Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.
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PMID:Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes. 12 74

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H2 oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H2 oxidase by 50% and solubilizes the bulk of the ATPase. 2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa3, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm). 3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2--3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H2 oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20-100% stimulation of oxygen uptake). 4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.
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PMID:Respiratory components and oxidase activities in Alcaligenes eutrophus. 18 46

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.
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PMID:Functional mosaicism of membrane proteins in vesicles of Escherichia coli. 19 Feb 12

The spheroplasts were prepared from the gram-negative bacterium Pseudomonas aeruginosa IBFM-13 upon incubation with lysozyme, but without EDTA. The following conditions were found to be optimal: 20 percent sucrose; lysozyme, 54 mcg/mg protein of the cells; pH 7.6. The spheroplasts, as well as the cells, have a high rate of malate oxidation, but do not oxidize NADH2, the latter fact suggesting that the cytoplasmic membrane remains intact.
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PMID:[Production of spheroplasts from Pseudomonas aeruginosa]. 80 84

1. The crude envelope preparation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was separated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a single trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped structures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. The outer membrane fraction consisted of protein (similar to 40%), lipopolysaccharide (similar to 36%) and lipid (similar to 18%) and had a density of about 1.22 g/cm3. The cytoplasmic membrane fraction consisted mostly of protein (similar to 56%) and lipid (similar to 38%), had a density of about 1.16 g/cm3, and contained almost all the NADH oxidase, succinate and D-lactate dehydrogenase activities of the crude envelope preparation. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6-7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.
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PMID:The outer membrane of Proteus mirabilis. I. Isolation and characterization of the outer and cytoplasmic membrane fractions. 109 Dec 89

Subcellular distribution study of cytoplasmic organelles was performed on human polymorphonuclear leukocytes after homogenization in 0.34 molar sucrose by differential centrifugation and sucrose density gradient centrifugation of the homogenate. The whole homogenate and each fraction was assayed for nitroblue tetrazolium (NBT)-reductase with and without 1 mM potassium cyanide, and the distribution of this enzyme was compared to the distribution of lysozyme, peroxidase, beta-glucuronidase, and acid and alkaline phosphatase. Enzyme recovery was 97 per cent and ranged between 74 and 124 per cent. Latent activity of all enzymes except NBT-reductase, acid, and alkaline phosphatase was demonstrated by observing a four- to sixfold increase in activity after the addition of Triton-X 100. Maximal relative specific activity using either DPNH or without cyanide for NBT-reductase was found in the 100,000 x g differential centrifugation fraction and was concentrated in the less dense top fraction of the sucrose density gradient. The distribution pattern was similar to acid and alkaline phosphatase. In contrast, the maximal concentration of beta-glucuronidase and peroxidase was found in the heavier 7,200 x g granule fraction and in the more dense bottom fractions of the sucrose density gradient. Maximal lysozyme activity was concentrated in the 30,000 x g granule fraction and in the fractions located between the heaviest and lightest fractions of the sucrose density gradient. The lack of latent activity and the similarity of subcellular distribution of NBT-reductase to acid and alkaline phosphatase, two enzymes associated with microsomes and plasmalemal membranes in human polymorphonuclear leukocytes (PMN), indicates that NBT-reductase is also a nonlysosomal enzyme located in microsomes or in plasmalemal membranes. These findings support the previously described histochemical observations that initial reduction of NBT to formazan occurs on the PMN plasmalemal surface membrane at the point of particle attachment. In addition, they suggest that alteration of the surface membrane of the PMN by particle attachment or other surface forces may activate NBT-reductase, leading to an accumulation of formazan in the region of the altered membrane as the phagocytic vacuole is formed.
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PMID:Subcellular distribution of nitroblue tetrazolium reductase (NBT-R) in human polymorphonuclear leukocytes (PMN). 118 38

