sodium azide and yeast inhibition
Deva
I have found that when sodium azide is added to a culture of
Saccharomyces cervisiae, for about the first ten hours, there is no
difference in yeast growth from a culture grown without the sodium
azide added. But after about ten hours, the yeast growth decreases.
I am wondering why it takes so long for the sodium azide to inhibit
the yeast enzymes. I know it has something to do with the fact that
enzymes are proteins which have life spans, but I can't quite make the
connection. Any suggestions? Thanks!
------------
Deva Freeborne
de...@humboldt1.com
http://www.humboldt1.com/~deva/wedding.html
Michael Walter
Deva wrote:
If sodium azide would e.g. directly inhibit an essential enzyme the
reaction would stop immediately and the cell would die at once.
So it is likely that sodium azide inhibits protein synthesis (either
transcription or translation). So the cell is able to live with the
proteins it already had when the azide is added. When the existing
proteins are degraded (normal protein turnover) no new protein can be
synthesized and the cell dies.
But don't quote me, since this is only a suggestion.
Mike
--
Michael Walter
Allgemeine Botanik
Universitaet Ulm
Albert-Einstein-Allee 11
89069 Ulm
Germany
phone: +49-731-502-4098
FAX: +49-731-502-2626
J.R. Pelmont
Michael Walter <michael...@biologie.uni-ulm.de> wrote:
> Deva wrote:
>
> > I have found that when sodium azide is added to a culture of
> > Saccharomyces cervisiae, for about the first ten hours, there is no
> > difference in yeast growth from a culture grown without the sodium
> > azide added. But after about ten hours, the yeast growth decreases.
> > I am wondering why it takes so long for the sodium azide to inhibit
> > the yeast enzymes. I know it has something to do with the fact that
> > enzymes are proteins which have life spans, but I can't quite make the
> > connection. Any suggestions? Thanks!
> > ------------
> > Deva Freeborne
> > de...@humboldt1.com
> > http://www.humboldt1.com/~deva/wedding.html
>
> If sodium azide would e.g. directly inhibit an essential enzyme the
> reaction would stop immediately and the cell would die at once.
> So it is likely that sodium azide inhibits protein synthesis (either
> transcription or translation). So the cell is able to live with the
> proteins it already had when the azide is added. When the existing
> proteins are degraded (normal protein turnover) no new protein can be
> synthesized and the cell dies.
> But don't quote me, since this is only a suggestion.
>
I am afraid it is not quite the right answer.
1) The main target of sodium azide is the cytochrome c oxidase (cyt.
a.a3) of the mitochondrial respiratory chain, at 0.1 to 5 mM. It is not
quite as effective as cyanide, especially at alkaline pH. There is a
drop in O2 consumtion and ATP in the cell. Other classical inhibitors
are rotenone (at the NADH-dehydrogenase complex level), and antimycin at
the cyt. b.C1 level). About these drugs, care should be taken that the
stock solutions are fressh and properly prepared, that they are not
destroyed in the culture (stupid comment of mine, but never know ;-)
2) Poisoned yeasts still can make ATP, although at a much reduced rate,
by fermentation, according to the carbon source. Glucose will be
fermented, but not succinate or citrate, I believe. Therefore a
different result is to be expected according to the composition of the
medium. Some workers have described the occurence in yeasts of side
respiratory chains, that are not poisoned by cyanide or azide easily. I
have no data at hand here, and it may be not true for Saccharomyces
anyway. Maybe a Medline search could answer.
3) Azide is a potent inhibitor of catalase (at 0.1 mM). It may block
some other Fe enzymes, but I do not remember the details. It is known to
inhibit O2 evolution in photosynthesis by plants.
4) The effect of protein synthesis is due mostly to the shortage of
energy. Remember that the building of each peptide bond costs 4 ATP on
the average (assuming that the aminoacids are readily available from the
medium).
5) Each time inhibition of living cells is examined using various drugs,
permeability barriers are to be considered. I do not know if it involved
here with azide, but sometimes it may delay the response. Therefore some
drugs that are supposed to be extremely active on an enzyme target
happens sometimes to be poorly effective on the cells, especially when
multiplying fast, because they cannot get easily into. But I am not sure
it has anything to do with the experiment discussed here.
I am not sure either that I answered properly to the question, I just
tried...
A philosophical comment : see and admire how these little yeast cells
are tough and how they fight adverse conditions, anyway ! Aren't they ?
;-)) Cheers
--
J. Pelmont, Biochimie
Univ. Grenoble I
Phil
Deva wrote:
>
> I have found that when sodium azide is added to a culture of
> Saccharomyces cervisiae, for about the first ten hours, there is no
> difference in yeast growth from a culture grown without the sodium
> azide added. But after about ten hours, the yeast growth decreases.
> I am wondering why it takes so long for the sodium azide to inhibit
> the yeast enzymes. I know it has something to do with the fact that
> enzymes are proteins which have life spans, but I can't quite make the
> connection. Any suggestions? Thanks!
> ------------
> Deva Freeborne
> de...@humboldt1.com
> http://www.humboldt1.com/~deva/wedding.html
As some of the other posters mentioned, azide will force the yeast into
fermentative growth. If memory serves me, this is ok but (perhaps among
other similar phenomena) lipid desaturase enzymes that indirectly
require oxidative phosphorylation fail to funtion. The existing lipids
are eventually depleted such that membrane integrity suffers and growth
slows and stops. Similar phenomena are noted with petite (mitochondrial
lesion) mutants in S. cerevisiae where desaturated lipids must be
provided for continued growth.
