Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 37
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Protein kinases catalyze phosphorylation,and phosphatases catalyze dephosphorylation. Althoughboth reactions are essentially irreversible, the counteractingactivities of kinases and phosphatases provide cells with a“switch” that can turn on or turn off the function of various proteins (Figure 3-30). Phosphorylation changes a protein’s charge and generally leads to a conformational change;these effects can significantly alter ligand binding by a protein, leading to an increase or decrease in its activity.Proteolytic Cleavage Irreversibly Activatesor Inactivates Some ProteinsThe regulatory mechanisms discussed so far act as switches,reversibly turning proteins on and off.
The regulation ofsome proteins is by a distinctly different mechanism: the irreversible activation or inactivation of protein function byproteolytic cleavage. This mechanism is most common in regard to some hormones (e.g., insulin) and digestive proteases. Good examples of such enzymes are trypsin andchymotrypsin, which are synthesized in the pancreas and secreted into the small intestine as the inactive zymogenstrypsinogen and chymotrypsinogen, respectively. Enterokinase, an aminopeptidase secreted from cells lining the smallintestine, converts trypsinogen into trypsin, which in turncleaves chymotrypsinogen to form chymotrypsin.
The delayin the activation of these proteases until they reach the intestine prevents them from digesting the pancreatic tissue inwhich they are made.86CHAPTER 3 • Protein Structure and FunctionHigher-Order Regulation Includes Controlof Protein Location and Concentrationactivity state into another or to the release of active subunits (see Figure 3-27).The activities of proteins are extensively regulated in orderthat the numerous proteins in a cell can work together harmoniously.
For example, all metabolic pathways are closelycontrolled at all times. Synthetic reactions take place whenthe products of these reactions are needed; degradative reactions take place when molecules must be broken down.All the regulatory mechanisms heretofore described affect aprotein locally at its site of action, turning its activity onor off.Normal functioning of a cell, however, also requires thesegregation of proteins to particular compartments such asthe mitochondria, nucleus, and lysosomes. In regard to enzymes, compartmentation not only provides an opportunityfor controlling the delivery of substrate or the exit of productbut also permits competing reactions to take place simultaneously in different parts of a cell.
We describe the mechanisms that cells use to direct various proteins to differentcompartments in Chapters 16 and 17.In addition to compartmentation, cellular processes areregulated by protein synthesis and degradation. For example,proteins are often synthesized at low rates when a cell has little or no need for their activities. When the cell faces increased demand (e.g., appearance of substrate in the case ofenzymes, stimulation of B lymphocytes by antigen), the cellresponds by synthesizing new protein molecules. Later, theprotein pool is lowered when levels of substrate decrease orthe cell becomes inactive. Extracellular signals are often instrumental in inducing changes in the rates of protein synthesis and degradation (Chapters 13–15). Such regulatedchanges play a key role in the cell cycle (Chapter 21) and incell differentiation (Chapter 22).Two classes of intracellular switch proteins regulate avariety of cellular processes: (1) calmodulin and relatedCa2-binding proteins in the EF hand family and (2) members of the GTPase superfamily (e.g., Ras and G), whichcycle between active GTP-bound and inactive GDP-boundforms (see Figure 3-29).KEY CONCEPTS OF SECTION 3.5Common Mechanisms for RegulatingProtein FunctionIn allostery, the binding of one ligand molecule (a substrate, activator, or inhibitor) induces a conformationalchange, or allosteric transition, that alters a protein’s activity or affinity for other ligands.■In multimeric proteins, such as hemoglobin, that bindmultiple ligand molecules, the binding of one ligand molecule may modulate the binding affinity for subsequent ligand molecules.
Enzymes that cooperatively bind substratesexhibit sigmoidal kinetics similar to the oxygen-bindingcurve of hemoglobin (see Figure 3-26).■■The phosphorylation and dephosphorylation of aminoacid side chains by protein kinases and phosphatases provide reversible on/off regulation of numerous proteins.■Nonallosteric mechanisms for regulating protein activity include proteolytic cleavage, which irreversibly convertsinactive zymogens into active enzymes, compartmentationof proteins, and signal-induced modulation of protein synthesis and degradation.■3.6 Purifying, Detecting,and Characterizing ProteinsA protein must be purified before its structure and themechanism of its action can be studied.
