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Imagine that you are working as a postdoctoral researcher in a laboratory that studies how heart lipid metabolism in rats varies during the onset of type 2 diabetes. As part of your work, you are characterizing how the activities of three different types of acyl CoA dehydrogenase (ACAD) change with disease progression. The three ACAD types are long chain ACAD (LCAD), medium chain ACAD (MCAD) and short chain ACAD (SCAD). At the end of an ACAD purification protocol, that started with purified rat heart mitochondria, you collect the protein eluting from each of five separate peaks from a high-resolution anion exchange chromatography column. One of these peaks is likely to be mitochondrial LCAD, another is mito MCAD while a third is mito SCAD.   How would you obtain initial rate data from an ACAD activity assay? Describe an assay, describe how it works, provide an example of the expected raw data and explain how you obtain the initial rates. What are the units of the initial rates? Describe enzyme kinetic experiments to determine which of the chromatographic peaks is LCAD, which is MCAD, and which is SCAD. Using this information, describe kinetic experiments to determine the relative amounts of each of the ACADs changes during disease progression.

Imagine that you are working as a postdoctoral researcher in a laboratory that studies how heart lipid metabolism in rats varies during the onset of type 2 diabetes. As part of your work, you are characterizing how the activities of three different types of acyl CoA dehydrogenase (ACAD) change with disease progression. The three ACAD types are long chain ACAD (LCAD), medium chain ACAD (MCAD) and short chain ACAD (SCAD). At the end of an ACAD purification protocol, that started with purified rat heart mitochondria, you collect the protein eluting from each of five separate peaks from a high-resolution anion exchange chromatography column. One of these peaks is likely to be mitochondrial LCAD, another is mito MCAD while a third is mito SCAD.   How would you obtain initial rate data from an ACAD activity assay? Describe an assay, describe how it works, provide an example of the expected raw data and explain how you obtain the initial rates. What are the units of the initial rates? Describe enzyme kinetic experiments to determine which of the chromatographic peaks is LCAD, which is MCAD, and which is SCAD. Using this information, describe kinetic experiments to determine the relative amounts of each of the ACADs changes during disease progression.

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Imagine that you are working as a postdoctoral researcher in a laboratory that studies how heart lipid metabolism in rats varies during the onset of type 2 diabetes. As part of your work, you are characterizing how the activities of three different types of acyl CoA dehydrogenase (ACAD) change with disease progression. The three ACAD types are long chain ACAD (LCAD), medium chain ACAD (MCAD) and short chain ACAD (SCAD). At the end of an ACAD purification protocol, that started with purified rat heart mitochondria, you collect the protein eluting from each of five separate peaks from a high-resolution anion exchange chromatography column. One of these peaks is likely to be mitochondrial LCAD, another is mito MCAD while a third is mito SCAD.  

  1. How would you obtain initial rate data from an ACAD activity assay? Describe an assay, describe how it works, provide an example of the expected raw data and explain how you obtain the initial rates. What are the units of the initial rates?
  2. Describe enzyme kinetic experiments to determine which of the chromatographic peaks is LCAD, which is MCAD, and which is SCAD.
  3. Using this information, describe kinetic experiments to determine the relative amounts of each of the ACADs changes during disease progression.

 

 

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