Figure 5.1 Rapid Diffusion of Membrane Proteins
The fluid mosaic model of cell membranes, described by Singer and Nicolson (1972), was critical to understanding biological membranes as proteins floating in a phospholipid matrix. Integral to this model was earlier work by Frye and Edidin (1970). These researchers examined the movement of proteins within the cell membrane by constructing heterokaryons, cells comprised of nuclei from both mice and humans. By using fluorescent stains (red or green) that were specific either to the mouse or human proteins (antigens), Frye and Edidin observed that after 40 minutes, the antigens were totally intermixed. To explain these results, the researchers hypothesized four mechanisms including new protein synthesis, movement of proteins from cytoplasm into the cell membrane, movement of proteins from the cell membrane into cytoplasm and then to a different location on the cell membrane, and diffusion of proteins within the cell membrane. In order to test these various mechanisms, the researchers conducted additional treatments involving inhibition of protein or ATP synthesis. The researchers also used a temperature gradient to test the effect of temperature on the intermixing of human and mouse proteins. The inhibitory treatments had no effect on the rate at which the proteins were intermixed, suggesting that proteins were neither being newly created nor actively transported. By contrast, decreased temperature reduced the rate of protein intermixing, as would be expected if the proteins were moving by diffusion. Thus, it was shown that the membrane surface is fluid, allowing for free diffusion of proteins within this surface.
Original paper
Frye, L. D., and M. Edidin. 1970. The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons. Journal of Cell Science 7: 319–335.
http://jcs.biologists.org/cgi/reprint/7/2/319
Links
Singer, S. J. and G. L. Nicolson. 1972. The fluid mosaic model of the structure of cell membranes. Science 175: 720–731.
http://www.jstor.org/pss/1733071
Martin, L.: The Fluid Mosaic Model - The Fluid Lipid Matrix
http://cnx.org/content/m15257/latest/
The Johns Hopkins University: Diffusion Process
http://www.jhu.edu/~virtlab/diffus/diff_txt.htm
Georgia State University: Diffusion and Osmosis
http://hyperphysics.phy-astr.gsu.edu/hbase/kinetic/diffus.html
Home page of Dr. Michael Edidin
http://www.bio.jhu.edu/Faculty/Edidin/Default.html
Figure 5.5 Aquaporins Increase Membrane Permeability to Water
Although diffusion can account for limited water movement across cell membranes, researchers noted that simple diffusion was unlikely to explain the considerable water movement in kidney and red blood cells. By happenstance, Agre and colleagues discovered a protein shared by these two types of cells and determined the DNA sequence of this protein. Unfortunately, this sequence did not offer any clues as to the function of this protein, although the researchers established that similar proteins were found in plant cells. The fact that this protein seemed to occur in cells that moved significant amounts of water led the researchers to hypothesize that this protein, initially named CHIP28, was responsible for cell membrane water transport. In an elegant experiment, Agre and colleagues tested this hypothesis using frog egg cells (oocytes) that normally have low water permeability. Experimental oocytes were injected with the CHIP28 protein RNA, whereas control oocytes were injected with water. Agre and colleagues observed that the control oocytes maintained their volume and shape, whereas the experimental oocytes became swollen and ruptured after 3 min. To further confirm CHIP28's role in water transport, the researchers added a chemical inhibitor to water transport and found that the experimental oocytes no longer became swollen. However, when a second chemical was added that reversed the inhibitory effect of the first chemical, the experimental oocytes again absorbed water and ruptured. Taken together, these data provided strong support for the role of CHIP28 in water transport across cell membranes. CHIP28 was later renamed “aquaporin,” and its structure and mechanism of activity has been intensely studied. Further, several other members of the aquaporin protein family have been identified, including proteins in humans that, when defective, are implicated in disorders related to vision and kidney function. Given the great importance of the discovery of aquaporins, Agre was awarded the Nobel prize in Chemistry in 2003.
