One possible is that two regulators interact to each others and loose their regulatory ability.

May be due to the interaction between binding sites of these two regulators.Is binding sites of regulators are adjacent ? Or any biochemical changes observed in the structure of transporter during the binding of regulators?

good luck

When you say they 'do not show any additive regulatory effect on the transporter' what is the actual result (because it would mean different things). For instance, when you add both, is there no change relative to transporter alone, does Vmax change while Km is constant, does Km change while Vmax is constant, or do they both change? If they both change, how much? This is a bit vauge - is this a hypothetical question or an experiment you're doing? As long as it isn't a homework or exam question, I'll be happy to add more answers...

Thank you all for the suggestions,

@Amin - Both the regulators down regulate the transporter, and no further down regulation was observed on adding both to check on the transporter activity.

@ Solem - This is an experiment that I performed. Both the regulators are down-regulators, i.e, they decrease the activity of the transporter. However in case of the first regulator - The Vmax changes but no change in Km, in the second regulator the Km changes however the effect on Vmax was inconclusive.

Awaiting further discussion. Thank you for helping

let 's asume that regulator 1 decrease Vmax to 60% and regulator 2 increase Km 50%. So when you add both did you see the same changes for Vmax and Km?

let me know this first.

I agree - to give ideas, we need to know what happened in your experiment with both inhibitors relative to those with each individually as this could significantly change the interpretation.

So when you add both, is there:

1. no inhibition at all

2. A decrease in Km that is a) the same as the second alone b) more than the second alone c) less than the second alone

3. A decrease in vmax that is a) the same as the first alone b) more than the first alone c) less than the first alone

4. A change in both Km and vmax. a b or c for each.

If there is no inhibition at all, the two inhibitors may interact with each other and no longer bind the transporter.

If only Km or only vmax decreases and it looks exactly like one of the inhibitors alone, then it is likely that only one of the inhibitors can bind first and it blocks the other.

If there is some change in Km or vmax, but not as much as each one alone, it is likely that only one inhibitor can bind at a time, but they bind with similar rate constants so you have a mixture of some transporters with one inhibitor and some with the other.

You can see how we need more information!

When you are referring to down-regulation of a transporter, you may not be talking about Vmax or Km, because these are kinetic properties of the expressed protein.

I agree with Martin. down regulation should be discussed based on other parameters.

When she said "regulator" I assumed she meant "inhibitor" as in something acting directly on a transporter as opposed to something indirectly affecting the transporter by changing the level of gene expression - Aruna can you clarify exactly what you are doing here?

Thank you all for your suggestions and help.

I have attached a ppt file to simplify what I want to say.

Elaborating my explaination:

I study the activity of a transporter in the presence of two regulators. These regulators are not substrate/inhibitors. They are actually already present within the cell under normal conditions.

So for checking the effect of the first regulator, the gene expressing this first regulator is knocked out and the regulator is added on the cell to check its specific effect on the transporter. It has been ensured and checked that this addition does enable the transport and activity of the regulator on the transporter.

For analyzing the effect of the second regulator an activator is added, which specifically will enhance the production of the second regulator within the cell.

The following attachement shows the results in more description.

Coming to my question now is , when both regulators have different mechanism of action and when regulator 2 is independent of regulation 1 action, then what could be the reason for no additive effect.

@martin - thank you for the suggestion.

However which other parameters would you consider to explain down-regulation. Why shouldn't kinetic parameters of the transport be considered as a factor for regulation, when both the regulators affect the kinetic properties of the transporter?

Your ppt slide suggests that your experiments are measuring activity of the transporter indirectly in a cell-based assay. In this case, to make strong conclusions about activity of the transporter in terms of kinetic parameters, you would need to directly determine these parameters in you experiments. If you cannot, you would atleast need to measure expression of the transporter to be sure that a decrease in actvity is not due to a decrease in gene expression.

So theoretically, if these regulators:

1. Do not change the expression level of the transporter

2. Are not substrates or inhibitors

How are they affecting tranporter kinetics?

I would add that transport in native cell-based systems often depends on cofactors/coregulators which could be closely associated with the transporter, but be targets themselves for effectors. It is not very practical to make useful suggestions without knowing much more about the specifics of the system. There are many potentially informative examples of such complex regulated transporter systems, such as CFTR or ferroportin. The latter is an example where effectors (such as hepcidin) control removal from its active location in the membrane by internalization.

