Eastern Illinois University The Keep Masters Theses Student Theses & Publications 1975 Studies of Acetylcholinesterase in Synaptosomes Syed Abbas Rizvi Eastern Illinois University This research is a product of the graduate program in Chemistry at Eastern Illinois University. Find out more about the program. Recommended Citation Rizvi, Syed Abbas, "Studies of Acetylcholinesterase in Synaptosomes" (1975). Masters Theses. 3502. https://thekeep.eiu.edu/theses/3502 This is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. PAPER CERTIFICATE #2 TO: Graduate Degree Candidates who have written formal theses. SUBJECT: Permission to reproduce theses. 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Osmania University Hyderabad, India THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science IN THE GRADUATE SCHOOL, ILLINOIS UNIVERSITY EASTERN CHARLESTON, ILLINOIS 1975 YEAR I HEREBY RECOMMEND THIS THESIS BE ACCEPTED AS FULFILLING OF V THIS PART THE GRADUATE DEGREE CITED ABO E �};197) \ ..uL �3.lf7( ? [f\TE I '-' ACKNOWLEDGEMENT The author expresses his sincere apprecia­ tion to Dr. C. D. Foote for suggesting the problem and for providing guidance, inspiration and assistance throughout the investigation. The author also wishes to thank other members of the faculty and graduate students for their active interest and help. ii TABLE OF CONTENTS Page INTRODUCTION • • • • • • • • • • • • • • • • • 1 MATERIALS AND METHODS. • • • • • • • • • • • 6 A. Crude Homogenate. • • • • • • • • • • 6 B. Partially Purified Enzyme • • • • • • 6 c. Synaptosomal Preparation. • • • • • • 7 D. Enzyme Assay. • • • • • • • • • • • • 7 E. Detergent Treated Pel let. • • • • • • 8 RESULTS. • • • • • • • • • • • • • • • • • • • 9 DISCUSSION • • • • • • • • • • • • • • • • • 15 BIBLIOGRAPHY • • • • • • • • • • • • • • • • 27 iii LIST OF FIGURES Figure Page 1 Detergent Treated Pellet •• • • • • • • • 23 2 Graph of Reciprocal Velocity vs Reciprocal of Dif ferent Concentra­ tions of Substrate • • • • • • • • • • • • 24 3 Percent Inhibition vs Different Concentrations of SDS •••••• • • • • • 25 4 Percent Inhibition vs Percent Concentration of Non-Ionic Detergent. • • • • • • • • • • • • • • • • 26 iv LIST OF TABLES Table Page 1 Absorbance Due to S DS in Detergent Treated Pellet • • • • • • • • • 22 v ABSTRACT Acetylcholinesterase from rat brain was studied in crude homogenates, partia lly puri fied en zyme fractions, and in synaptosomes, to observe modification of the enzyme as a consequence of the modification of the membrane containing it. Sodium dodecyl sulfate decreases the absorbance due to particles in the homogenate. This change in absorbance was proportional to both the concentration of so dium dodecyl sulfate and the concentration of the particles. Further, sodium dodecyl sulfate caused sulfhydryl groups to ap pear in the crude homogenate and in partially purified enzyme frac , - tions. Synaptosomal preparations did not give such resu lts. At concentrations of sodium dodecyl sulfate sufficient to cause absorbance changes, and therefore to modify the mem- branes in the preparation, the en zyme acetylcholinesterase was completely inhibited. A non-ionic detergent in the same range of con centra- tions as for sodium dodecyl su l fate, neither inhibited the enzyme nor caused any noticeable change in abso rbance due to the membranous particles. INTRODUCTION There are millions of nerve cells in the brain which require special enzymes for the synthesis and metabolism of substances which allow communication between cells. Nerve impulses are transmitted from one cell to another throu gh a synapse. The transmission of information across the synapse occurs by the release of tra nsmitter substance from the pre- synaptic membrane and its interaction wi th the receptor in the postsynaptic membrane (1). Acetylcholine is one of sev- eral transmitter su bstances involved in the transmission of information across synapses. Interaction of acetylcholine with its receptor in the postsynaptic memb rane leads. to I excitation of the cell whereupon the acetylcholine is destroyed by hydrolysis catalyzed by the enzyme acetyl- cholinesterase (2). The components of neuronal membranes· are importan t for understanding of the structure and function of this membrane. Proteins are obviously