scholarly journals Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer

1999 ◽  
Vol 338 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jinping DU ◽  
Barbara H. KNOWLES ◽  
Jade LI ◽  
David J. ELLAR
Keyword(s):  
Biochemical Characterization ◽  
Bound State ◽  
Phospholipid Bilayer ◽  
Membrane Pore ◽  
Glucose Release ◽  
System A ◽  
Molecular Masses ◽  
Release Assay ◽  
Membrane Bound

The interaction of two Bacillus thuringiensiscytolytic toxins, CytA and CytB, with a phospholipid bilayer and their structure in the membrane-bound state were investigated by proteolysis using phospholipid vesicles as a model system. A toxin conformational change upon membrane binding was detected by comparing the proteolytic profile of membrane-bound toxin and saline-solubilized toxin. When membrane-bound toxin was exposed to protease K or trypsin, novel cleavage sites were found between the α-helical N-terminal half and β-strand C-terminal half of the structure at K154 and N155 in CytA and at I150 and G141 in CytB. N-terminal sequencing of membrane-protected fragments showed that the C-terminal half of the toxin structure comprising mainly β-strands was inserted into the membrane, whereas the N-terminal half comprising mainly α-helices was exposed on the outside of the liposomes and could be removed when liposomes with bound toxin were washed extensively after proteolysis. The C-termini of the membrane-inserted proteolytic fragments were also located by a combination of N-terminal sequencing and measurement of the molecular masses of the fragments by electrospray MS. Using a liposome glucose-release assay, the membrane-inserted structure was seen to retain its function as a membrane pore even after removal of exposed N-terminal segments by proteolysis. These data strongly suggest that the pores for glucose release are assembled from the three major β-strands (β-5, β-6 and β-7) in the C-terminal half of the toxin.

Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus Phellinus pomaceus

1998 ◽  
Vol 64 (8) ◽  
pp. 2831-2835 ◽  
Author(s):  
Deepti Saxena ◽  
Saleh Aouad ◽  
Jihad Attieh ◽  
Hargurdeep S. Saini
Keyword(s):  
Atmospheric Chemistry ◽  
Biochemical Characterization ◽  
Active Form ◽  
Methyl Chloride ◽  
Bound State ◽  
Ph Optimum ◽  
Methyl Donors ◽  
Membrane Bound

ABSTRACT Many wood-rotting fungi, including Phellinus pomaceus, produce chloromethane (CH3Cl). P. pomaceus can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH3Cl. The biosynthesis of CH3Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent Km for Cl− (ca. 300 mM) was much higher than that for I− (250 μM) or Br− (11 mM). A comparison of theseKm values to the relative in vivo methylation rates for different halides suggests that the realKm for Cl− may be much lower, but the calculated value is high because the CH3Cl produced is used immediately in a coupled reaction. Among various methyl donors tested, S-adenosyl-l-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.

Biochemical characterization of different genotypes of Paenibacillus larvae subsp. larvae, a honey bee bacterial pathogen

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2381-2390 ◽  
Author(s):  
Sandra Neuendorf ◽  
Kati Hedtke ◽  
Gerhard Tangen ◽  
Elke Genersch
Keyword(s):  
Honey Bee ◽  
Reference Strain ◽  
Biochemical Characterization ◽  
Bacterial Pathogen ◽  
Paenibacillus Larvae ◽  
Bacterial Disease ◽  
System A ◽  
Biolog System ◽  
Far Future

Paenibacillus larvae subsp. larvae (P. l. larvae) is the aetiological agent of American foulbrood (AFB), the most virulent bacterial disease of honey bee brood worldwide. In many countries AFB is a notifiable disease since it is highly contagious, in most cases incurable and able to kill affected colonies. Genotyping of field isolates of P. l. larvae revealed at least four genotypes (AB, Ab, ab and α B) present in Germany which are genotypically different from the reference strain DSM 7030. Therefore, based on these data, five different genotypes of P. l. larvae are now identified with genotype AB standing out with a characteristic brown-orange and circled two-coloured colony morphology. Analysing the metabolic profiles of three German genotypes (AB, Ab and ab) as well as of the reference strain using the Biolog system, a characteristic biochemical fingerprint could be obtained for each strain. Cluster analysis showed that while genotypes Ab, ab and the reference strain DSM 7030 are rather similar, genotype AB is clearly different from the others. Analysis of all isolates for plasmid DNA revealed two different plasmids present only in isolates belonging to genotype AB. Therefore, genotype AB is remarkable in all aspects analysed so far. Future analysis will show whether or not these differences will expand to differences in virulence.

