyphosphate was found to be the only macromolecular polyanion in vacuoles of normal or phosphate-depleted cells. Gel filtration experiments revealed that about half the polyphosphate of normal vacuoles is bound strongly by vacuolar spermidine, Mg2+, and Ca2+. The polyphosphate thus occupied was not available for basic amino acid binding. We have identified about 90% of the cations of isolated vacuoles; in addition to spermidine, Mg2+, and Ca2+, the cation pool consists mainly of arginine, ornithine, histidine, lysine, and Na+, with a small amount of K+. Isolated vacuoles appear to be almost wholly impermeable to all these ions, and in vivo, vacuoles appear to be highly selective in ion uptake by an active process. The interaction of basic amino acid with the available polyphosphate was found to reduce the chemical activity of the former. In keeping with this effect, cells with abnormally high basic amino acid-polyphosphate ratios displayed greatly swollen vacuoles, indicating considerable osmotic activity of the basic amino acids and their counterions under these conditions.