important in the metabolism of neurotransmitters, the structure of the membranes, as well as other vital components of the chemical transmission from one cell to another. Several experiments have confirmed the classical observation of Weiss and Hiscoe (1948) (3) that cytoplasmic components may migrate down the axon. There are data suggesting that proteins may be also synthesized in 1 2 cytoplasmic and mitochondri al components of axons (3). The lipid classes present in sy naptic plasma membrane are very similar to whole brain (4). The rat synaptic plasma membrane is abou t two times richer in lipids than plasma membranes from rat liver. The most striking feature o f synaptic plasma membrane is the higher proportion of longer cha in fatty acids with a high degree of unsaturation in the synaptic plasma membrane tha n in whole brain . These fatty ac ids are primarily moieties of phosphatidylethanolamine and phosphatidylserine (4). It is assumed that membrane bound proteins interact directly with some of the membrane lipids and are involved with the lipids in making up the structural matrix of the membrane (5) . Since the hydrophobic groups of the proteins are thought to penetrate into the interior of the membrane, th is interaction would lead to the co nformation of t he pro­ teins that accommodates th ese deep associations (6). The important consequence of the physical arrangement of li pids and proteins in synaptic plasma mem brare is that the presence of long chain unsaturated fatty ac ids would lead to steric restricti ons on the conformation of the membrane boun::i proteins. The organization of the synaptic region is rather complex (7 ). Of particular interest are the thickenings o f the synaptic membranes, that is the presence of the inter­ synaptic filaments wh ich join the two membranes across the cleft; and a system of filaments, the presynaptic web, 3 projecting into th e postsynaptic region. Such components constitute what may be called the junctional complex of the synapse {7). Most of the acetylcholinesterase is membrane bound and can be isolated in synaptosomes (3). Synaptosomes are artificially produced durinJ homogenization by a cl osing of synaptic membranes to form sm all globules containinJ most of the junctional complex of the synapse (3). Solubilization of pr oteins that are loosely associ­ ated with membranes can be c:bne partially, or selectively, by methods that involve chelating agents, manipulation of ionic strength and pH. These methods, however, do not lead to solubili zation of all proteins, especially those whi ch are more strongly bound to the lipid matri� of membranes. For such protei ns, the use of detergents serves the best purpose of solubilization (8). As the concentration of the detergent increases in a sus pension of liposomes, detergent is incorporated into the bilayer and hence causes changes in its physical prop­ erties by forming mixed micelles. The for mation of mixed micelles gives rise to phase transitions after wh ic h in­ crease in th e detergent to phospholipid ratio causes a decrease in micelle si ze ( 8). From the above knowledge, it is pr oposed that long chain unsaturated lipids and the ir deep penetration into the interior of the membrane give ri se to hydrophobic interactions to form a sterical ly restricted structural 4 matrix co ntaining the en zyme acetylcholinesterase. Sl ight disruption of the lipid matrix sh ould force the enzyme into a different environment free of the original forces. In this new en vironment it is expected th at th e en zyme would exhibit altered chemical and physical properties. Two possible ways to study the modi fication in the characteristics of the en zyme by modifying the membrane which holds it in a rigid con formation are either to solubilize the membrane and observe whether the en zyme was modified when released from the structural matrix of the membrane, or to treat the membrane wit h a detergent which would modify th e membrane but keep the enzyme bound to the membrane. We have attempted th e latter. Using light scattering as a measure of the integrit y of the synaptosomal membranes, we have sought conditions under which the membranes have been modified an d under \Jiich the enzyme property of catalysis could be measured. We have sought changes in