Biochemistry and site-directed mutational analysis of Δ7-sterol-C5(6)-desaturase

2000 ◽  
Vol 28 (6) ◽  
pp. 799-803 ◽  
Author(s):  
A. Rahier ◽  
P. Benveniste ◽  
T. Husselstein ◽  
M. Taton
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Enzyme System ◽  
Conserved Residues ◽  
Catalytic Role ◽  
Membrane Bound ◽  
Haem Iron ◽  
Oxygen Site

This report describes recent work on the process of desaturation at C5(6) of sterol precursors in plants. Biochemical characterization of the plant Δ7-sterol C5(6)-desaturase (5-DES) indicates that the enzyme system involved shows important similarities to the soluble and membrane-bound non-haem iron desaturases found in eukaryotes, including cyanide and hydrophobic chelators sensitivity, CO resistance and a requirement for exogenous reductant and molecular oxygen. Site-directed mutational analysis of highly conserved residues in 5-DES indicated that eight histidine residues from three histidine-rich motifs were essential for the catalysis, possibly by providing the ligands for a putative Fe centre. This mutational analysis also revealed the catalytic role of the functionally conserved Thr-114.

scholarly journals GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

1999 ◽  
Vol 10 (1) ◽  
pp. 119-134 ◽  
Author(s):  
Siew Heng Wong ◽  
Yue Xu ◽  
Tao Zhang ◽  
Gareth Griffiths ◽  
Stephen Loucian Lowe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Golgi Apparatus ◽  
Amino Acid Sequence ◽  
Northern Blot Analysis ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Polyclonal Antibodies ◽  
Sequence Identity ◽  
Membrane Bound

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

scholarly journals The morphological and biochemical characterization of a line of rat promegakaryoblasts

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 110-121
Author(s):  
R Weinstein ◽  
MB Stemerman ◽  
DE MacIntyre ◽  
HN Steinberg ◽  
T Maciag
Keyword(s):  
Biochemical Characterization ◽  
Stage Iv ◽  
In Vitro Study ◽  
Stage Iii ◽  
Immunofluorescent Staining ◽  
Morphological Evidence ◽  
Specific Proteins ◽  
Membrane Bound

Biochemical and morphological evidence is presented to support the characterization of a rat bone-marrow-derived cell line (RPM) as an analog of the promegakaryoblast. The conditions for in vitro growth and maturation of the RPM line are described. Rapid proliferation of the RPM line is readily achieved when cultures are supplemented with moderate levels of fetal bovine serum (FBS). The proliferative compartment is a small blast-like cell. Immunofluorescent staining demonstrates that the RPM cells contain factor VIII:antigen and fibrinogen in their cytoplasm. The cells secrete, into their conditioned medium, a potent mitogenic activity for rat aortic smooth muscle cells. When incubated under conditions of relative serum deprivation, the cells stop proliferating and undergo a process of maturation. The sequence of maturation is described as stage I (promegakaryoblast--the proliferative compartment); stage II (immature megakaryocyte or promegakaryocyte); stage III (mature megakaryocyte). The stage III cells release, from their cytoplasm, small membrane-bound vesicular bodies containing lavender granules and cytoplasmic organelles. These have been designated stage IV. The RPM line may provide a useful model for the in vitro study of megakaryocyte maturation and the synthesis of megakaryocyte-specific proteins.

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

FEBS Journal ◽  
2005 ◽  
Vol 272 (21) ◽  
pp. 5584-5594 ◽  
Author(s):  
Filomena A. Carvalho ◽  
Luis M. Graca ◽  
Joao Martins-Silva ◽  
Carlota Saldanha
Keyword(s):  
Endothelial Cell ◽  
Cell Membrane ◽  
Biochemical Characterization ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Membrane Bound

scholarly journals Biochemical Characterization of a Membrane-bound Manganese-containing Superoxide Dismutase from the CyanobacteriumAnabaenaPCC 7120

2002 ◽  
Vol 277 (46) ◽  
pp. 43615-43622 ◽  
Author(s):  
Günther Regelsberger ◽  
Werner Atzenhofer ◽  
Florian Rüker ◽  
Günter A. Peschek ◽  
Christa Jakopitsch ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Biochemical Characterization ◽  
Membrane Bound

scholarly journals Further characterization of the plasma membrane- and intracellular membrane-associated platelet Ca2+ transport systems

1989 ◽  
Vol 263 (2) ◽  
pp. 547-552 ◽  
Author(s):  
J Enouf ◽  
R Bredoux ◽  
N Bourdeau ◽  
B Sarkadi ◽  
S Levy-Toledano
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Ph Dependence ◽  
Transport Systems ◽  
Intracellular Membrane ◽  
Activity Maximum ◽  
Membrane Enzyme ◽  
Molecular Masses

Biochemical characterization of the Ca2+-ATPases isolated from human platelet intracellular and plasma membranes is reported. A comparative study of the previously partly described plasma membrane Ca2+-ATPase [Enouf, Bredoux, Bourdeau & Levy-Toledano (1987) J. Biol. Chem. 261, 9293-9297] and the intracellular membrane Ca2+-ATPase obtained simultaneously shows differences in the following parameters: (1) different kinetics of the two enzymes; (2) similar apparent affinity towards Ca2+ (10(-7) M), though the intracellular membrane enzyme was inhibited at Ca2+ concentrations above 10(-6) M; (3) different pH dependence with an activity maximum at pH 7 for the intracellular membrane Ca2+-ATPase and no detectable pH maximum for the plasma membrane Ca2+-ATPase; (4) a 10-fold difference in the ATP requirement of the two Ca2+-ATPases; (5) different patterns of inhibition by vanadate. Finally, the possible regulation of the Ca2+-ATPases was examined by studying the effect of chlorpromazine on the two Ca2+-ATPase activities, with only the plasma membrane enzyme being inhibited. It is concluded that the two platelet Ca2+ transport systems show biochemical differences in spite of the previously shown similarity in the molecular masses of their Ca2+-ATPases, thus conferring a definite specificity to the platelet system.

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