LOCALIZATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN <i>ESCHERICHIA COLI</i> USING COMBINED TECHNIQUES OF CYTOCHEMISTRY AND ELECTRON MICROSCOPY

Sedar, Albert W.; Burde, Ronald M.

doi:10.1083/jcb.24.2.285

Cited by 29 publications

(10 citation statements)

References 17 publications

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“…Tho inhibition of dehydrogenaso activity in whole cell and cell free extracts occurs around the MIC value for each organism, although the enzyme systems are inhibited by l o w t~ drug coiiccntrationR than is 0011 viability. The dehydrogenase enzymes syst.ems arc located in the cell membrane as demonstrated by Sedar & Burde (1965) and Hirano, Gcorgi & Militzer (1969) and alteration of these enzymes may change membrane structure and permit leakage. There is no stimulation of enzyme activity by low concentrations of bronopol, and both the aerobic and anaerobic metabolism of glucose are affected by approximately equal concentrations.…”

Section: Discussionmentioning

confidence: 95%

“…For E . coli the succinate and formate dehydrogenase activities were also examined using the methods of Sedar & Burde (1965) and Billen & Lichstein (1950), respectively. Dehydrogenase activity was also determined by measuring the reduction of triphenyltetrazolium chloride (TTC) in open tubes by the method of Hugo (1954) in a system containing 6 mg of cells, 250 mg of TTC, 0.01 M phosphate buffer (pH 7.2) and 0.05 M glucose.…”

Section: A Nal Yticul Methodsmentioning

confidence: 99%

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Some Aspects of the Mode of Action of the Antibacterial Compound Bronopol (2‐bromo‐2‐nitropropan‐1,3‐diol)

Stretton

Manson

1973

Journal of Applied Bacteriology

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A series of nitro compounds studied inhibited Eecherichia coli, ~taphylococcua aureue and P 8 e U d O V l O W aeruginosa, bronopol being the mout active derivative. Bronopol causes some membrane damage. A consideration of its action on enzyme activity, reaction with cell constituents and chemical reaction with cysteine, glutathiono and proteins suggests that the oxidation of thiol groups t o disulphides may be its primary action. ZSOLNAI (1961) examined a number of alkyl and aralkyl nitro compounds and demonstrat,eci the antifungal activity of this group, particularly where the structural feature, CBrNO,, was present. Eckstcin et al. (1963) described the antifungal activity of some 2-nitropropan-l,3-01 derivatives and showed that 2-bromo-2-nitropropan-1,3 tliol was one of the more active compounds. Croshaw, Groves & Lessel (1964) studied the activity of 2-bromo-2-nitropropan-l,3-diol (bronopol) and found it to be active against both Gram positive and Gram negative bacteria, being particularly active against Pseudomonas aeruginosa, and exhibiting activity against yeasts and fungi. The bacteriostatic action was decreased in the presence of cysteine hydrochloride, serum or horse blood. The wide spectrum of activity makes it suitable for use as a preservative in pliarmaceutical products (Sykes, 1969), and because of its lack of irritancy combined with stability in aqueous solution, Bryce & Smart (1965) recommended its use in shampoo formulations. Brown (1966) showed that bronopol was rapidly active against Ps. aeruginosa and unaffected by the presence of nonionic detergents. Materials and Methods OrganismsEscherichia coli NCTC 9001 , Staphylococcus aureus NCIB 8626 and Pseudomom aeruginosa NCIB 8295 were used throughout, and Bacillus megaterium NCIB 9521 for the preparation of protoplasts. MediaThe organisms were maintained in a nutrient broth containing (g/l): Oxoid beef extract, 10; Oxoid neutralized peptone, 10; sodium chloride, 6. Solid media were * Present address, Rexford Ltd, Manningtree, Essex. 1611 62 R. J. Stretton and T. W. Manson prepared by adding 1.5% w/v of Oxoid agar No. 3. A drug neutralizing medium was prepared by adding 0-1 1 yo of sodium thioglycollate. The pH value of all media was adjusted to 7.2. Chemicals Bronopol (2-bromo-2-nitropropan-l,3-diol) is a white, odourless, crystalline solid, map. 121 O, and is readily soluble in water. The sample used was a gift from The Boots Co. Ltd, Nottingham, England. The other diol nitro compounds examined, 2-nitro propan-1,3-diol and 2-chloro-2-nitropropan-l,3-diol were prepared by the method of Eckstein et al. (1963). 2-Dinitropropane, 2,3-nitro-2-methylbutane, 2-bromo-2nitropropane, 2-chloro-2-nitropropane and 2-bromo-2-nitro-3,3-dimethylbutane were prepared by Dr W. R. Bowman of this Department. Sterile solutions of the nitro compounds were prepared by filtration through a membrane filter of 0.2 pm porosity (Sartorius Membrane filter, V.

