Simultaneous formation of menaquinones and demethylmenaquinones byMicrococcus varians IAM 12146 depending on cell growth media

Changes in the isoprenoid quinone composition ofMicrococcus varians IAM 12146 in response to growth in different media were investigated. When the bacterium was growth in an ordinary complex medium, it produced menaquinones as the sole quinones, with a dihydrogenated menaquinone with seven isoprene units as the major component, at all growth stages. On the other hand, cells grown in a chemically defined medium containing glutamate and pyruvate as carbon sources produced both menaquinones and demethylmenaquinones. The major demethylmenaquinone homologs produced were the unsaturated and dihydrogenated types with seven isoprene units. The demethylmenaquinone/menaquinone ratio in cells varied during a batch growth in the chemically defined medium. The highest ratio was found in cells at the mid-exponential phase of growth.     

Role of metal ions in oxidant cell injury

This paper represents a short review of recent data on the molecular mechanism(s) of H₂O₂ cytotoxicity. The role of metal ions has been discussed in light of their ability to drive a reaction of the Fenton type. Hydroxyl radicals play a key role in mediating the deleterious effects of the oxidant, although these species do not seem to directly produce the lethal lesions. It still remains unclear whether or not DNA represents a critical target for H₂O₂-induced cytotoxicity.     

Slow adaptive changes in urease levels of tobacco cells cultured on urea and other nitrogen sources

Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea as the sole source of nitrogen have urease activities about four times higher than cells which have been cultured on nitrate. When cells which had always been grown on nitrate were transferred to urea, the urease activity in these cells remained at a lower level for eight transfers (40 generations), then gradually increased 4-fold during the next seven to 10 transfers. Cells with high urease activity multiplied 19% more rapidly and accumulated less urea than cells with low urease activity. These findings suggest that elevated urease accelerates urea assimilation; therefore, urea limited growth. Clones of cells with low urease activity responded in the same way as uncloned populations when transferred from nitrate to urea, indicating that high urease cells originate from low urease cells, rather than from a preexisting subpopulation of high urease cells. The urease levels in clones of cells from a population with high urease activity were three to seven times the low urease level. The observed dependence of urease activity on generations of growth on urea was matched with a model in which high urease cells originated at mitosis of low urease cells at a frequency of 8 × 10⁻⁵, then multiplied 19% more rapidly than low urease cells. This frequency is about 10³ greater than that of other biochemical variants previously isolated from XD cells. The high urease activity gradually declined in cells transferred from urea to other nitrogen sources, but rose rapidly when such cells were returned to urea, indicating the existence within the cells of some form of record of their ancestors' growth on urea. The data indicate the existence of a mechanism for generation, at unusually high frequency, of metastable variants with high urease activity. This mechanism, coupled with enrichment for the variants' progeny by virtue of their higher multiplication rate on urea, can account for the observed slow increase in urease activity of the population. It is suggested that the molecular basis of the urease increase may be gene amplification, based on animal cell models. An alternative hypothesis, namely a specific response induced in all cells by urea and manifested as a very slow adaptive increase in urease, has not been ruled out.     

Role of carbon and nitrogen sources in bacterial growth and sporulation

[This corrects the article on p. 131 in vol. 22.].     

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.     

Heavy metal ions inhibition of jack bean urease: potential for rapid contaminant probing

The kinetics of heavy metal ions inhibition of jack bean urease was studied by progress curve analysis in a reaction system without enzyme-inhibitor preincubation. The inhibition was found to be biphasic with an initial, small inhibitory phase changing over the time course of 5-10 min into a final linear steady state with a lower velocity. This time-dependent pattern was best described by mechanism B of slow-binding inhibition, involving the rapid formation of an EI complex that subsequently undergoes slow conversion to a more stable EI* complex. The kinetic parameters of the process, the inhibition constants Ki and Ki* and the forward k5 and reverse k6 rate constants for the conversion, were evaluated from the reaction progress curves by nonlinear regression treatment. Based on the values of the overall inhibition constant Ki*, the heavy metal ions were found to inhibit urease in the following decreasing order: Hg2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > Pb2+ > Co2+ > Fe3+ > As3+. With the Ki* values as low as 1.9 nM for Hg2+ and 7.1 nM for Cu2+, 100-1000 times lower than those of the other ions, urease may be utilized as a bioindicator of the trace levels of these ions in environmental monitoring, bioprocess control or pharmaceutical analysis.     

Template-directed synthesis of oligoguanylates in the presence of metal ions

We have studied the condensation reaction of ImpG² on a poly(C) template, in the presence of various metal ions. With Mg²⁺ as co-catalyst we confirmed that Pb²⁺ and Zn²⁺ are effective catalysts. A catalytic effect was also observed for Bi³⁺, Sb³⁺ and Mn²⁺. Bi³⁺ and Sn²⁺, like Pb²⁺, favored the formation of 2′-5′ linkages. With Mn²⁺ a rather complex mixture of oligomers is formed, some of which contain pyrophosphate linkages. None of the metal ions investigated behaved like Zn²⁺ in favoring the formation of the naturally occurring 3′-5′ linkages.     

