Magnesium Starvation of Aerobacter aerogenes III. Protein Metabolism

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.     

The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei

1. The loss of nucleic acids and protein from isolated HeLa-cell nuclei was studied. During 4hr. incubation at 37 degrees DNA was conserved, but appreciable amounts of RNA and protein were lost. 2. Two classes of nuclear RNA were distinguished: at least 75% of the RNA was lost from the nuclei relatively slowly through degradation to acid-soluble fragments; the rest of the RNA was lost much more rapidly, not only through degradation to acid-soluble fragments but also through diffusion of RNA out of the nuclei into the incubation medium. 3. The RNA that was preferentially lost was the fraction of nuclear RNA that was rapidly labelled when intact HeLa cells were grown in a medium containing radioactive precursors of RNA. 4. The RNA appearing in the incubation medium was apparently partially degraded and had a sedimentation coefficient of about that of transfer RNA. 5. Both the degradation of RNA and the loss of RNA from the nuclei were sensitive to bivalent cations. Low concentrations of Mg(2+) and Mn(2+) greatly increased the rate of degradation of the rapidly labelled RNA to acid-soluble fragments, and produced a corresponding decrease in the amount of RNA diffusing into the medium. At higher concentrations they suppressed both degradation and diffusion of RNA. The cations Ca(2+), Cu(2+), Zn(2+) and Ni(2+) all progressively inhibited both forms of loss of RNA. 6. Salts of univalent cations produced appreciable effects only at ionic strengths of about 0.2, when degradation to acid-soluble fragments was preferentially inhibited. 7. Both ADP and ATP inhibited loss of RNA at about 30mm. 8. It was concluded that the diffusion of rapidly labelled RNA out of the isolated nuclei was not related to the movement of RNA from nucleus to cytoplasm in vivo, but reflected the ease with which the rapidly labelled RNA detached from the chromatin and the permeability of the membranes of isolated nuclei.     

Effect of natural zeolite on methane production for anaerobic digestion of ammonium rich organic sludge

The effect of an inorganic additive on the methane production from NH(4+)-rich organic sludge during anaerobic digestion was investigated using different kinds of inorganic adsorbent zeolites (mordenite, clinoptilolite, zeolite 3A, zeolite 4A), clay mineral (vermiculite), and manganese oxides (hollandite, birnessite). The additions of inorganic materials resulted in significant NH4+ removals from the natural organic sludge ([NH4+]=1, 150 mg N/l), except for the H-type zeolite 3A and birnessite. However, an enhanced methane production was only achieved using natural mordenite. Natural mordenite also enhanced the methane production from the sludge with a markedly high NH4+ concentration (4500 mg N/l) during anaerobic digestion. Chemical analyses of the sludge after the digestion showed considerable increases in the Ca2+ and Mg2+ concentrations in the presence of natural mordenite, but not with synthetic zeolite 3A. The effect of Ca2+ or Mg2+ addition on the methane production was studied using Na(+)-exchanges mordenite and Ca2+ or Mg(2+)-enriched sludge. The simultaneous addition of Ca2+ ions and Na(+)-exchanged mordenite enhanced the methane production; the amount of produced methane was about three times greater than that using only the Na(+)-exchanged mordenite. In addition, comparing the methane production by the addition of natural mordenite or Ca2+ ions, the methane production with natural mordenite was about 1.7 times higher than that with only Ca2+ ions. The addition of 5% and 10% natural mordenite were suitable condition for obtaining a high methane production. These results indicated that the Ca2+ ions, which are released from natural mordenite by a Ca2+/NH4+ exchange, enhanced the methane production of the organic waste at a high NH4+ concentration. Natural mordenite has a synergistic effect on the Ca2+ supply as well on the NH4+ removal during anaerobic digestion, which is effective for the mitigation of NH4+ inhibition against methane production.     

Mechanisms of protein degradation in growing and non-growing L-cell cultures.

L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20--30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.     