However, becauseproteins vary in size, charge, and water solubility, no singlemethod can be used to isolate all proteins. To isolate oneparticular protein from the estimated 10,000 different proteins in a cell is a daunting task that requires methods bothfor separating proteins and for detecting the presence of specific proteins.Any molecule, whether protein, carbohydrate, or nucleicacid, can be separated, or resolved, from other molecules onthe basis of their differences in one or more physical orchemical characteristics.
The larger and more numerous thedifferences between two proteins, the easier and more efficient their separation. The two most widely used characteristics for separating proteins are size, defined as either lengthor mass, and binding affinity for specific ligands. In this section, we briefly outline several important techniques for separating proteins; these techniques are also useful for theseparation of nucleic acids and other biomolecules. (Specialized methods for removing membrane proteins from membranes are described in the next chapter after the uniqueproperties of these proteins are discussed.) We then considergeneral methods for detecting, or assaying, specific proteins,including the use of radioactive compounds for trackingbiological activity.
Finally, we consider several techniquesfor characterizing a protein’s mass, sequence, and threedimensional structure.Several allosteric mechanisms act as switches, turningprotein activity on and off in a reversible fashion.Centrifugation Can Separate Particles andMolecules That Differ in Mass or DensityThe binding of allosteric ligand molecules may lead tothe conversion of a protein from one conformational/The first step in a typical protein purification scheme iscentrifugation. The principle behind centrifugation is that■■3.6 • Purifying, Detecting, and Characterizing Proteinstwo particles in suspension (cells, organelles, or molecules) with different masses or densities will settle to thebottom of a tube at different rates.
Remember, mass is theweight of a sample (measured in grams), whereas densityis the ratio of its weight to volume (grams/liter). Proteinsvary greatly in mass but not in density. Unless a proteinhas an attached lipid or carbohydrate, its density will notvary by more than 15 percent from 1.37 g/cm3, the average protein density.
Heavier or more dense molecules settle, or sediment, more quickly than lighter or less densemolecules.A centrifuge speeds sedimentation by subjecting particlesin suspension to centrifugal forces as great as 1,000,000times the force of gravity g, which can sediment particles assmall as 10 kDa. Modern ultracentrifuges achieve theseforces by reaching speeds of 150,000 revolutions per minute(rpm) or greater. However, small particles with masses of5 kDa or less will not sediment uniformly even at such highrotor speeds.Centrifugation is used for two basic purposes: (1) as apreparative technique to separate one type of material fromothers and (2) as an analytical technique to measure physical properties (e.g., molecular weight, density, shape, andequilibrium binding constants) of macromolecules.
The sedimentation constant, s, of a protein is a measure of its sedimentation rate. The sedimentation constant is commonlyexpressed in svedbergs (S): 1 S 1013 seconds.Differential Centrifugation The most common initial step inprotein purification is the separation of soluble proteins frominsoluble cellular material by differential centrifugation.
Astarting mixture, commonly a cell homogenate, is pouredinto a tube and spun at a rotor speed and for a period of timethat forces cell organelles such as nuclei to collect as a pelletat the bottom; the soluble proteins remain in the supernatant(Figure 3-31a). The supernatant fraction then is poured offand can be subjected to other purification methods to separate the many different proteins that it contains.Rate-Zonal Centrifugation On the basis of differences intheir masses, proteins can be separated by centrifugationthrough a solution of increasing density called a density gradient. A concentrated sucrose solution is commonly used toform density gradients. When a protein mixture is layered ontop of a sucrose gradient in a tube and subjected to centrifugation, each protein in the mixture migrates down the tubeat a rate controlled by the factors that affect the sedimentation constant.
All the proteins start from a thin zone at thetop of the tube and separate into bands, or zones (actuallydisks), of proteins of different masses. In this separation technique, called rate-zonal centrifugation, samples are centrifuged just long enough to separate the molecules ofinterest into discrete zones (Figure 3-31b). If a sample is centrifuged for too short a time, the different protein moleculeswill not separate sufficiently.
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