Stimulation of mitochondrial protein synthesis by metal ions.

1--10 muM Cu2+, Ag+, and Au3+ were found to stimulate rat liver mitochondrial protein synthesis in vitro. Cu2+ and Ag+ also produced an increase in mitochondrial volume ("swelling"). Thus, thyroid hormones and their analogs are not unique, as suggested previously (Buchanan, J.L., Primack, M.P. and Tapley, D.F. (1970) Endocrinology 87, 993--999), in stimulating both mitochondrial protein synthesis and swelling. Furthermore, the data suggest a role for Cu2+ in the regulation of mitochondrial protein synthesis.     

Role of some trace metal ions in placental membrane lipid peroxidation

The antioxidative and/or pro-oxidative potential of three trace metal ions, namely aluminum (Al), manganese (Mn), and selenium (Se), has been studied. The effect of Al and Mn was found to be anion independent. The pro-oxidative potential of Al was more prominent than its antioxidative potential. This may be due to its redox inert nature. The increase in lipid peroxidation rates in placental syncytiotroblast membranes may contribute to the etiology of aluminum toxicity. Selenium had an antioxidative potential only in the whole-cell homogenate. This appears to be mediated by glutathione peroxidase of which Se is a cofactor. Manganese proved to be the trace metal ion of choice. It decreased the production of thiobarbituric acid reactive substances (TBARS). This may be due to its capacity to quench the superoxide anion and hydroxyl radicals and also due to its chain-breaking capacity. During the present course, ferrous-ascorbate mediated lipid peroxidation has been studied using various combinations of FeSO₄ and ascorbic acid. Extrapolating the combined ratio of the individual combination as substrate concentration ([S]) and treating the observed amount of malondialdehyde (MDA) produced equivalent to initial velocity (v _i ), as in the case of enzymatic studies, the data were treated according to Michaelis-Menten kinetics and the values of k _c and C _max have been calculated.     

Role of oxygen and metal ions in the instability of streptolysin O.

Thiol-containing preparations of streptolysin O (SLO) and pure cysteine generate superoxide radicals in alkaline buffer on autoxidation of the thiol groups. Autoxidation is stimulated by cupric ions. Reconstituted SLO preparations accumulate hydrogen peroxide with a concomitant loss of activity on storage at room temperature. Short-term protection of hemolytic activity was achieved by inclusion of catalase in the preparation; no apparent protection was observed by superoxide dismutase, whereas 1,10-O-phenanthroline offered long-term protection of the hemolysin.     

Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.     

Inhibition of urease by cyclic beta-triketones and fluoride ions

Competitive inhibition of soybean urease by 11 cyclic beta-triketones was studied in aqueous solutions at pH 7.4 and 36 degrees C. This process was characterized quantitatively by the inhibition constant (Ki), which showed a strong dependence on the structure of organic chelating agents (nickel atoms in urease) and varied from 58.4 to 847 microM. Under similar conditions, the substrate analogue (hydroxyurea) acted as a weak urease inhibitor (Ki = 6.47 mM). At 20 degrees C, competitive inhibition of urease with the ligand of nickel atoms (fluoride anion) was pH-dependent. At pH 3.85-6.45, the value of Ki for the process ranged from 36.5 to 4060 microM. Three nontoxic cyclic beta-triketones with Ki values of 58.4, 71.4, and 88.0 microM (36 degrees C) were the most potent inhibitors of urease. Their efficacy was determined by the presence of three >C=O- groups in the molecule and minimum steric hindrances to binding with metal sites in soybean urease.     

Role of metal ions in the tetraloop-receptor complex as analyzed by NMR

Metal ions are critical for the proper folding of RNA, and the GAAA tetraloop-receptor is necessary for the optimal folding and function of many RNAs. We have used NMR to investigate the role of metal ions in the structure of the tetraloop-receptor in solution. The NMR data indicate native tertiary structure is formed under a wide range of ionic conditions. The lack of conformational adaptation in response to very different ionic conditions argues against a structural role for divalent ions. Nuclear Overhauser effects to cobalt hexammine and paramagnetic relaxation enhancement induced by manganese ions were used to determine the NMR structures of the tetraloop receptor in association with metal ions, providing the first atomic-level view of these interactions in the solution state. Five manganese and two cobalt hexammine ions could be localized to the RNA surface. The locations of the associated metal ions are similar, but not identical to, those of previously determined crystal structures. The sites of association are in general agreement with nonlinear Poisson-Boltzmann calculations of the electrostatic surface, emphasizing the general importance of diffusely associated ions in RNA tertiary structure.     

Role of divalent metal ions in the hammerhead RNA cleavage reaction.

A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur.(ABSTRACT TRUNCATED AT 250 WORDS)