Ribosomal complexes from an extremely halophilic bacterium and the role of cations

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

Fructokinase (Fraction IV) of Pea Seeds

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Polyamine replacement by magnesium ions in BHK-21/C13 cells

Cultures of BHK-21/C13 cells, whose growth was inhibited by deprivation of serum, were stimulated to grow by addition of serum to the culture medium. Addition of MgCl(2) to the medium, to increase the concentration of Mg(2+) ions by 15mm, 30min before addition of serum, had no effect on the stimulation of cell growth, but inhibited the accumulation of cellular spermidine, so that the spermidine/spermine molar ratio was lower in these cultures than in cultures that had received no additional cations. The increase in the activity of ornithine decarboxylase that occurs 4-5h after serum ;step-up' was substantially diminished by increasing the concentration of Mg(2+) ions, but not of Na(+) or K(+) ions, in the medium by 30mm, 30min before addition of serum, and this inhibition was maintained for at least 24h. Methylglyoxal bis(guanylhydrazone), added to serum-deprived cultures to a concentration of 20mum, 30min before addition of serum, severely inhibited the increase in cell growth. The inhibitory effects of the drug were prevented by simultaneous addition of spermidine to the medium (to 100mum), and were partly prevented by the simultaneous addition of Mg(2+) ions (to 30mm). Mg(2+) ions were particularly effective in overcoming the inhibitory effect of methylglyoxal bis(guanylhydrazone) on the synthesis of DNA. Thus although a certain lack of specificity for cations exists in BHK-21/C13 cells, in that Mg(2+) ions can be substituted for polyamines, particularly spermidine, to some extent, there are cellular processes for which the requirement for polyamines as cations is specific.     

K(+) and Mg(2+) ions promote the self-splicing of the td intron RNA inhibited by spectinomycin

The effects of Mg(2+) and K(+) ions on the self-splicing inhibition of the td (thymidylate synthase gene) intron RNA by spectinomycin were investigated. The maximum splicing activity occurred at 20 mM KCl. The K(m) and V(max) values for GTP in the presence of 5 mM Mg(2+) are 2.25 microM and 0.55 min(-1), whereas those for GTP both in the presence of 5 mM Mg(2+) and 5 mM K(+) are 1.23 microM and 0. 46 min(-1), respectively. Spectinomycin at 10 mM concentration inhibited the splicing by about 10%, but at 20 mM concentration, the splicing rate was inhibited by about 63%. The splicing inhibition by the low concentration of spectinomycin was overcome markedly as the concentration of Mg(2+) ion was raised. At 30 mM spectinomycin, however, the splicing inhibition was not significantly affected by increasing the concentration of Mg(2+). A similar activation of the splicing rate was observed as the concentration of K(+) ion was increased. The concentration of K(+) ion required for the normal recovery of the splicing was much higher than that of Mg(2+) ion. Unlike Mg(2+) ion, 30 mM K(+) ion effectively alleviated the splicing inhibition by spectinomycin at its high concentration. The results indicate that K(+) and Mg(2+) ions may show mechanistically different interactions with spectinomycin in the self-splicing reaction of the td intron RNA.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

Nutritional regulation of degradation of aspartate transcarbamylase and of bulk protein in exponentially growing Bacillus subtilis cells.

The rate of degradation of aspartate transcarbamylase in exponentially growing Bacillus subtilis cells was determined by measurement of enzyme activity after the addition of uridine to repress further enzyme synthesis and by specific immunoprecipitation of the enzyme from cells grown in the presence of [3H]leucine. Aspartate transcarbamylase was degraded with a half-life of about 1.5 h in cells growing on a glucose-salts medium with NH4+ ions as the sole source of nitrogen. Replacement of NH4+ in this medium with a combination of the amino acids aspartate, glutamate, isoleucine, proline, and threonine reduced the degradation rate to an undetectable level. Various other amino acids and amino acid mixtures had smaller effects on the rate of degradation. The carbon source also influenced the degradation rate, but to a smaller extent than the nitrogen source. The effects of these nutritional variables on the rate of bulk protein turnover in growing cells were generally similar to their effects on degradation of aspartate transcarbamylase. Since the degradation of aspartate transcarbamylase has been shown to be 10 to 20 times faster than bulk protein turnover, the results suggest that a substantial portion of protein turnover in growing cells represents regulable, rapid degradation of a number of normal proteins, of which aspartate transcarbamylase is an example.     

Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase.

Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it. From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose. A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments. The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-). If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited. Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.     

Formation of aerobic granules by Mg2+ and Al3+ augmentation in sequencing batch airlift reactor at low temperature

Aerobic granules technology (AGS) was difficult to cultivate at low temperature, and the treatment efficiency of domestic sewage was remarkably low because of low temperature, which greatly limits its development and application. AGS formation time significantly decreased for 43 days by adding 19.0 mg/L Mg(2+) and 21.0 mg/L Al(3+), moreover, AGS possessed better simultaneously chemical oxygen demand, NH(4) (+)-N, TP removal efficiencies at low temperature, which the respective removal efficiencies were 85.6, 88.8, and 91.9%. The content of total polysaccharides was 8.23 mg/gMLSS as well as the content of total protein was 8.52 mg/gMLSS, consequently, the total proteins/total polysaccharides ratio was 1.04, which the relatively high protein content induced by Mg(2+) and Al(3+) presented an essential feature for AGS formation. In addition, the affinity among Mg(2+), Al(3+) and -OH may drive the stretching vibration of -OH band which led to the infrared motion of functional groups in AGS and accelerate AGS formation as well.     

Influence of growth condition on the concentration of potassium in Bacillus subtilis var. niger and its possible relationship to cellular ribonucleic acid, teichoic acid and teichuronic acid

1. Mg(2+)-limited Bacillus subtilis var. niger, growing in a chemostat in a simple salts medium, contained considerably more potassium and phosphorus than Mg(2+)-limited Aerobacter aerogenes growing in a similar medium at corresponding dilution rates. 2. Growth of the bacillus in a K(+)-limited environment did not lower the cellular potassium and phosphorus contents, the molar proportions of cell-bound magnesium, potassium, RNA (as nucleotide) and phosphorus being approximately constant at 1:13:5:13 (compared with 1:4:5:8 in Mg(2+)-limited or K(+)-limited A. aerogenes). 3. Growth of B. subtilis in a phosphate-limited environment caused the cellular phosphorus content to be lowered to a value similar to that of Mg(2+)-limited A. aerogenes, but the potassium content was not correspondingly lowered; the molar potassium:magnesium ratio varied from 14 to 17 with changes in dilution rate from 0.4 to 0.1hr.(-1). 4. Whereas over 70% of the cell-bound phosphorus of Mg(2+)-limited or K(+)-limited A. aerogenes was contained in the nucleic acids, these polymers accounted for less than 50% of the phosphorus present in similarly limited B. subtilis; much phosphorus was present in the walls of the bacilli, bound in a teichoic acid-type compound composed of glycerol phosphate and glucose (but no alanine). 5. Phosphate-limited B. subtilis cell walls (from organisms grown at a dilution rate of 0.2hr.(-1)) contained little phosphorus and no detectable amounts of teichoic acid, but 40% of the cell-wall dry weight could be accounted for by a teichuronic acid-type compound; this contained a glucuronic acid and galactosamine, neither of which could be detected in the walls of Mg(2+)-limited B. subtilis grown at a corresponding rate. 6. It is suggested that the high concentration of potassium in growing B. subtilis (compared with A. aerogenes) results from the presence of large amounts of anionic polymer (teichoic acid or teichuronic acid) in the bacillus cell walls.     

