Detachment and Chemical Characterization of the Regularly Arranged Subunits from the Surface of an Acinetobacter

Acinetobacter sp. strain MJT/F5/199A carries an array of tetragonally arranged subunits on its outer surface. The subunits can be detached from isolated cell walls by incubation with 1 M urea or by washing with water after treatment with 10 mM ethylenediaminetetraacetic acid or ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid. After removal of the urea, they reaggregate into the same ordered array at air-water interfaces in the presence of MgCl(2). The detached subunits were characterized as an acidic protein of molecular weight 65,000. They represent one-fifth of the total cell wall protein.     

Biochemical requirements for PCBCK1 kinase activity, the Pneumocystis carinii MEKK involved in cell wall integrity

Fungal cell wall assembly is a complicated process involving multiple enzymes and coordinated signaling pathways. The cell wall integrity MAPK pathway acts to stabilize the fungal cell wall during conditions of elevated temperature by regulation of glucan synthesis. The upstream kinase, BCK1, is a critical component of this pathway. Pneumonia is a significant cause of death from the fungal opportunistic pathogen Pneumocystis in immunocompromised states, especially with HIV infection. We have previously shown that PCBCK1 functions in the cell wall integrity pathway in yeast as a functional protein kinase. Kinases have specific requirements for enzymatic function which have not been investigated in fungi. Here we examine the biochemical requirements for PCBCK1 kinase activity expressed in Saccharomyces cerevisiae bck1Delta yeast. PCBCK1 requires 10 mM MgCl(2), pH 6, temperature 30 degrees C, and 10 microM ATP for kinase activity. Interference of the Pneumocystis cell wall integrity pathway is an attractive target for drug development since glucan synthesis machinery is not present in humans.     

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.     

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.     

Loss of primary alkylsulfatase and secondary alkylsulfatases (S-1 and S-2) from Pseudomonas C12B: effect of culture conditions, cell-washing procedures, and osmotic shock.

Secondary alkylsulfatases (S-1 and S-2) were detected in Pseudomonas C12B cultured in minimal media lacking alkylsulfatases. The synthesis of these enzymes was not repressed by SO4-2- and L-cysteine or derepressed by L-methionine. Growth on 4% sodium citrate caused a 98% loss in the secondary alkylsulfatase activity of cells and 9% of this activity was detected in the culture medium. Citrate (20 mM) inhibited the activity of cell extracts (18%) but the inhibition was reversible by dialysis. The primary alkylsulfatase content of cells was not diminished by growth on citrate. The MgCl2 concentration of the medium also influenced the cellular levels of secondary alkylsulfatase. Bacteria grown on MgCl2 (0.05 mM - 40 mM) exhibited progressively increasing activity while the converse distribution was observed for activity present in the medium after growth at each MgCl2 concentration. Both primary and secondary alkylsulfatases were released when cells were either subjected to osmotic shock or treated for cell wall removal. Cells washed with 0.085 M sodium citrate-10 mM Tris-20%sucrose released roughly 87% of both enzymes and MgCl2 (0.04 M) inhibited the release of primary alkylsulfatase by 11% and secondary alkylsulfatase by 50%. It is suggested that citrate chelates divalent cations necessary for the attachment of secondary alkylsulfatase at the cell periphery.     

PEG和盐对棕色固氮菌细菌铁蛋白和钼铁蛋白晶体生长的影响

棕色固氮菌（ＯＰ）体内的固氮酶钼铁（ＭｏＦｅ）蛋白和细菌铁蛋白均为重要的生物功能蛋白。前者为生物固氮的关键酶［１］,后者则可为生物代谢贮存丰富而又可溶的铁原子［２］。因而都得到了广泛而深入的研究。Ｋｉｍ［３］报道了ＭｏＦｅ蛋白衍射结果。赵宝光等［２］...     