Original paper
Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385–387.
http://www.jstor.org/stable/2877088
Links
Agre, P. 2006. The aquaporin water channels. Proc. Am. Thorac. Soc. 3: 5–13.
http://www.atsjournals.org/doi/full/10.1513/pats.200510-109JH#.U_qIO0vLy0w
Structure, Dynamics, and Function of Aquaporins
http://www.ks.uiuc.edu/Research/aquaporins/
Aquaporins - Water Channels
http://www.bio.miami.edu/~cmallery/150/memb/water.channels.htm
Wikipedia: Aquaporin
http://en.wikipedia.org/wiki/Aquaporin
Aquaporins.org
http://www.aquaporins.org/
Dr. Peter Agre: The Nobel Prize in Chemistry 2003
http://nobelprize.org/nobel_prizes/chemistry/laureates/2003/agre-autobio.html
The New York Times: Using a Leadership Role to Put a Human Face on Science
http://www.nytimes.com/2009/01/27/science/27agre.html?_r=1
From the Nobel Prize to Third World Medicine: An Interview With Peter Agre
http://blogs.sciencemag.org/sciencecareers/2010/08/from-the-nobel.html
Stephen Colbert chats with Dr. Agre
http://www.colbertnation.com/the-colbert-report-videos/76990/october-19-2006/peter-agre?videoId=76990
Dr. Agre’s home page at the Johns Hopkins Bloomberg School of Public Health
http://faculty.jhsph.edu/Default.cfm?faculty_id=1953
Transport across cell membranes
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/Diffusion.html
Figure 5.15 The Discovery of a Second Messenger
While studying the action of glycogen phosphorylase, Earl Sutherland and colleagues determined that this enzyme could only be activated by epinephrine when the entire contents of liver cells, including membrane fragments, were present. The researchers hypothesized that a cytoplasmic messenger must transmit 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 into cytoplasmic 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 to centrifugation to remove the membranes, leaving only the soluble portion in the supernatant. A small sample of the membrane-free solution was added to the cytoplasmic fraction, which was then assayed for the presence of glycogen phosphorylase activity. The assay showed that active glycogen phosphorylase was indeed present in the cytoplasmic fraction. Thus, these results confirmed the hypothesis that a soluble second messenger was produced in response to epinephrine binding to its receptor in the membrane, and then it diffused into the cytoplasm to activate the enzyme. Later research by Sutherland identified cAMP as the second messenger involved in the mechanism of action of epinephrine as well as many other hormones. Sutherland’s research was highly regarded in the scientific community, and in 1971 he received the Nobel Prize in Physiology or Medicine for his discoveries concerning “the mechanisms of the action of hormones.” This work, however, led to the question of how hormone binding leads to the formation of cAMP in the cell. Additional studies revealed that cAMP is formed from ATP through the action of an enzyme called adenylyl cyclase. Thus, in the experiment discussed above, one could confirm that cAMP, and not ATP, is the second messenger in this system by incubating the membrane-free solution with activated adenylyl cyclase prior to its addition to the cytoplasmic fraction. The ATP would be converted to cAMP, thereby interfering with any ATP-dependent processes.
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: 463–475.
http://www.jbc.org/cgi/reprint/224/1/463
Links
Nobelprize.org: The Nobel Prize in Physiology or Medicine 1971
http://nobelprize.org/nobel_prizes/medicine/laureates/1971/press.html
TIME.com: The Second Messenger
http://www.time.com/time/magazine/article/0,9171,877333,00.html
Journal of Biological Chemistry: Earl W. Sutherland’s Discovery of Cyclic Adenine Monophosphate and the Second Messenger System
http://www.jbc.org/content/280/42/e39.full
University of Miami: Cyclic AMP and Its Action
http://fig.cox.miami.edu/~lfarmer/BIL265/CAMP.HTM
Second Messengers
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/Second_messengers.html