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Section: Discussionmentioning

confidence: 95%

Section: A Nal Yticul Methodsmentioning

confidence: 99%

Some Aspects of the Mode of Action of the Antibacterial Compound Bronopol (2‐bromo‐2‐nitropropan‐1,3‐diol)

Stretton

Manson

1973

Journal of Applied Bacteriology

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“…A number of similar experiments concerning dehydrogenase localization have been carried out on bacterial cells (2,6,9,14,15,16,18,19). A relationship between esterases and structures of the bacterial cell has not been studied by electron microscopy.…”

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confidence: 96%

Electron Microscopy of Alkaline Phosphatase of Escherichia Coli

Kushnarev¹,

Смирнова²

1966

Can. J. Microbiol.

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A method is described for determining the localization of alkaline phosphatase in the cells of E. coli B with the electron microscope. Enzyme activity, determined by deposition of inorganic phosphate, is located in the exterior layer of the cell wall.Electron microscopy is of great importance in the study of the relationship of enzymes and cell ultrastructures and this application has permitted the localization of some esterases and dehydrogenases (1,4,11). A number of similar experiments concerning dehydrogenase localization have been carried out on bacterial cells (2,6,9,14,15,16,18,19). A relationship between esterases and structures of the bacterial cell has not been studied by electron microscopy.The present paper concerns the distribution of alkaline phosphatase in the cells of Escherichia coli B. Materials a n d MethodsThe cells used were E . coli B. An 18-hour culture of E. coli B on nutrient agar was inoculated into a salts medium having limited inorganic phosphate (17) and grown for 24 to 48 hours. All the salts were recrystallized. Sodium P-glycerophosphate for determination of phosphatase was obtained from the Merck Company. No trace of free orthophosphate was found in this preparation.Coloration.-After growth, the cells were centrifuged and placed in a glycerophosphate medium (3) a t pH 9.0. The incubation was continued a t 37 OC for 10 minutes to 2 hours. After that the cells were again centrifuged and suspended in a 2y0 cobalt nitrate soiution, b u t in some experiments, suspension in cobalt solution was omitted. After 2-3 minutes the cell suspension in cobalt solution was centrifuged a t 3000 r.p.m. for 3 minutes and the supernatant was placed on formvar-coated grids for electron microscopy.Ultrathin sections.-The material obtained after cobalt nitrate treatment was fixed according to Ryter et al. (13), dehydrated in alcohols, and embedded in the mixture of butyl-and methyl-metacrylates (4: 1). The polymerization was performed a t 55' for 24-48 hours. The sections were obtained on the "Ultratome 4800" with glass knives and collected on formvar-covered copper grids strengthened with carbon. The material obtained was contrasted in 5yo uranyl acetate solution a t room temperature for 1 hour.Electron microscopy.-The preparations of whole cells and sections were looked over in UEMV-100B electron microscope a t 50 and 75 kV with a 40-p objective and recorded with photoplates MR. Electron optical magnification, 8000-62,000 X.

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“…Flasks containing medi~im optimal for vegetative growth of the isolate (see Nolan 19756) were inoculated and then incubated for 3 d at 16°C on a reciprocating shaker (100 strokes per minute, model R-27, Psycrotherm Incubator Shaker, New Brunswick Scientific Co. Inc., New Jersey). Following a wash in 0.1 M phosphate buffer (pH 7.2) the myceliuni was inc~ibated for 30 min under the four sets of conditions as described by Sedar and Burde (1965). The conditions included (i) fixation control, (ii) experimental conditions, (iii) substrate control, and (iv) inhibition control.…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural localization of succinate dehydrogenase in a self-parasitic isolate of Saprolegnia megasperma

Murrin¹,

Nolan²

1977

Can. J. Microbiol.

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The enzyme succinate dehydrogenase (SDH, succinate: (acceptor) oxidoreductase, EC 1.3.99.1) was localized by the combined techniques of cytochemistry and electrom microscopy in the hyphae of a self-parasitizing isolate of Saprolegnia megasperma Coker. The enzyme was localized in the mitochondrial membranes; its activity was inhibited by malonate. Electron-dense deposits, whose formation was not prevented by the addition of malonate, appeared outsided of the hyphal cell walls. No evidence was found at the ultrastructural level within the vegetative hyphae for any abnormalities which could be linked to the phenomeonon of self-parasitism.

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LOCALIZATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN ESCHERICHIA COLI USING COMBINED TECHNIQUES OF CYTOCHEMISTRY AND ELECTRON MICROSCOPY

Cited by 29 publications

References 17 publications

Some Aspects of the Mode of Action of the Antibacterial Compound Bronopol (2‐bromo‐2‐nitropropan‐1,3‐diol)

Some Aspects of the Mode of Action of the Antibacterial Compound Bronopol (2‐bromo‐2‐nitropropan‐1,3‐diol)

Electron Microscopy of Alkaline Phosphatase of Escherichia Coli

Ultrastructural localization of succinate dehydrogenase in a self-parasitic isolate of Saprolegnia megasperma

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