Low Concentrations Of MgCl2 Research Articles

Pyrophosphat-abhängige ᴅ-Fructose-6-phosphat Phosphotransferase in Rhodospirillaceae / Pyrophosphate-Dependent ᴅ-Fructose-6-phosphate-Phosphotransferase in Rhodospirillaceae

A pyrophosphate-dependent fructose-6-phosphate phosphotransferase from the photosynthetic bacterium Rhodospirillum rubrum was partially purified and characterized in respect to kinetic and regulatory properties. The enzyme had a molecular weight of about 95 000 dalton and required Mg2+-ions for catalysis and maintenance of activity. The phosphotransferase was specific for fructose-6-phosphate (F-6-P) and inorganic pyrophosphate (PP) as substrates of the fructose-1,6-bisphosphate-forming reaction (forward reaction). In the phosphate (Pi)-dependent back reaction, the preferred substrate was fructose-1,6-bisphosphate (FBP). At optimal pH (7.2 for the forward, and 8.6 for the back reaction) the back reaction had a slightly higher Vmax than the forward reaction. The substrate-saturation curves of the enzyme were all hyperbolic with intersecting kinetic pattern. The Km-values (in mM) at saturating MgCl2-concentration were: 0.38 (F-6-P); 0.25 (PP); 0.02 (FBP) and 0.82 (Pi). The forward reaction was inhibited by ADP. The inhibition by ADP (Ki = 0.18 mM) was of the mixed type in respect to F-6-P, but independent of the PP-concentration. The inhibition by AMP (Ki = 0.017 mM) was of a more complex type, because AMP not only decreased the Vmax of the F-6-P- or PP-saturation curve, but also increased the Hill-coefficient from nH = 1 to nH = 2.5 of the F-6-P-saturation curve. The inhibition of the back reaction by the two adenylates was less pronounced. ATP (at 2.5 mM), like citrate, inhibited the back reaction only at low MgCl2 concentration (1 mM) indicating that the inhibitory effect was due to the chelation of Mg2+. Out of 5 other species of the Rhodospirillaceae tested, the PP-dependent phosphofructokinase was only shown to present in Rhodopseudomonas gelatinosa.

Partial purification and characterization of DNA polymerases from the cauliflower inflorescence.

Two distinct DNA polymerases, designated A and B, have been partially purified from rapidly growing apical tissues of the cauliflower inflorescence (Brassica oleracea, var. botrytis). They were readily separable from each other, because enzyme A was adsorbable on an anion-exchanger, but enzyme B was not. The effects of divalent rations and ionic strength on both enzyme activities were very different. Enzyme A utilized activated DNA well at low concentration of MgCl2 or at low ionic strength, but heat-denatured DNA was utilized more effectively than was activated DNA at high concentration of MgCl2. Enzyme B also utilized much more heat-denatured DNA and poly (rA)•(dT)10 at high ionic strength. Gel chromatography with Sephadex G-200 revealed that enzyme A has a higher molecular weight (approx. 100, 000) than enzyme B (approx. 75, 000). It has been also described that some points of properties in enzyme A and B resemble to those of the two DNA polymerase -α and -β from mammalian cells, respectively. The existence of the third enzyme, designated C, in the fraction with enzyme A was suggested with an assay system using poly (rA)•(dT)10 as the template.

Role of Alkaline Endonucleases in the Release of Soluble Chromatin from Thymus, Spleen and Liver Nuclei of Normal and Irradiated Mice

Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.

Lipoamide Dehydrogenase Regulation in Rat Brain

The Pyruvate dehydrogenase multienzyme complex (PDHC) purified from rat brain is phosphorylated in the presence of low concentrations of ATP and MgCl2. The phosphorylated PDHC is incapable of catalyzing the oxidative decarboxylation of pyruvate. In the presence of high concentrations (10 mM) of MgCl2, the phosphorylated (inactive) PDHC is converted back to the dephospho-form of PDHC which is catalytically active. The dihydrolipoyl dehydrogenase (LAD) component, E3, of PDHC is inactivated by pyridoxal phosphate (PLP) and the PLP-inactivated LAD can be reactivated by an amino acid, taurine. These results indicate the reversible formation of Schiff base between PLP and LAD. They also provide clear evidence for the involvement of LAD (E3) in the previously reported inactivation of PDHC by PLP.

