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Objective: Becoming familiar with the purification and kinetic analysis of an enzyme that produces a product important in cell survival. Note: You should do Task 12 first, since it has the background information needed to complete this task. The investigators then carried out several additional chromatographic steps and finally obtained a fraction that contained pure sphingosine kinase, as determined by SDS- PAGE. The molecular weight was determined by comparing the migration of the enzyme with the migration of standard molecular weight markers in Lane 1 of the SDS-PAGE gel to the right. The investigators also carried out an additional experiment in which one batch of the enzyme was treated with the reducing agent ß-mercaptoethanol (Lane 2). A second batch was not treated (Lane 3). 3. a. Estimate the molecular weight of sphingosine kinase in kiloDaltons. Mwt markers (Kd) 102 78 50 ohi 16.0 eve 50 34.2 28.3 1 2 3

Objective: Becoming familiar with the purification and kinetic analysis of an enzyme that produces a product important in cell survival. Note: You should do Task 12 first, since it has the background information needed to complete this task. The investigators then carried out several additional chromatographic steps and finally obtained a fraction that contained pure sphingosine kinase, as determined by SDS- PAGE. The molecular weight was determined by comparing the migration of the enzyme with the migration of standard molecular weight markers in Lane 1 of the SDS-PAGE gel to the right. The investigators also carried out an additional experiment in which one batch of the enzyme was treated with the reducing agent ß-mercaptoethanol (Lane 2). A second batch was not treated (Lane 3). 3. a. Estimate the molecular weight of sphingosine kinase in kiloDaltons. Mwt markers (Kd) 102 78 50 ohi 16.0 eve 50 34.2 28.3 1 2 3

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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3. a. Estimate the molecular weight of sphingosine kinase in kiloDaltons.

b. Interpret the results of the experiment with the reducing agent. What does the result of Lane 2 and Lane 3 migrations tell you about the primary, secondary or tertiary structure of the sphingosine kinase?

Objective: Becoming familiar with the purification and kinetic analysis of an enzyme that produces a product important in cell survival. Note: You should do Task 12 first, since it has the background information needed to complete this task. The investigators then carried out several additional chromatographic steps and finally obtained a fraction that contained pure sphingosine kinase, as determined by SDS- PAGE. The molecular weight was determined by comparing the migration of the enzyme with the migration of standard molecular weight markers in Lane 1 of the SDS-PAGE gel to the right. The investigators also carried out an additional experiment in which one batch of the enzyme was treated with the reducing agent ß-mercaptoethanol (Lane 2). A second batch was not treated (Lane 3). 3. a. Estimate the molecular weight of sphingosine kinase in kiloDaltons. Mwt markers (Kd) 102 78 Hev 50 34.2 28.3 1 2 3 b. Interpret the results of the experiment with the reducing agent. What does the result of Lane 2 and Lane 3 migrations tell you about the primary, secondary or tertiary structure of the sphingosine kinase?
Transcribed Image Text:Objective: Becoming familiar with the purification and kinetic analysis of an enzyme that produces a product important in cell survival. Note: You should do Task 12 first, since it has the background information needed to complete this task. The investigators then carried out several additional chromatographic steps and finally obtained a fraction that contained pure sphingosine kinase, as determined by SDS- PAGE. The molecular weight was determined by comparing the migration of the enzyme with the migration of standard molecular weight markers in Lane 1 of the SDS-PAGE gel to the right. The investigators also carried out an additional experiment in which one batch of the enzyme was treated with the reducing agent ß-mercaptoethanol (Lane 2). A second batch was not treated (Lane 3). 3. a. Estimate the molecular weight of sphingosine kinase in kiloDaltons. Mwt markers (Kd) 102 78 Hev 50 34.2 28.3 1 2 3 b. Interpret the results of the experiment with the reducing agent. What does the result of Lane 2 and Lane 3 migrations tell you about the primary, secondary or tertiary structure of the sphingosine kinase?
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