Studies on the endogenous metabolism and senescence of starved Sarcina lutea

1. When washed suspensions of Sarcina lutea are starved aerobically in phosphate buffer at the growth temperature of 37 degrees , the rate of endogenous oxygen consumption decreases to very low values after 10hr., although many of the cells survive for 40hr. If starvation is prolonged further, the bacteria die at a rate of approximately 1.5% of the initial viable population per hour. 2. Oxidation of intracellular free amino acids accounts for most of the observed endogenous oxygen uptake but RNA is also utilized and a portion of the component bases and pentose is degraded and presumably oxidized. Ammonia appears in the supernatant and some pentose and ultraviolet-absorbing nucleotide are released from the cells. DNA, protein and polysaccharide are not measurably degraded. 3. Survival can be correlated with the ability of aerobically starved bacteria to oxidize exogenous l-glutamate and glucose. When starved under nitrogen for 40hr. cells continue to oxidize their endogenous reserves at undiminished rates when transferred to aerobic conditions; on prolonging anaerobic starvation the rate of oxidation declines during the period of most rapid loss of viability. 4. In the presence of Mg(2+), RNA degradation during aerobic starvation is almost completely suppressed without affecting the period for which the bacteria survive. 5. Cells grown in peptone supplemented with glucose accumulate reserves of polysaccharide which are metabolized in aerobic starvation, together with free amino acids. Ammonia is evolved and RNA is degraded to a greater extent than in peptone-grown suspensions. Bacteria rich in polysaccharide survive less well than those which are deficient in the polymer; the reason for this phenomenon has yet to be established. 6. In peptone medium, endogenous oxygen uptake and the concentration of intracellular free amino acids decline as growth progresses and they continue to decrease when the organism is held in stationary phase. Under the conditions used, the endogenous Q(o2) and free amino acid pool of cells grown in peptone with 2% (w/v) glucose did not decline so markedly and the bacteria contained large amounts of polysaccharide at all stages of growth.     

Translation of poly U by ribosomes from rel+ and rel- E. coli strains]

A translation of polyU in a cell-free system from CP78 (relA+) and CP79 (relA-) E. coli strains is investigated. The strains studied no not differ in misreading at Mg2+ concentration of 20 mM and concentrations of 16 mM (optimal for phenylalanine incorporation) and 18-22 mM (optimal for leucine incorporation) respectively. It is found that leucine incorporation increases similarly in both strains under conditions simulating an amino acid deficience (in phenylalanine-free incubation medium): the ratio leucine(-phenylalanine)/leucine (+phenylalanine) is 3.5-4 at a concentration of enzymatic fraction protein being 15-30 mkg per example, and it is 2 st a concentration of enzymatic fraction of 60-180 mkg of protein. It is suggested that differences in activities of a number of enzymes under amino acid starvation in Rel+ and Rel- cells are not due to differences in the precision of mRNA translation, but depend on the activity of respective genes.     

Characterization of Mg(2+) efflux from rat erythrocytes non-loaded with Mg(2+).

Non-Mg(2+)-loaded rat erythrocytes with a physiological level of Mg(2+)(i) exhibited Mg(2+) efflux when incubated in nominally Mg(2+)-free media. Two types of Mg(2+) efflux were shown: (1) An Na(+)-dependent Mg(2+) efflux in NaCl and Na gluconate medium, which was inhibited by amiloride and quinidine, as was Na(2+)/Mg(2+) antiport in Mg(2+)-loaded rat erythrocytes; and (2) an Na(+)-independent Mg(2+) efflux in sucrose medium and choline Cl medium, which may be differentiated into SITS-sensitive Mg(2+) efflux at low Cl(-)(o) (in sucrose) and into SITS-insensitive Mg(2+) efflux at high Cl(-)(o) (in 150 mmol/l choline Cl).     

Identification of the intracellular polyhydroxyalkanoate depolymerase gene of Paracoccus denitrificans and some properties of the gene product

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene (phaZ) was identified near the PHA synthase gene (phaC) of P. denitrificans. Cell extract of Escherichia coli carrying lacZ--phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D(--)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg(2+) ion inhibited PHB degradation. However, the inhibitory effect by Mg(2+) ion was not observed using the cell extract of P. denitrificans.