�ber den enzymatischen Abbau von Tripolyphosphat und Diphosphat in zerkleinertem Fleisch

The diphosphatase of comminuted muscle tissue is activated by high MgCl2 concentrations. CaCl2, EDTA and low concentrations of MgCl2 lower the activity. The addition of diphosphate inhibits even after its complete enzymatic hydrolysis the breakdown of additionally added diphosphate; addition of MgCl2 increases the diphosphatase activity again.

Studies on the physiology and metabolism of hydrocarbon-utilizing microorganisms. XVII. Partial purification and some kinetic properties of NAD-linked and NADP-linked isocitrate dehydrogenases from Candida tropicalis.

NAD- and NADP-linked isocitrate dehydrogenases were purified 280-fold and 54-fold, respectively, from the cell-free extract of n-alkane-grown Candida tropicalis by means of ammonium sulfate fractionation, and column chromatography on Sephadex G-200 and DEAE- cellulose. NAD-linked isocitrate dehydrogenase was activated by AMP, ADP or citrate, whereas the NADP-linked enzyme was not affected by these compounds. The NAD-linked enzyme showed the kinetics of Michaelis-Menten type at a low concentration of MgCl2 (3.3 mm), while allosteric nature of the enzyme was observed at a high MgCI2 concentration (33.3 mm). Concomitant presence of glyoxylate and oxalacetate inhibited the reactions of both dehydrogenases. Especially, the NADP-linked enzyme was completely inhibited by these acids at a concentration of as low as 1 mm each, showing a mixed pattern of inhibition.

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.

The removal of non-collagen components from newborn calf dermis with magnesium chloride solution.

1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concentrations of MgCl2 from 0.5 to 1.0 M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0 M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2. 2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0 M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0 M MgCl2 solution by electron microscopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0 M MgCl2. The results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen. 3. The non-collagenous protein was purified by DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimentation analysis showed a single peak with 1.8 S and the molecular weight was approx. 43,000 as determided by SDS polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.

Length distribution of F-actin transformed from Mg-polymer.

Abstract The length distribution of F-actin produced from Mg-polymer formed in the presence of β-actinin was investigated in detail by electron microscopy. When Mg-polymer was formed at a low MgCl2 concentration (approx. 1 mM), where a fairly large amount of G-actin coexisted with Mg-polymer, and was transformed into F-actin by the addition of KCl at a fairly high temperature (45 °C), the length distribution of F-actin was of a Poisson type. The number average length was approximately 0.6 μm. On the other hand, the formation of Mg-polymer at a high MgCl2 concentration (approx. 2mM), where only a small amount of G-actin remained, resulted in a simple exponential distribution of particle length of the transformed F-actin and its average lengths became shorter. Moreover, when the amount of G-actin coexisting with Mg-polymer was increased by the addition of G-actin to the Mg-polymer solution and transformation then induced, the average length of the resultant F-actin was increased and the length distribution became more homogeneous. These results are explained by the theories of polymerization in systems with a fixed number of polymers. The amount of G-actin coexisting with Mg-polymer also increased with increase of total actin concentration. However, in this case, the average lengths were not significantly changed after its transformation into F-actin. When the transformed F-actin solution was left standing for a long time, the length distribution gradually changed from the Poisson type to the simple exponential type. This was probably due to the fact that a re-arrangement of the preformed F-actin took place. The relaxation time for such redistribution was about 25 h at 35 °C. Both β-actinin and ATP were indispensable for the transformation of Mg-polymer into the F-actin particles having a homogeneous length distribution.