@ Solem: Yes I checked the kinetic parameters, The Km of the transporter is changed only by one of the regulator. However the same method was not considered good to measure Vmax. About the level of transporter protein, this level is altered due to exocytosis by the other regulator which DOES NOT change the Km.

So based on these results, one can deduce that the regulator affecting the Km may cause a transporter modification to affect its activity while the second one not affecting the Km causes down activation by inhibiting exocytosis. This is why I expected an additive effect when both are added, sadly that did not happen, even when the experiment was performed many times.

Please help.

thanks

@martin: thank you for the suggestion. Yes it is a complex regulated transporter system that I work on. Lot of regulators are known to effect it in-vivo and all these end up with a cascade of protein network activation finally doing the required regulation. However this was the first time that there is this particular regulator which showed an effect on the Km value. The other as you said only affects the exocytosis. Hence the doubt arised as why when both are put together and are affecting the transporter by different means, why NO ADDITIVE EFFECT.

thanks

Hmmm... how can you measure Km but not vmax? Your Km is by definition is 1/2 Vmax. Therefore, if your Vmax changes, the "rate" of the reaction at the Km also changes... Can you explain more about this? You are just so vague in what you are doing and what the results are, I can't say much useful. Regulation can happen in so many ways! It is very common to see effects that are not additive - you could see the effect of only one, slightly more with two, or you could see way more effect with two together than you would expect from their individual contributions.

The regulator that causes exocytosis is now clear. This means that it is NOT causing a change in Vmax of the transporter, it is causing a change in the amount of transporter which then changes the maximum you observe in your assay. So although you are indeed changing the maximum observed rate in your assay, I would not call this a Vmax in a classical enzyme characterization way. So your Vmax and Km for the transporter is actually constant, you are just changing the amount protein and so observe a different rate of activity.

So some other things to think about...

Does the "Km" regulator bind the transporter directly? Could it be exocytosed with the transporter due to the effects of the other inhibitor?

I agree to the question that how is it possible to measure Km without measuring the Vmax. Assisting the condition; that the expression of the transporter differs in each cell expressed, hence the Vmax would therefore be an inconclusive one. However under a given defined condition, the affinity (Km) irrespective of the transporter concentration, should be the same. Keeping this logic, the Km was assessed. The Vmax obtained was ignored as it changed in each preparation, even when the conditions were maintained constant.

Under such conditions, in the presence of Regulator 1 exposure, the Km remained a constant. However this is the regulator which regulates the transporter by exocytosis. Hence it brings a change in the no. of transporter expressed on the surface. This is the reason for our saying that regulator 1 changes the Vmax. However we could not prove the same.

Using the same conditions, when Regulator 2 was exposed, the Km changed. Hence we state that regulator 2 has a kinetic effect on the transporter. Although direct binding has been cited in papers, we never measured it ourselves.

Based on the above information, it was difficult to understand, why when both are applied, no additive effect was observed.

The regulator 2 is capable of causing down-regulation in the activity of the transporter even when regulator 1 is knocked out of the cell. Thereby indicating that the exocytosis of Reg2 with the transporter, even if occurs may not be an effect of Reg1.

Thanks - those details are useful. I can see how you would expect to have an atleast partially additive effect when one is reducing the amount of transporter and the other is decreasing transporter activity. However, in systems of high complexity it is hard to predict. I have never had to deal with such a complex system, but I'll try to brainstorm for you.

- Direct interaction between Reg 1 and 2

- When Reg 1 is activated, it also turns off Reg 2 (directly or indirectly)

- Reg 1 causes exocytosis of Reg 2 (when I asked if Reg 2 could be exocytosed with the transporter, I meant basically could Reg 1 remove Reg 2 from the cell by exocytosing them both together)

[for the 2 above, measure levels of the regs?]

- When Reg 1 causes exocytosis of the transporter, it changes the overall stucture and such of the membrane which affects interaction of Reg 2 with the transporter

- When Reg 1 decreases the amount of protein in the membrane, it decreases the amount of Reg 2 binding

- Crazy regulatory networks [ie you have no idea, but maybe you can figure it out later]

These may not apply to your system at all - but maybe they'll help you think of something else!

@ Solem: Thank you :)