© 2004 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 32, No. 1, pp. 45–48, 2004 Laboratory Exercises The Zymogen-Enteropeptidase System: A Practical Approach to Study the Regulation of Enzyme Activity by Proteolytic Cleavage* Received for publication, July 1, 2003, and in revised form, November 14, 2003 Joa˜ o M. Pizauro Junior‡, Jesus A. Ferro, Andre´ a C. F. de Lima, Karina S. Routman, and Maria Ce´ lia Portella From the Departamento de Tecnologia, Faculdade de Cieˆ ncias Agra´ rias e Veterina´ rias, UNESP, 14884-900 Jaboticabal SP, Brazil The present research describes an efficient procedure to obtain high levels of trypsinogen and chymo- trypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisti- cated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly “switched on” by specific proteolytic cleavage. Keywords: Zymogen, trypsin, chymotrypsin, enteropeptidase, enzyme regulation, proteolytic cleavage. The enormous variety of biochemical reactions that peptide bond present in trypsinogen. This hydrolysis ren- comprise the metabolism of nutrients and their assimila- ders the trypsin active, which in turn also activates tryp- tion in the tissues depends on thousands of enzymes for sinogen by cleaving Arg and Lys residues and then gen- which catalytic activity is regulated by various mecha- erates more trypsin. Trypsin also triggers the activation of nisms. One of these mechanisms is the proteolytic cleav- other proteolytic enzymes from their zymogens, such as age of an inactive protein precursor. Examples of this chymotrypsin, elastase, and carboxypeptidases A and B model of regulation are found in physiological processes [1, 2, 4, 5]. such as digestion, blood clotting, hormone action, and The major aim of this laboratory class is to attract stu- vision [1–3]. dents attention to the importance of the “switch on” mech- The digestion process of proteins in the duodenum re- anism of enzymatic activity at the right time and place in quires concurrent action of several proteolytic enzymes. animals through inexpensive experiments by using pan- Each enzyme is specific for the cleavage of a limited creatic and duodenal extracts. These experiments might number of specific amino acid sequences. Because these be performed to point out the biochemical and physiolog- enzymes are dangerous to cells, their production requires ical importance of pancreatic enzymes. specialized functions in the tissue. The proteolytic en- zymes that function in gastric and intestinal digestive pro- BACKGROUND THEORY AND PRE-LABORATORY PREPARATION cesses are first synthesized as zymogens inside the glan- The metabolism of nutrients is a tightly regulated proc- dular cells and then secreted into the gastrointestinal tract. ess in which different types of tissues, cells, organelles, The synthesized zymogen accumulates in the acinar cells and biomolecules participate in the coordination and reg- of the pancreas and is released into the duodenum by a ulation of metabolism. In higher animals, the activity of the hormonal signal or a nerve impulse [1–3]. regulating enzyme of a biochemical pathway may be reg- After secretion into the duodenum, trypsinogen is con- ulated by short- and long-term mechanisms. Depending verted to trypsin by enteropeptidase, originally named en- on physiological needs, the genetic control of enzyme terokinase, which hydrolyze a unique lysine-isoleucine synthesis may regulate the amount of enzyme available by a long-term mechanism [6–8]. The enzymes involved in the metabolism of protein, * This work was supported in part by the Fundac¸a˜ o de Amparo carbohydrates, and lipids may also be regulated by short- a` Pesquisa do Estado de Sa˜ o Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico (CNPq). term mechanism. One of these mechanism is the allosteric A. C. F. L. was recipient of a scholarship from FAPESP; K. S. R. regulation, where the binding of small molecules, which was recipient of a scholarship from CNPq; M. C. P., J. M. P., and may be an activator, inhibitor, or substrate (modulators), J. A. F. were CNPq-supported Research Fellows. increases or decreases enzyme activity. Thus, enzyme ‡ To whom correspondence should be addressed: Departa- mento de Tecnologia, Faculdade de Cieˆ ncias Agra´ rias e Veteri- activity can be regulated according to physiological needs na´ rias, UNESP, 14884-900 Jaboticabal SP, Brazil. E-mail: that may arise during the cell life [6, 7]. [email protected]. Enzyme activities can also be regulated by reversible This paper is available on line at http://www.bambed.org 45 46 BAMBED, Vol. 32, No. 1, pp. 45–48, 2004 FIG.1. Schematic representation for the activation process of pan- creatic proteolytic enzymes trig- gered by