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

The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane.
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PMID:Localization of D-lactate dehydrogenase in membrane vesicles prepared by using a french press or ethylenediaminetetraacetate-lysozyme from Escherichia coli. 80 22

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

Ozone (O3) adaptation is a well-known, but poorly understood phenomenon that has been demonstrated in humans and laboratory animals. This study examined pulmonary function and bronchoalveolar lavage fluid (BALF) parameters in O3-adapted F-344 rats to explore possible mechanisms of adaptation. Of particular interest was ascorbic acid (AA), an antioxidant reported to be protective against O3 injury and found to be increased in O3-adapted rats. Adaptation was induced by exposure to 0.25 ppm O3, 12 hr/day for 6 or 14 weeks and evaluated with a challenge test, one that reexposed rats to 1.0 ppm O3 and measured attenuation in the O3 effect on frequency of breathing. Pulmonary function was assessed 1 day postexposure and adaptation and BALF were evaluated 1, 3, and 7 days postexposure. Results showed that forced vital capacity increased over time but decreased due to exposure and that the 14-week, O3-exposed rats had an increase in forced expiratory flow rate. All of the O3-exposed rats that were tested demonstrated adaptation on Postexposure Days 1, 3, and 7, but it was diminished on Day 7. Adaptation was also more pronounced in rats exposed for 14 weeks. Except for AA, BALF levels of total protein, potassium, lysozyme, uric acid, and alpha-tocopherol were unaffected by O3 exposure. Lactic acid dehydrogenase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, and total glutathione were also assayed but were always below detectable limits. Ascorbic acid concentrations were elevated on Days 1, 3, and 7, showing postexposure patterns similar to those found for adaptation. Significant correlation was found between AA concentration and the magnitude of adaptation (r = 0.91, p < 0.002). We conclude that AA may play an important role in mechanisms associated with O3 adaptation in rats.
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PMID:Adaptation to ozone in rats and its association with ascorbic acid in the lung. 899 53

N-Acetyl-D-glucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an intermediate in the biosynthesis of the enterobacterial common antigen in E.coli and some O-antigen chains in gram-negative bacteria, is formed by the transfer of GlcNAc 1-P from UDP-GlcNAc to Und-P, analogous to the reaction forming GlcNAc-P-P-dolichol (GlcNAc-P-P-Dol) in mammalian cells. Since the microsomal enzyme from animal cells exhibits a strong preference for Dol-P, which contains a saturated alpha-isoprene unit, the polyisoprenyl phosphate specificity of the homologous bacterial enzyme was characterized. The enzyme remained bound to the membrane fraction when spheroplasts, formed by lysozyme-EDTA treatment, were lysed in hypotonic buffer. GlcNAc-P-P-Und synthase (GPT) activity was elevated in a strain of E.coli bearing the rfe gene, which encodes GPT on a multicopy plasmid, and virtually absent from rfe null mutants. GPT actively utilized fully unsaturated polyprenyl phosphate (Poly-P) substrates with maximal activity seen with (C55) Und-P, but was unable to utilize (C55)Dol-P. This substrate specificity contrasts with the microsomal GPT from pig brain, which actively utilized (C55)Dol-P, but not Und-P, as substrate. GPT activity bound to particulate fractions from three strains of bacilli also exhibited a strict preference for fully unsaturated Poly-P substrates. Unexpectedly, E.coli GPT activity cofractionated with the cytosolic marker enzyme, beta-galactosidase, and not the membrane-bound enzyme, D-lactate dehydrogenase, in cells disrupted in a French pressure cell. The properties and polyisoprenyl phosphate specificity of the soluble form of GPT were identical to the activity associated with the membrane preparations obtained from spheroplasts. The evolutionary and functional significance of the use of polyisoprenyl glycosyl carrier lipids with saturated alpha-isoprene units in eukaryotes remains uncertain.
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PMID:Polyisoprenyl phosphate specificity of UDP-GlcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E.coli. 913 38