Sphingolipid metabolites have recently been shown to be involved in cell viability. Ceramide...

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Sphingolipid metabolites have recently been shown to be involved in cell viability. Ceramide stimulates apoptosis whereas sphingosine-P promotes cell survival. Thus understanding the kinetics of sphingosine metabolism is critical to understanding these vital cell processes.
The following data was collected for the purification and kinetic analysis of Sphingosine kinase

The graphs above show the kinetic data for sphingosine kinase for both substrates :ATP and sphingosine Using these graphs calculate the Km sand Vmax values for both substrates. (show all units)

The graph below shows the kinetic data for this enzyme in the presents of a stereoisomer of sphingosine, threo-sphingosine. Determine what type of inhibitor threo-sphingosine is and explain your answer . (show all units)

Many kinase enzymes use a phospho-enzyme intermediate and proceed through a ping-pong mechanism. Describe what experiment needs to be done to determine if this enzyme follows this general pattern. Draw a sketch (scanned hand drawn image is acceptable) of the expected result.

Below is an abbreviate purification table from the purification of sphingosine kinase. Please answer the following questions about the purification process.
1. Why would the authors do two precipitations with ammonium sulfate?
2. The elution of the protein from the DEAE was very broad (see Figure 1 below); note the activity eluded very late in this profile. What can you conclude about the charge of sphingosine kinase from this data?

Table 1 Purification of Sphingosine Kinase

Step	Activity (units x 10³)	Protein(mg)	Specific activity (units/mg)	% Recovery	Fold purification
Crude homogenate	12,654	163,000	78	100	1
Ammonium sulfate 25-45%	8584	15,100	568	68	7
DEAE cellulose	7400	3550	2085	58	27
Blue A sepharose	4569	215	21,253	36	273
Calmodulin-Sepharose	670	0.94	71.3 X 10⁴	5.3	9141
Suerdex 75	77	0.001	50 X 10⁶	0.6	641,026

The final stage of the purification was gel exclusion of the active enzyme and proof of purity by SDS gel electrophoresis. Estimate the size of the sphingosine kinase from the gel exclusion chromatography. Compare the gel exclusion size estimate and the SDS size. What can you conclude about the subunit structure of sphingosine kinase?

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Robert · Accepted Answer

Sphingolipid metabolites have recently been shown to be involved in cell viability. Ceramide stimulates apoptosis whereas sphingosine-P promotes cell survival. Thus understanding the kinetics of sphingosine metabolism is critical to understanding these vital cell processes.
The following data was collected for the purification and kinetic analysis of Sphingosine kinase
1. The graphs above show the kinetic data for sphingosine kinase for both substrates :ATP and sphingosine Using these graphs calculate the Km sand Vmax values for both  substrates. (show all units)
Ans 1)-  the lineweaver graph tells us the Km and Vm values as the X and Y intercept on the axes respectively 
So in the graph 1 for sphingosine as a substrate the -1/Km = -1/-1.5 µM = 0.666 µM and the 1/Vm = 1/0.0075 = 133.33 mg/min/µ mol 
In the graph 2 for ATP as a substrate the -1/Km = -1/-17.5 µM = 0.05714 µM
And the 1/Vm = 1/ 0.008 = 125 mg/min/µ mol . 
2. The graph below shows the kinetic data for this enzyme in the presents of a stereoisomer of sphingosine, threo-sphingosine. Determine what type of inhibitor threo-sphingosine is and explain your answer. (show all units)
Ans)-  The stereoisomer of sphingosine i.e. threo sphingosine is a competitive inhibitor of the enzyme since structurally it resembles the sphingosine which is the substrate of the enzyme and the inhibition only occur by an inhibitor which is structurally similar to the substrate due to which the actual binding of the substrate can’t take place .
3.

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