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

Isolation of Outer Membranes with an Ordered Array of Surface Subunits from Acinetobacter

A method has been developed for the isolation of outer membranes from Acinetobacter sp. strain MJT/F5/199A. Washed cells were broken in a French press and, after deoxyribonuclease and ribonuclease treatment, removal of intact cells, and four washes in 20 mosmol phosphate buffer, pH 7.4, with centrifugation at 25,000 x g for 10 min, preparations of cell wall fragments from which almost all pieces of plasma membrane had been removed resulted. Treatment of the cell walls with lysozyme and further washing, in the presence of 20 mM MgCl(2), yielded preparations of outer membranes. Electron microscopy of freeze-etched preparations shows that a regular pattern of subunits is present on the outer surfaces of intact cells. After negative staining, these subunits are visible on isolated walls and outer membranes; they can be removed by brief treatment with papain. In section, the cell wall structure is that typical of gram-negative bacteria, but the subunits are not detectable on the surface of the outer membrane. The outer membrane retains the appearance of a "unit membrane" in the cell wall, isolated outer membrane, and papain-treated outer membrane fractions. Both cell walls and outer membranes contain a high percentage of protein (76 and 84%, respectively) and not more than 5% carbohydrate, of which glucose and galactose are constitutents. The outer membranes of this Acinetobacter thus differ in structure and composition from those of bacteria in the Enterobacteriaceae.     

Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods

Multiple methods have been developed to isolate the intestinal microvillus membrane and facilitate the study of its composition and function. Variations in membrane composition and fluidity may result from different preparative techniques. This study shows that the use of MgCl2 and/or KSCN in vesicle preparation alters phospholipid and protein composition of the membrane compared to CaCl2 precipitation. The use of MgCl2 in membrane preparation increased phosphatidylethanolamine and decreased phosphatidylinositol content. The use of KSCN in membrane preparation decreased the protein content. The structural changes seen with the use MgCl2 alone are accompanied by an increase in both static and dynamic membrane fluidity. These results suggest that different methods of membrane vesicle preparation affect membrane phospholipid and protein content as well as membrane fluidity.     

Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions.

The ability of different metal ions to promote transformation of Pseudomonas aeruginosa by deoxyribonucleic acid of the plasmid RP1 was examined. CaCl2, MgCl2, and MnCl2 were found to promote such transformation, although at different frequencies and with the optimum response at different concentrations. Only MgCl2 promoted transfection of P. aeruginosa by the linear deoxyribonucleic acid of phage F116. CaCl2 was demonstrated to allow adsorption and entry into the cell of F116 deoxyribonucleic acid such that it became resistant to exogenous deoxyribonuclease, but phage production occurred only when MgCl2 was provided. Inactivation of linear phage deoxyribonucleic acid taken up in the absence of MgCl2 was observed. The transfection frequencies at various concentrations of MgCl2 were compared, and the optimum response occurred at the concentration which promoted the highest frequency of transformation by RP1 deoxyribonucleic acid.     

Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.     

Magnesium deficiency suppresses cell cycle progression mediated by increase in transcriptional activity of p21(Cip1) and p27(Kip1) in renal epithelial NRK-52E cells

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.     

Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.     

Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.     

A long acidic domain affects the chromatographic behaviour of a neuronal adaptor protein on DEAE-Sepharose

The stepwise chromatographic behaviour on DEAE-Sepharose of rat Fe65, a neuronal protein, was tested, using as eluants KCl, CaCl2, and MgCl2. Assays by western blot showed that Fe65 was eluted by CaCl2, at a ionic strength 20% lower than that of MgCl2 or KCl. Interestingly, in the case of a truncated Fe65, lacking a glutamic acid rich region at the N-terminus, the ionic strengths of the various eluants were almost identical. These results suggested a possible inhibitory role of calcium ions in the binding of the protein to DEAE and a specific affinity of these ions for long acidic stretches.     

Release of alkaline phosphatase from cells of Pseudomonas aeruginosa by manipulation of cation concentration and of pH

Pseudomonas aeruginosa ATCC 9027 contains an inducible alkaline phosphatase. The enzyme is readily removed from 14-hr cells by washes in 0.2 m MgCl(2), pH 8.4. Similar washes in tris(hydroxymethyl)aminomethane buffer, 20% sucrose, monovalent ions, or water partially release enzyme from the cells. The release of alkaline phosphatase is correlated with an increased release of protein and retention of internal enzymes. The effect of 0.2 m MgCl(2) washing upon the cells is minimal since both viability and growth rates remain unchanged as compared to water washing. Although cells are plasmolyzed in both 0.2 m MgCl(2) and 20% sucrose, it is evident that plasmolysis alone is unable to account for total enzyme release and that a divalent metal, i.e. Mg(2+), augments the release pattern. Growing cells in the presence of increasing concentrations of MgCl(2) or at increased pH values results in an almost total secretion of the enzyme to the culture filtrate. The findings suggest that P. aeruginosa alkaline phosphatase is linked to the exocytoplasmic region through divalent metal ion, presumably Mg(2+), bridges.