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2,2'-azo-bis-2-amidinopropane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 microM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals. Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD+ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 microM (tryptophan and Tempol) to 5 mM (NAD+).
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PMID:Effect of additives on the inactivation of lysozyme mediated by free radicals produced in the thermolysis of 2,2'-azo-bis-(2-amidinopropane). 177 8

Legionella pneumophila, the etiologic agent of Legionnaires' disease, is phagocytized in an unusual way and multiplies in human mononuclear phagocytes in a novel phagosome. As a first step toward understanding these L. pneumophila-phagocyte interactions, we have studied the envelope of L. pneumophila Philadelphia 1 strain. We isolated cell envelopes by treating whole bacterial cells with lysozyme and EDTA to convert them to spheroplasts, then lysing the spheroplasts osmotically or sonically. We resolved the cell envelopes into two membrane fractions by isopycnic centrifugation. We localized NADH oxidase to the fraction of buoyant density 1.145, which we designated cytoplasmic membrane, and lipopolysaccharide (LPS) to the fraction of density 1.222, which we designated outer membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the L. pneumophila outer membrane contains a single major protein species migrating at 28,000 mol wt; this is the major protein of the bacterium. The cytoplasmic membrane also contains a single major protein species migrating at 65,000 mol wt. Surface iodination of the bacteria and agglutination and immunofluorescence studies with rabbit antibody produced against the purified major outer membrane protein (MOMP) revealed that this protein is exposed at the cell surface. We isolated LPS from L. pneumophila membranes by SDS-EDTA treatment. The pattern obtained by subjecting the LPS to SDS-PAGE and staining the gel with silver nitrate suggests that L. pneumophila LPS might be atypical. We studied patient serologic responses to cell envelope components of L. pneumophila Philadelphia 1, a serogroup 1 organism. Sera from patients with evidence of infection with serogroup 1 L. pneumophila contained large amounts of antibody to this strain. Few of these antibodies recognized the MOMP of L. pneumophila. In contrast, greater than 98% of these antibodies were directed against the LPS. This indicates that LPS is the dominant serogroup antigen and the major antigen responsible for the reactivity of patient sera in the indirect fluorescent antibody assay, currently the principal diagnostic assay for Legionella infection.
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PMID:Isolation and characterization of the cytoplasmic and outer membranes of the Legionnaires' disease bacterium (Legionella pneumophila). 388 79

The energy-transducing, Mg-Ca activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of E. coli is located on the inner surface of the cytoplasmic membrane. Antibody to purified ATPase has now been used to demonstrate that membrane vesicles as ordinarily prepared by the lysozyme-EDTA method consist of two distinct populations. About half the vesicles are everted, and thus readily agglutinated by antibody to ATPase, while half are right-side out. NADH oxidase (reduced NAD:O(2) oxidoreductase EC 1.6.99.3) activity is associated almost entirely with everted vesicles, while the ability to concentrate proline is a property of the right-side out vesicles. The results explain the failure of previous workers to observe the energization of membrane vesicles by oxidation of NADH.
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PMID:Heterogeneity of membrane vesicles from Escherichia coli and their subfractionation with antibody to ATPase. 415 73

1. The liberation of the vesicles of the mesosomes from protoplasts of Bacillus licheniformis strain 6346 is controlled by the Mg(2+) concentration present during the removal of the walls by lysozyme. 2. The functioning of the cytoplasmic membranes is also critically controlled by the Mg(2+) concentration. 3. The isolated mesosomes and the cytoplasmic membranes differ in their enzymic activities. Both succinate dehydrogenase and NADH oxidase activities are very low or absent from the mesosomes. 4. The cytoplasmic membranes can also be separated into materials of different density. 5. Distribution of the membranes between these fractions is controlled by the Mg(2+) concentration and the growth conditions of the microorganisms.
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PMID:Some enzymic activities and chemical properties of the mesosomes and cytoplasmic membranes of Bacillus licheniformis 6346. 430 39


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