Try adding some complex lipids sources to media or even some of the
Tweens.
Phil
Graham Shepherd
Jeff E. Janes wrote in message
<354900...@silibone.cchem.berkeley.edu>...
>J.R. Pelmont wrote:
>>
>> I am afraid it is not quite the right answer.
>> 1) The main target of sodium azide is the cytochrome c oxidase (cyt.
>> a.a3) of the mitochondrial respiratory chain, at 0.1 to 5 mM. It is not
>> quite as effective as cyanide, especially at alkaline pH. There is a
>> drop in O2 consumtion and ATP in the cell. Other classical inhibitors
>> are rotenone (at the NADH-dehydrogenase complex level), and antimycin at
>> the cyt. b.C1 level). About these drugs, care should be taken that the
>> stock solutions are fressh and properly prepared, that they are not
>> destroyed in the culture (stupid comment of mine, but never know ;-)
>>
>We put azide into our protein solutions (after isolating from E. coli)
>for storage. Does azide also inhibit growth of prokaryotes by
>inhibiting their
>equivalent of the mitochondrian cytochrome? Of course, they still have
>fermentation. Perhaps azide is not as effective as a preservative as I
>thought.
>
>Jeff
You can use sodium azide in culture media to select for facultative
anaerobes - culture the organisms on azide agar aerobically - the oxygen
stops the obligate anaerobes and the aside stops the obligate aerobes.
At normal concentrations (0.1%) it's a reasonably effective bacteriostatic
preservative but a few species bugs will still be able to grow, although
probably more slowly. I always thought that azide was more potent thatn
cyanide, and that it acts at a second point in the respiratory chain in
addition to cytochrome c oxidase.
GS
J.R. Pelmont
Graham Shepherd <muh...@globalnet.co.uk> wrote:
>
> You can use sodium azide in culture media to select for facultative
> anaerobes - culture the organisms on azide agar aerobically - the oxygen
> stops the obligate anaerobes and the aside stops the obligate aerobes.
>
> At normal concentrations (0.1%) it's a reasonably effective bacteriostatic
> preservative but a few species bugs will still be able to grow, although
> probably more slowly. I always thought that azide was more potent thatn
> cyanide, and that it acts at a second point in the respiratory chain in
> addition to cytochrome c oxidase.
>
Well, I tried to check this again. Sorry if my post is a little bit
long, just trying to help. I could find no target for azide at a point
of the respiratory chain other than the cytochrome c oxidase. It seems
to be fairly specific. However, some interference between azide and
iron-sulfur clusters is possible, probably at much hicher dose.
Generally when using inhibitors in excess, secondary effects are
commonly observed.
Azide like cyanide acts on bacterial respiratory chains when cytochrome
c oxidase is the terminal cerrier to dioxygen. Using these stuffs myself
on a bacterial system from Proteus mirabilis, I observed than cyanide
was more efficient than azide on the same concentration basis, but this
may not be general, and in some instances, we can expect than azide is
more potent. I just do not know exactly. Possibly ferredoxins can be
botherd by high azide.
Azide is used instead of cyanide for protecting protein solutions or
chromatographic adsorbents, because it is much more stable in solution
at pH 7 than cyanide. The latter is very alkaline, and if neutralized,
emits slowly HCN. Therefore protection by KCN is expected to vanish with
time. Even solid KCN decomposes slowly on exposure to air. Remember
Raspoutin surviving after eating a poisoned cake : this was a possible
explanation !
Not all bacteria have respiratory chains sensitive to azide or cyanide.
These reagents act on heminic proteins having the sixth ligand position
of the iron free. This is not the case for cytochrome c, cytochrome b
and others. In cytochrome a.a3, the reagents bind at the a3 heme, which
normally reacts with O2 (with a copper ion present). Catalase reacts
with azide in a complex way : the Fe(III) binds azide at the sixth
position (instead of H2O2), and the iron is reduced to Fe(II) with a
distinct spectroscopic signal. Azide is also able to interact with
hemoglobin.
Many bacteria develop several respiratory chains. Those having
cytochrome c oxidase (a.a3 as in Paracoccus denitrificans) are "oxidase
positive", and give a blue color with tetramethyphenylene diamin (the
dye is reduced at the cytochrome c1, c2 ... level (Pseudomonas,
Rhodobacter). The Paracoccus chain works much on the same principle than
the mitochondrial chain in eukaryotic cells. These respiratory chains
are sensitive to cyanide or azide. Respiratory chains called
"cyanide-resistant" are common. They are inhibited by higher levels of
cyanide and azide (about 50 times higher). E. coli has two main
pathways. The cyanide sensitive respiratory chain has cyt.bo as the
terminal oxidase, carries electron from ubiquinol to O2. This pathway is
present in well-aerated cultures. A second pathway is present in aging
cultures or when O2 is limiting : cytochrome bd (or d) is the terminal
carrier, easy to identify by its absorption band in the reduced state at
630 nm. This is inhibited by much higher doses of cyanide. E. coli has
no cytochrome c (unless it is genetically modified), just because it is
not able to export cytochrome and link the heminic group covalently to
it in the periplasm.
Anaerobic respiratory chains, for instance the nitrate reducing chain in
E. coli, is not blocked by azide, if I am not mistakened. The terminal
carrier is nitrate reductase with a Mo cofactor, completely foreign to
cyt. c oxidase. Obviously, according to what I tried to explain,
anaerobic bugs should be unbothered by azide as a general rule.
Cheers to all.