Reactions of Lipolytic Enzymes An Undergraduate Biochemistry Experiment Kelly A. Clingman and Joseph Hajdu' California State University, Northridge, Northridge, CA 91330 Lipolytic enzymes represent a unique class of esterases hydrolyzing in vivo water-insoluble substrates. Although they do catalyze in vitro hydrolysis of lipids in molecular dispersions as well, the same substrate present in an organized lioid-water interface is deeraded at 1Oz-lo3 times higher rites ( I ) . Specifically, all n&al substrates of phosphoiipases are long-chain phospholipids.
8 JPhos~holipaae
R ~ U ~ - H Phospholipase Az
2
I C H 2 ~ ~ - O - X /
D
A-
Phospholipase C where X is choline, serine, or ethanolamine and RI and RZ are long-chain alkyl and alkenyl groups. These phospholipids occur in three main organizational forms: (1) in membranes, (2) in the serum as lipoproteins or liposomes, and (3) as mixed mieelles in conjunction with bile salts in the intes-
tine. Although the strong influence of the physicochemical state of the suhstrate on phospholipase activity has long delayed elucidation of the enzyme mechanisms, a wide variety of physiologically important processes have been shown to involve catalytic participation of these enzymes (2-6). Phospholipase Ap,for example, is involved in platelet aggregation (2), cardiac contraction and excitation (3), prostaglandin biosynthesis (4,5)as well as aldosterone-dependent sodium transport (6). The enzyme is also known to interfere with calcium-dependent ATPase activity (7), with opiate receptor binding (8)and with neurotransmitter release a t motoric nerve terminals (9). Development of convenient in vitro assay systems utilizing biologically occurring long-chain phosphoglycerides seemed to be a key,step toward understanding the kinetic behavior and catalytic mechanisms of phospholipases. During the past decade considerable progress has been made toward that goal, and i t appears that micellar dispersions of phospholipids in conjunction with nonionic (10)and ionic (11) surf&ants were employed with most success in attempts aimed at the chararterization of enzymatic catalyses. Most sirnificantlv. ".mixed-micellar substrates fulfill the two basic requirements for conducting enzyme kinetic studies:
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(1) incorporation of the water-insoluble phospholipids into well-defined macromolecular aggregates and (2) the ability to vary the substrate concentration at the water-lipid interface. Despite the initial controversy that surrounded these systems for some time (ZZ), recent success of employing mixed micelles for detailed kinetic studies of phospholipases, including direct determination of inhibition constants (131, has allowed us to introduce the subject to students as a part of an instructional hiochemistry laboratory course. In this experiment two types of phospholipases are emoloved: A". (from hee venom. as reoresenta. . ohosoholioase . . . . tive of the insect enzyme, being compared with mammalian, oancreatic oorcine PLA?) and . ohos~holioase . . C. a bacterial ;hosphodiisterase isola&d from H. cereus. The enzymes are reacted with two kinds of mixed micelles utilizing nhosoholipid aggregates sequestered by the nonionic det;igent;rriton X-100 and the bile salt, sodium cholate, respectively. The products of enzymatic lipolyses are then characterized by thin-layer chromatography, and finally the specificities of phospholipases for the respective substrates are demonstrated to illustrate the underlying differences between the enzymes. The experimental work requires a two-part, three-hour time frame: the first oeriod is dedicated to the isolation and purification of egg p'hosphatidylcholine, and the second is devoted to enzymology. The latter focuses on elucidation of the catalytic reactions and characterization of the products obtained on enzymatic hydrolyses of the highly purified phospholipid substrate. In addition to acquainting the students with modern techniques in lipid hiochemistry, the objectives of the experiment include delineation of the specificity of each enzyme in terms of (1) the site of attack, (2) the nature of the reaction catalyzed (carboxyesterase vs. phosphodiesterase activity), and (3) the preference for the type of mixed-micellar aggregates incorporating the phospholipid substrate depending on the source (mammalian vs. insect) of the phospholipase.
Materials
Pancreatic narcine nhmoholinase . . . A?. bee venom ohosoholi~ase . . .
A2. and barterla1 phospholipase C from B. cereus were obtained
from Hochringer Mannheim Biochemicals and were used as re. reiwd. Srork adutiuns of the enzymes were prepared in 0.05 M Tris
' Auihor to whom correspondence should be addressed.
pH 8.5 and diluted into the reaction mixture as required. Triton X100 and sodium cholate were obtained from Sigma Chemical Compan". Ileogmt-grodr urganic and inorganic chemivalu were used throughout the cxprrimrnt. Yor thin-layer chron~atographyM'hatm m MKRY f2.5- X 7.5-cnu and KBF (5- X 20-cml silica re1 ~ l a t e s were used. Standard solutions (5 mM in chloroform) of piosphatidvlcholine.. lvsoohosohatidvlcholine. , . oalmitic acid. and 1.2-dioal. mitin were pr~paredjust prior I I I the ~xperiment.Column rhromaturraphv was carrlrd a Teflon stopcock, having 1.0cm of level sand Ihth hehu and ahore the ndsorbmt. 'l'hr crude ph~,=phsrrdylch, ,,r 50 s:: bsrterial phuiphdiparr C from H. cerrur wm added 103-mLasiay mixture. The reactiuns were run at 40 for 4 0 min using a Lauda Brmkman R.\IS6
