From owner-metabolic-reg@net.bio.net Wed Jun 05 23:00:00 1996
Path: biosci!student.dtu.dk!okjans
From: okjans@student.dtu.dk (Jan Schripsema)
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Date: 6 Jun 1996 08:27:24 -0700
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First of all, I would like to thank all those who sent comments
and suggestions for further reading, especially Martin Brand who
discussed my experimental procedure. I would like to continue
discussing my experiment.

When I added loganin to my cultures, I wanted to simulate the
effect of an increased flux to this compound on the flux of the
pathway after loganin. I see it as if I had increased an enzyme
in the biosynthetic pathway to loganin which did result in an
increased flux to loganin.

My questions were: 
- Can the steps after loganin sustain a higher flux towards
alkaloids when the availability of this intermediate is
increased?
- If the flux is increased, is it because of higher enzyme
concentrations?

The experimental observations were:
- Flux to alkaloids did increase after addition of loganin.
- The activity of the measured enzymes (steps after loganin) was
the same with or without the addition of loganin.

My conclusions were:
- The levels of the enzymes measured are sufficient to sustain
higher fluxes; they do not limit alkaloid accumulation under
normal culture conditions (without the addition of loganin).
- Regarding the enzymatic steps I did not measure (most of them
unknown) they can also sustain higher fluxes after addition of
loganin but I can not exclude the possibility that (some) enzyme
levels increased.

Intuitively:
- From the observation that the same amount of enzyme can sustain
a higher flux, can I not conclude that small increases in the
concentration of this enzyme (as required in MCA) will have no
(hardly any) effect on flux?
- From the observation that no intermediates accumulated but only
the end product, can I not conclude that the flux increase
through each enzyme was proportional to the availability of their
substrate (enzyme by far not substrate saturated under normal
culture conditions) or in other words, that all enzymes between
loganin and the final alkaloid have high elasticity coefficients
towards their substrates (nothing is known about loganin acting
as an effector)?

Dear Martin,
Do the elasticities you mention in the first paragraph of your
reply refer to the effect of the concentration of loganin on the
activity of the separate enzymes?

H. Kacser and J.A. Burns in Biochem. Soc. Trans. (1979):7, 1149.
In page 1158 1st paragraph they discuss briefly when a metabolite
can be considered a parameter. I agree with you that when I add
loganin to the cultures I split the pathway in two.

In the same article they mention the double modulation method to
determine elasticities in vivo. I am now looking for articles
which use this method (preferably by addition of intermediates
not effectors).

I did not really understand why I can't conclude that steps in
a pathway have low flux control when their addition leads to
increases in flux (if I know enzyme amounts did not change). But
I'm certain I have a lot to read to really understand mca. I'll
keep on trying.
Thanks
Denise Dagnino
*************************************
  Denise Dagnino
  The Technical University of Denmark 
  Dept. of Organic Chemistry
  Building 201
  DK-2800  Lyngby
  Denmark
  tel: **-45-45252111
  fax: **-45-45933968
*************************************


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