scholarly journals High-yield purification of cytochrome aa3 and cytochrome caa3 oxidases from Bacillus subtilis plasma membranes

1995 ◽  
Vol 309 (1) ◽  
pp. 279-283 ◽  
Author(s):  
W Henning ◽  
L Vo ◽  
J Albanese ◽  
B C Hill
Keyword(s):  
Bacillus Subtilis ◽  
Plasma Membranes ◽  
Sequence Data ◽  
High Yield ◽  
Cytochrome Aa3 ◽  
Quinol Oxidase ◽  
Terminal Oxidases ◽  
Aerobic Culture ◽  
Conventional Procedure ◽  
Molecular Masses

When grown in aerated shaking culture, Bacillus subtilis expresses two different haem A-containing terminal oxidases: cytochrome aa3-quinol oxidase and cytochrome caa3 oxidase. This paper describes a high-yield conventional procedure for purifying the two haem A-containing oxidases from the same aerobic culture of Bacillus subtilis. Yields of close to 40% of the total haem A are achieved and about 6 mg of each of the purified oxidases is obtained from 4 litres of liquid culture. Both of the purified enzymes have two subunits, with apparent molecular masses of 71.6 kDa and 34.3 kDa for the cytochrome caa3 oxidase, and 67.6 kDa and 37.2 kDa for aa3-quinol oxidase. These features are in agreement with the sequence data for the corresponding structural genes in the aa3 and caa3 operons of B. subtilis. Some spectral and enzymic features of the two purified oxidases are reported that are consistent with the inclusion of both of these enzymes as members of the cytochrome oxidase superfamily.

Structure of CuB in the Binuclear Heme-Copper Center of the Cytochrome aa3-Type Quinol Oxidase from Bacillus subtilis: An ENDOR and EXAFS Study

Biochemistry ◽  
1995 ◽  
Vol 34 (32) ◽  
pp. 10245-10255 ◽  
Author(s):  
Yang C. Fann ◽  
Ishak Ahmed ◽  
Ninian J. Blackburn ◽  
John S. Boswell ◽  
Marina L. Verkhovskaya ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cytochrome Aa3 ◽  
Quinol Oxidase ◽  
Copper Center ◽  
Exafs Study

scholarly journals Terminal Oxidases of Bacillus subtilisStrain 168: One Quinol Oxidase, Cytochromeaa3 or Cytochrome bd, Is Required for Aerobic Growth

2000 ◽  
Vol 182 (23) ◽  
pp. 6557-6564 ◽  
Author(s):  
Lena Winstedt ◽  
Claes von Wachenfeldt
Keyword(s):  
Bacillus Subtilis ◽  
Sequence Analysis ◽  
Terminal Oxidase ◽  
Rich Medium ◽  
Aerobic Growth ◽  
Quinol Oxidase ◽  
Terminal Oxidases ◽  
Cytochrome Bd ◽  
Heme Copper Oxidases ◽  
Quinol Oxidases

ABSTRACT The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa 3 is a cytochromec oxidase, whereas cytochrome bd and cytochromeaa 3 are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochromeaa 3 or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa 3 is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa 3 or cytochromeaa 3, is required for efficient sporulation ofB. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa 3 makes the strain sporulation deficient.

scholarly journals The hyaluronate synthase from a eukaryotic cell line

1993 ◽  
Vol 290 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L Klewes ◽  
E A Turley ◽  
P Prehm
Keyword(s):  
Monoclonal Antibodies ◽  
Phase Separation ◽  
Affinity Chromatography ◽  
Cell Line ◽  
Aqueous Phase ◽  
Plasma Membranes ◽  
Eukaryotic Cell ◽  
Molecular Masses ◽  
Triton X ◽  
Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

scholarly journals Rapid isolation of plasma membranes in high yield from cultured fibroblasts

1977 ◽  
Vol 168 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D Thom ◽  
A J Powell ◽  
C W Lloyd ◽  
D A Rees
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
High Yield ◽  
Chemical Compositions ◽  
Differential Centrifugation ◽  
Rapid Preparation ◽  
Cultured Fibroblasts ◽  
Good Agreement

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2—lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.

Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis

Biochemistry ◽  
2015 ◽  
Vol 54 (32) ◽  
pp. 5030-5044 ◽  
Author(s):  
Sophia M. Yi ◽  
Alexander T. Taguchi ◽  
Rimma I. Samoilova ◽  
Patrick J. O’Malley ◽  
Robert B. Gennis ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Cytochrome Aa3 ◽  
High Affinity

scholarly journals Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat

2004 ◽  
Vol 186 (4) ◽  
pp. 1129-1135 ◽  
Author(s):  
Rachele Isticato ◽  
Giovanni Esposito ◽  
Rita Zilhão ◽  
Sofia Nolasco ◽  
Giuseppina Cangiano ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Posttranslational Modifications ◽  
Mother Cell ◽  
Spore Coat ◽  
Spore Surface ◽  
Subtilis Spore ◽  
Cell Compartment ◽  
Bacillus Subtilis Spore ◽  
Molecular Masses

ABSTRACT We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

The Terminal Oxidase in the Marine Bacterium Pseudomonas nautica 617

1997 ◽  
Vol 17 (3) ◽  
pp. 343-346 ◽  
Author(s):  
Helen Simpson ◽  
Michel Denis ◽  
Francesco Malatesta
Keyword(s):  
Hydrogen Peroxide ◽  
Carbon Monoxide ◽  
Oxygen Reduction ◽  
Epr Spectroscopy ◽  
Marine Bacterium ◽  
Large Family ◽  
Terminal Oxidase ◽  
Quinol Oxidase ◽  
Terminal Oxidases ◽  
Production Of Hydrogen

The molecular properties of a novel membrane quinol oxidase from the marine bacterium Pseudomonas nautica 617 are presented. The protein contains 2b hemes/mole which may be distinguished by EPR spectroscopy but not by optical spectroscopy and electrochemistry. Respiration, though being cyanide insensitive, is not inhibited by carbon monoxide and oxygen reduction is carried out only half-way with production of hydrogen peroxide. The terminal oxidase represents, therefore, a unique example in the large family of terminal oxidases known up to date.

scholarly journals Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement

1995 ◽  
Vol 1232 (1-2) ◽  
pp. 67-74 ◽  
Author(s):  
Gaetano Villani ◽  
Maria Tattoli ◽  
Nazzareno Capitanio ◽  
Philippe Glaser ◽  
Sergio Papa ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Quinol Oxidase
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