Purification and Biochemical Properties of Soluble Recombinant Human Bax

Lewis, Shareta; Bethell, Susanne S.; Patel, Sita Sharan; Martinou, Jean‐Claude; Antonsson, Bruno

doi:10.1006/prep.1997.0871

Cited by 27 publications

(27 citation statements)

References 26 publications

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“…We have shown previously that active C-terminally truncated Bax isolated in the presence of detergent from a glutathione Stransferase fusion protein is present as an oligomer in solution [38]. Equilibrium centrifugation analysis showed the complex to best fit a heptamer model [38]. The large conductance levels, up to 1.6 nS, recorded for the Bax channel in lipid bilayers would be consistent with a large channel [20].…”

Section: Discussionmentioning

confidence: 66%

“…The molecular mass of the Bax oligomer corresponds to a complex of six to eight molecules. We have shown previously that active C-terminally truncated Bax isolated in the presence of detergent from a glutathione Stransferase fusion protein is present as an oligomer in solution [38]. Equilibrium centrifugation analysis showed the complex to best fit a heptamer model [38].…”

Section: Discussionmentioning

confidence: 93%

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Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

Antonsson¹,

Montessuit²,

Lauper³

et al. 2000

Biochemical Journal

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468

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Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

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Section: Discussionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 93%

Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

Antonsson¹,

Montessuit²,

Lauper³

et al. 2000

Biochemical Journal

Self Cite

468

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show abstract

“…In contrast, the majority of proapoptotic Bax is located in the cytosol in monomeric inactive form (28,29). Upon apoptosis induction, cytosolic Bax translocates from the cytosol to the mitochondria, where it displays its apoptosis-inducing function (28 -30), which perhaps involves its heptamerization (43). Alternatively, mitochondrial Bax can heterodimerize with Bcl-2 (28,31), an association that is likely to involve interactions between their BH1, BH2, and BH3 domains.…”

Section: Discussionmentioning

confidence: 99%

Interleukin-3 Induces the Phosphorylation of a Distinct Fraction of Bcl-2

Poommipanit

Chen

Oltvai

1999

Journal of Biological Chemistry

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Bcl-2-related proteins (i.e. Bcl-2 and Bax) regulate the effector stage of apoptosis and can modulate the entry of quiescent cells into the cell cycle. Phosphorylation of Bcl-2 is presumed to modify its apoptosis-inhibitory function. By utilizing an interleukin-3 (IL-3)-dependent hematopoietic cell line, we examined the structural requirements of Bcl-2 phosphorylation and the correlation of this post-translational modification with its function. In the presence of IL-3, constitutively expressed Bcl-2 was phosphorylated on serine residue(s), and phosphorylated Bcl-2 lost its capacity to heterodimerize with Bax. Whereas the majority of Bcl-2 resided in mitochondria, phosphorylation only affected a minor pool of total Bcl-2 that selectively partitioned into a soluble fraction. Cytosolic targeting of Bcl-2 greatly increased its ratio of phosphorylation. Bcl-2 phosphorylation was reduced during IL-3 deprivation, and its phosphorylation was also delayed after transient cytokine deprivation. This pattern of phosphorylation temporally correlated with the accelerated exit and delayed reentry of Bcl-2-expressing cells into the cell cycle upon transient IL-3 deprivation and subsequent cytokine restimulation. Thus, IL-3-induced phosphorylation of a distinct pool of Bcl-2 may contribute to the inactivation of its antiproliferative function.

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“…Subsequently it was demonstrated that recombinant BCL-X L , BCL-2, and BAX can form ion channels in artificial membranes with distinct characteristics (Antonsson et al 1997;Schendel et al 1997Schendel et al ,1998Schlesinger et al 1997). Perhaps the tendency of BCL-2 family members to form multimers in vitro [e.g., recombinant BAX oligomerizes in solution (Lewis et al 1998)] and detectable dimers and multimers in vivo ) may relate to their ability to form channels. The surprising similarity in the structure of BID to the BCL-X L structure emphasizes the conserved importance of this structure including the two central hydrophobic core helices (Fig.…”

Section: At the Mitochondria: Mechanisms Of Action Of Pro-/anti-apoptmentioning

confidence: 99%