EFFECT OF SALINITY ON POLLEN I. POLLEN VIABILITY AS ALTERED BY INCREASING OSMOTIC PRESSURE WITH NaCl, MgCl2, and CaCl2

Petunia (Petunia hybrida Vilm. cv. ‘Snowstorm') plants were grown in saline solution (NaCl, MgCl2, and/or CaCl2) of 0, 1, 2, and 3 bars osmotic pressures. Pollen viability was tested by tetrazolium chloride staining and by germination (by the hanging drop method, using 15 % sucrose and 0.01 % boric acid as the nutrient medium, at 27 ± 1 C). Pollen viability decreased with increased salinity. Pollen from plants grown in single salt solutions of NaCl, MgCl2, and CaCl2 (each at 0, 1, 2, or 3 bars osmotic pressure) was germinated in base culture medium. Pollen viability decreased more with NaCl than with MgCl2 or CaCl2. In vitro studies of the effects of three salts, viz., NaCl, MgCl2, and CaCl2, on pollen germination and tube growth showed that NaCl inhibited germination and pollen tube growth more than did MgCl2 or CaCl2. MgCl2 was least injurious, and even promoted tube growth at 0.5 and 0.75 bars osmotic pressure. Adding low concentrations of MgCl2 reduced the toxic effect of NaCl and increased the percentage of germination. CaCl2 reduced the effect of NaCl less than did MgCl2. We conclude that specific ion effects were more important than osmotic pressure.

Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: III. MECHANISM OF THE EFFECT OF SALT ON MENADIONE REDUCTASE

Abstract Halobacterium cutirubrum extracts exhibit no menadione reductase activity in the absence of salt; activity increases with salt concentration to a maximum at 2 m or higher concentrations of sodium chloride. The enzyme activity lost at low salt concentrations can be restored by adding 2 m NaCl. In addition to this reversible loss of activity, the enzyme also exhibits a time-dependent irreversible inactivation at salt concentrations below 2 m. The effects of salt on the initial enzyme activity and on the irreversible inactivation have a reciprocal relationship to one another. A mechanism for the salt effect is proposed, on the basis of kinetic data, whereby the active, stable form of the enzyme is in a salt-dependent equilibrium with an inactive, unstable form. The effect of NaCl, according to this model, is not to prevent the irreversible denaturation of the enzyme but rather to shift the equilibrium in favor of the active, stable form. The pH dependence of the inactivation rate constant and the reversibility of the pH effect indicate that there is an acid-base equilibrium, with pK = 9.3, between a less and a more stable intermediate form of the enzyme. Low concentrations of MgCl2, CaCl2, and some polyamines also have activating and protecting effects on menadione reductase. The mode of action of these substances is shown to be unlike that of NaCl but to consist in the alteration of the intermediate form of the enzyme in such a way as to make it more stable and partially active.

Deacylated tRNA-phe binding to a reticulocyte ribosomal site for the initiation of polyphenylalanine synthesis.

The initiation of polyphenylalanine synthesis at low MgCl(2) concentration in the reticulocyte transfer system has been found to have a stringent requirement for deacylated tRNA(Phe). An initial poly-U-directed complex between deacylated tRNA(Phe) and ribosomes is formed. The onset of polyphenylalanine synthesis causes the rapid release of tRNA(Phe) from the complex. Thermal dissociation studies indicate that deacylated tRNA(Phe) and phenylalanyl-tRNA are bound to the same ribosomes. A total of three ribosomal tRNA binding sites is indicated.

The subcellular distribution and nucleotide specificities of Na+, K+-stimulated adenosine triphosphatase and [14C]adenosine diphosphate-adenosine triphosphate exchange reactions in rat brain.

Abstract—(1) The subcellular distributions of Na‐K ATPase and [14C]ADP‐ATP exchange activities were studied in rat brain. The data presented are not consistent with a discrete localization of these enzymes in any given fraction, but nerve endings and microsomes had similar specific activities. The supernatant fraction had the highest exchange and the lowest Na‐K ATPase activities, measured at a concentration of 3 mm‐MgCl2.(2) Nucleotide specificity of the Na‐K ATPase was determined in all fractions, and this enzyme system showed an absolute requirement for ATP. The [14C]ADP‐ATP exchange, measured at 3mm‐MgCl2, possessed broader specificity and also was active toward ITP, UTP and GTP; this serves to differentiate it from the Na‐K ATPase.(3) Treatment of nerve ending fractions with NaI medium removed the bulk of the [14C]ADP‐ATP exchange activity without loss in Na‐K ATPase activity.(4) The exchange activity in NaI‐insoluble fractions was insensitive to NaCl in the presence of 3 mm‐MgCl2, but it was stimulated 502‐820 percent at low MgCl2 concentrations, a finding which may be consistent with the postulated role of this exchange reaction in the Na‐K ATPase system.

