7/27/2019 WwD 7.1 Discovery of a Second Messenger

http://slidepdf.com/reader/full/wwd-71-discovery-of-a-second-messenger 1/2

Life: The Science of B iology , Tenth Edition 

Sadava • Hillis • Heller • Berenbaum 

Working with Data 7.1: The Discovery of a Second Messenger 

Original Paper

Rall, T. W., E. W. Sutherland, and J. Berthet. 1957. The relationship of epinephrine and glucagon to liver  phosphorylase. Journal of Biological Chemistry 224: 463.http://www.jbc.org/cgi/reprint/224/1/463 

Analyze the Data

While studying the action of glycogen phosphorylase, Earl Sutherland and his colleaguesdetermined that this enzyme could be activated by

epinephrine only when the entire contents,including membrane fragments, of liver cells were

 present (see textbook Figure 7.11). The researchershypothesized that a cytoplasmic messenger musttransmit the message from the epinephrine receptor at the membrane to glycogen phosphorylase,

located in the cytoplasm. To test this idea, liver tissue was homogenized and separated intocytoplasmic and membrane components,

containing the enzyme and epinephrine receptors,respectively. Epinephrine was added to the

membrane fraction and incubated for a period of 

time. This fraction was then subjected tocentrifugation to remove the membranes, leaving

only the soluble portion in the supernatant. A smallsample of the membrane-free solution was added

to the cytoplasmic fraction, which was thenassayed for the presence of glycogen

 phosphorylase activity. The assay showed that

active glycogen phosphorylase was indeed presentin the cytoplasmic fraction. These resultsconfirmed the hypothesis that a soluble secondmessenger was produced in response toepinephrine binding to its receptor in the

membrane, and then diffused into the cytoplasm toactivate the enzyme. Later research by Sutherland

identified cAMP as the second messenger involvedin the mechanism of action of epinephrine, as well

as many other hormones. Sutherland’s researchwas highly regarded in the scientific community,and in 1971 he won the Nobel Prize in Physiology or Medicine for his discoveries concerning “the

mechanisms of the action of hormones.” 

7/27/2019 WwD 7.1 Discovery of a Second Messenger

http://slidepdf.com/reader/full/wwd-71-discovery-of-a-second-messenger 2/2

 

Question 1

As part of Sutherland’s research, the activity of glycogen phosphorylase was measured in va rious liver cell fractions, with or without incubation with epinephrine. The table shows the results. Explain how

these data support the hypothesis that there is a soluble second messenger that activates the enzyme.

Question 2

Propose an experiment to test whether the factor (second messenger) that activates the enzyme is stable

on heating (and therefore probably not a protein), and give predicted data.

Question 3

The second messenger, cAMP, was purified from the hormone-treated membrane fraction. Propose

experiments to show that cAMP could replace the membrane fraction and hormone treatment in theactivation of glycogen phosphorylase, and create a table to show possible results.

Condition  Enzyme activity (units) 

Homogenate 0.4

Homogenate + epinephrine 2.5

Cytoplasm 0.2

Cytoplasm + epinephrine 0.4

Membranes + epinephrine 0.4

Cytoplasm + membranes +

epinephrine2.0