enteropeptidase as de- scribed by Orten et al. [5]. ⅐ ϳ covalent modifications, such as the addition of phosphate CaCl2 2H2Oin 900 ml of CO2 free water and adjust to pH 8.2 groups to serine, threonine, or tyrosine residues. Covalent with diluted HCl. Finally, using a volumetric flask, make up to 1 modification is a highly efficient pathway to control not liter with distilled water. Bring to 37 °C immediately before using. only enzyme in metabolic reactions, such as glycogen BAPNA Solution—Dissolve 40 mg of benzoyl-DL-arginine-p- nitroanilde hydrocloride (BAPNA) in 1 ml dimethylsufoxide. This synthesis and break down, but also many other cellular solution is stable at 4 °C for 2 wk. Prepare fresh daily by dilution functions, including cell growth and differentiation [6–8]. of 1 ml of this solution to 100 ml with 0.05 M Tris buffer, pH 8.2, Another important mechanism to regulate protein func- containing 50 mM CaCl2. tion is the proteolytic activation that can act like switches, 0.2 M Tris Buffer, pH 7.6, Containing 50 mM of CaCl2—Dissolve ⅐ turning proteins “on” and “off.” The proteolytic activation is 24.228 g of tris(hydroxymetil)-aminometane and 7.351g CaCl2 2H O in 900 ml of CO free water, and adjust to pH 7.6 with a special form of covalent modification in which the inac- 2 2 diluted HCl. Finally, using a volumetric flask, make up to 1 liter tive precursor of the enzyme is activated by an irreversible with distilled water. Bring to 37 °C immediately before using. cleavage. Enzymes active in digestive and blood clotting GAPNA Solution—Dissolve 20 mg N-glutarayl-L-phenylala- processes are switched on by proteolytic cleavage. In nine-p-nitroanilde (GAPNA) in 1 ml dimethylformamide. This so- general, these proenzymes are synthesized as slightly lon- lution is stable at 4 °C for 2 wk. Prepare fresh daily by dilution of ger polypeptide chains than true active enzymes, and usu- 1 ml of this solution to 25 ml with 0.2 M Tris buffer, pH 7.6, ally they are part of a cascade of mechanisms. As ob- containing 50 mM CaCl2 [10]. served in Fig. 1, after secretion in the duodenum, Preparation of the 1 mM p-Nitroaniline Solution—Dissolve 13.814 mg p-nitroanilde (pNA) in 1 ml dimethylsufoxide. Finally, trypsinogen is converted to trypsin by enteropeptidase, using a volumetric flask, make up to 1 liter with Tris buffer. Prepare which hydrolyzes a unique lysine-isoleucine peptide bond fresh daily. in trypsinogen. The hydrolysis renders trypsin active, Standard Curve of p-Nitroaniline—Prepare a set of 3-fold num- which also has the ability to activate trypsinogen by cleav- bered tubes, place 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.4, and 1.6 ml of ing Arg and Lys residues and then generating more trypsin 0.1 mM p-nitroaniline and 0.2 ml of 30% acetic acid in each tube. [5]. In addition, trypsin also triggers a cascade activation of In each tube, adjust the total volume to 2.0 ml with the appropri- ate amount of Tris buffer, pH 8.2, containing 50 mM of CaCl to other proteolytic enzymes, and the resulting mixture of 2 give seven calibration standards and one blank tube with reagent enzymes includes endopeptidases (trypsin, chymotrypsin, grade water only. Measure the absorbance of each tube at 410 and elastase) and exopeptidases (carboxypeptidases A nm using control (water blank). Then plot absorbance (y-axis) and B). Thus, the formation of trypsin by enteropeptidase against the number of ␮mol p-nitroaniline (x-axis), labeling the is the essential activation step [2]. graph appropriately. Do a curve fitting by linear regression to Ϫ1 Ϫ1 General Note—In this experiment, we describe a meth- obtain the ⑀ value (x M cm ), which corresponds to the angular odology adapted to the use of trypsinogen, chymo- coefficient of the curve. trypsinogen, and enteropeptidase from broilers, pigs, fishes, and ruminants due to the facility of obtaining these Sources of Proteolytic Enzymes animals. Alternatively, other animals can be used such as The broilers were obtained from a commercial hatchery. In guinea pigs, mice, or rabbits. order to obtain pancreatic glands with high levels of proenzymes Generally, this experiment uses some hazardous re- and low levels of trypsin in the duodenal extract, animals were agents, like acetic acid, p-nitroanilide, N,N-dimethylform- starved overnight. amide, and dimethylsulfoxide. Adult Hubbard broilers of both sexes were slaughtered. The These reagents should be manipulated according to pancreas were removed, rinsed in ice-cold 0.9% (w/v) NaCl so- their handling instructions and disposed in accordance lution, and homogenized with 0.5 M Tris-HCl buffer, pH 8.0, containing 0.05 M CaCl (20 ml/g), in an OMNI-GLH-2511 tissue with local safety instructions.