Sodium stimulated [14C]adenosine diphosphate-adenosine triphosphate exchange activity in brain microsomes.

Abstract—An ADP‐ATP exchange reaction was studied in NaI‐treated microsomes from rat brain. At low concentrations of MgCl2 a nucleotide exchange reaction, elicited in the presence of Na+, had an absolute specificity for ATP. The reaction was stimulated by oligomycin and inhibited by ouabain and EDTA. It is probable that this Na+‐stimulated ADP‐ATP exchange reaction is a component of the Na‐K ATPase system.

Isolation of ribosome particles from meningopneumonitis organisms.

In ribonucleic acid (RNA) extracted by phenol and sodium dodecyl sulfate from purified reticulate bodies of meningopneumonitis (MP) organisms, 21S, 16S, and 4S RNA were found by sucrose density gradient sedimentation analysis. When purified reticulate bodies were homogenized by sonic treatment or by treatment with sodium deoxycholate and were fractionated by differential centrifugation, more than 50% of the RNA was recovered in the fraction which was sedimented by centrifugation at 105,000 x g for 2 hr, but not at 13,000 x g for 20 min. From homogenates prepared in this manner, 50S and 30S particles containing RNA were isolated by sucrose density gradient centrifugation. These 50S and 30S particles were also found in lysates of cytoplasmic fractions of infected cells which were labeled by (32)P during 17 to 17.5 hr or 15 to 18 hr after infection. The synthesis of 50S and 30S particles was not inhibited by actinomycin D. When infected cells were homogenized in the presence of 0.01 or 0.02 m MgCl(2), 70S particles were isolated instead of 50S and 30S particles. When dialyzed against low concentrations of MgCl(2), the 70S particles dissociated to 50S and 30S particles. The base ratio of the 70S particles is very similar to that of 16S plus 21S RNA. The characteristics of the 70S, 50S, and 30S particles suggest that these are ribosome particles, similar to bacterial ribosomes.

Ribosome-bound Polyriboadenylate Polymerase

Abstract The relationship in vitro of the enzyme polyriboadenylate polymerase to ribonucleoprotein particles was investigated. The enzyme was predominantly bound to 70 S ribosomes in solutions containing 10 mM MgCl2, and to 50 S ribosomes in 1 mM and 0.1 mM MgCl2. Little if any enzymic activity was found associated with 100 S ribosomes. Dissociation of the enzyme from ribosomes in low concentrations of MgCl2 was reversible. Purified polyriboadenylate polymerase was also found to bind to 70 S, 50 S, and 30 S particles. The polyriboadenylate product of the reaction catalyzed by the ribosomal enzyme was bound to ribonucleoprotein particles in a rapidly sedimenting complex. With increasing time of incubation the sedimentation rate of this complex increased. After 5 min, it was about 130 S; 20 min, 190 S; and 60 min, greater than 300 S. This increase in sedimentation velocity of the polyriboadenylate-ribosome complex appeared to be due to aggregation of polyriboadenylate rather than to chain prolongation; the average polymer size between 5 to 60 min of incubation remained constant at approximately 200 adenylate units. The sedimentation coefficient of polyriboadenylate dissociated from ribosomes in 0.1 mM MgCl2 was 5 to 8 S. The rapid sedimentation of the complex was not changed by pancreatic ribonuclease, but after limited incubation with micrococcal nuclease, polyriboadenylate sedimented with 70 to 100 S ribosomes. Although less than 1% of total ribosomes was present in the polyriboadenylate-ribosome complex, these ribosomes were associated with most of the enzymic activity.