Can enzyme kinetics be measured in whole cell extracts? - (Apr/07/2008 )
My enzyme of interest is present in whole cell extracts and I want to know if I can determine Km and Vmax values of this enzyme with whole cell extracts. I thought instead of giving Vmax as pmol/min/mg of purified enzyme I could use units/mg but I am not sure if that would give the right values.
-chalet2-
QUOTE (chalet2 @ Apr 7 2008, 05:03 AM)
My enzyme of interest is present in whole cell extracts and I want to know if I can determine Km and Vmax values of this enzyme with whole cell extracts. I thought instead of giving Vmax as pmol/min/mg of purified enzyme I could use units/mg but I am not sure if that would give the right values.
normally, chemical and physico-chemical properties are determined for purified enzymes under defined conditions;
In lysates, you have too much components which may interact with your enzymes; therefore, kinetics of enzymes in lysates are often strongly different from purified enzymes. Even if you try, you may not find the typical phases (initial phase, increase (slope), saturation) which you need to assess Km and Vmax
-The Bearer-
QUOTE (The Bearer @ Apr 7 2008, 08:57 AM)
QUOTE (chalet2 @ Apr 7 2008, 05:03 AM)
My enzyme of interest is present in whole cell extracts and I want to know if I can determine Km and Vmax values of this enzyme with whole cell extracts. I thought instead of giving Vmax as pmol/min/mg of purified enzyme I could use units/mg but I am not sure if that would give the right values.
normally, chemical and physico-chemical properties are determined for purified enzymes under defined conditions;
In lysates, you have too much components which may interact with your enzymes; therefore, kinetics of enzymes in lysates are often strongly different from purified enzymes. Even if you try, you may not find the typical phases (initial phase, increase (slope), saturation) which you need to assess Km and Vmax
In other words, you can in conditions where only your enzyme functions and not the 10000 other enzymes that are present in the same reaction.
-gorkin-
QUOTE (gorkin @ Apr 8 2008, 12:37 PM)
QUOTE (The Bearer @ Apr 7 2008, 08:57 AM)
QUOTE (chalet2 @ Apr 7 2008, 05:03 AM)
My enzyme of interest is present in whole cell extracts and I want to know if I can determine Km and Vmax values of this enzyme with whole cell extracts. I thought instead of giving Vmax as pmol/min/mg of purified enzyme I could use units/mg but I am not sure if that would give the right values.
normally, chemical and physico-chemical properties are determined for purified enzymes under defined conditions;
In lysates, you have too much components which may interact with your enzymes; therefore, kinetics of enzymes in lysates are often strongly different from purified enzymes. Even if you try, you may not find the typical phases (initial phase, increase (slope), saturation) which you need to assess Km and Vmax
In other words, you can in conditions where only your enzyme functions and not the 10000 other enzymes that are present in the same reaction.
which will be difficult to find and perform simulate in vitro; you have not only an ensemble of enzymes but scaffolding proteins, small molecular regulators etc which may influence enzyme activity by direct interaction
-The Bearer-