Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.4.24.3 (
collagenase
)
18,340
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Xenopus laevis
oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of
Xenopus laevis
frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog
Lithobates catesebeianus
, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief
collagenase
treatment and mechanical defolliculation for isolating individual oocytes from
Lithobates
ovaries. We find that they have a similar size, shape, and appearance to
Xenopus
oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (
P
f
) show that
Lithobates
oocytes can express AQPs to the plasma membrane and significantly increase the
P
f
of the oocytes. In fact, the
P
f
values are similar to historical values gathered from
Xenopus
oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the
P
f
of oocytes expressing human (h) AQP1, hAQP1
FLAG
[FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1],
hAQP8
, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys - hAQP1
C189S
mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 - were mercury insensitive. Contrary to previous studies with
Xenopus
oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider
Lithobates
oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to
Xenopus
oocytes.
...
PMID:
Lithobates catesbeianus
(American Bullfrog) oocytes: a novel heterologous expression system for aquaporins. 2953 Sep 31
