Cholecystokinin-converting enzymes in brain (cholecystokinin fragments/Michaelis constant/enzyme specificity/radioimmunoassay)

Crude extracts of porcine cerebral cortical tissue convert cholecystokinin (CCK) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8). The Sephadex G-75 void volume eluate of the crude extract cleaves the arginine-isoleucine bond and effects conversion only to CCK-12; the Sephadex G-50 void volume eluate of the same extract cleaves the arginine-aspartate bond as well, so that both CCK-12 and CCK-8 are end products. Thus, there are at least two enzymes; the one involved in the conversion to CCK-12 is of larger molecular radius than the other. The Km for the cleavage of CCK at the arginine-isoleucine bond by the Sephadex G-75 void volume eluate enzyme is 1.1 X 10-6 M; the Km for trypsin cleavage of the same bond is 4.7 X 10-6 M. The lower Vmax for the brain enzyme (1.5 X 10-11 mol/min per g of extract) compared with trypsin (66 X 10-11 mol/min per g of trypsin) simply reflects the lesser degree of purity of the brain extract than of the highly purified trypsin. We have previously partially purified an enzyme that is readily solubilized from extracts of mammalian brain and that converts porcine cholecystokinin (CCK-33) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8), yet fails to convert "big" gastrin (G-34) to heptadecapeptide gastrin (G-17) although in each case the bond hydrolyzed is one whose carboxyl residue is donated by either an arginine or a lysine residue (1). Thus, the enzyme is distinguishable from trypsin in substrate specificity. It also differs from trypsin in size, temperature sensitivity, and other physicochemical properties. The enzyme does not appear to be species specific in that extracts from the brains of pig, dog, rabbit, rat, and mouse convert CCK to the COOH-terminal fragments. The finding that there are two end products of the conversion, CCK-12 and CCK-8, suggested that there might be two enzymes involved (1). In this report we demonstrate that there are at least two enzymes which differ in molecular size and substrate specificity. The kinetic constants for the enzyme that converts CCK-33 to CCK-12 are compared with those of trypsin for the same conversion. MATERIALS AND METHODS Preparation of Partially Purified Enzyme. Extracts of porcine cerebral cortical tissue were prepared by a modification of a previously published method (1). Two grams of frozen porcine cerebral cortex were extracted at 4°C in 0.1 M barbital buffer at pH 8.6 with a Teflon grinder; the final concentration was 0.1 g (wet weight) of tissue per ml. The extract was then centrifuged at 10,000 X g for 15 min. For some studies, crude brain extract was used. For others, 10 ml of the supernatant was chromatogriaphed on a Sephadex G-75 column (2.5 X 85.cm) previously calibrated by application of 12'I-labeled gamma globulin and 131Ito mark the positions of the void volume and iodide peak. The column was eluted with the same buffer at a flow rate of 0.5 ml/min. The void volume fractions were pooled and diluted to a final protein concentration of 1 mg/ml; 1-ml portions were lyophilized and stored at -20°C. Immediately before use, the lyophilized material was reconstituted with 1.0 ml of 0.02 M barbital. On occasion, the pooled void volume eluates of a Sephadex G-50 column were also tested. The protein concentrations of the crude brain extract and of the void volume eluates were determined by the method of Henry (2) and all were adjusted to 1 mg/ml. Experimental Procedure for Kinetic Study. Purified porcine CCK-33, a gift from Victor Mutt (Karolinska Institute, Stockholm) received through the Gastrointestinal Hormone Research Service of the National Institute of Arthritis, Metabolism, and Digestive Diseases (Bethesda, MD), was used as a substrate in concentrations ranging from 0.13 to 36 Mg/ml in standard diluent (0.5 g of bovine serum albumin per 100 ml of 0.02 M barbital buffer at pH 8.6). Substrate and enzyme solutions were placed separately in a constant-temperature bath at 370C and allowed to reach temperature equilibrium. Then, 0.2 ml of enzyme solution was added to 0.3 ml of substrate solution and the mixture was vortexed. At various times between 5 and 60 min, 10to 100-ul portions were removed from each incubation mixture and pipetted into 2 ml of standard diluent. The diluted samples were immediately boiled for 5 min to inactivate the enzyme. Fractionation of immunoreactive CCK peptides was then carried out by differential absorption to QUSO G32 (Philadelphia Quartz, Philadelphia) as described (3); 10 mg of QUSO was added to 2-ml samples. After vortexing and centrifugation at 3000 rpm (2300 X g) for 15 min, the concentration of immunoreactive COOH-terminal fragments was measured by radioimmunoassay of the supernatant. This procedure permits distinction between CCK and the COOHterminal fragments but does not distinguish between CCK-12 and CCK-8. For control studies, similar enzyme preparations were boiled and then incubated under identical conditions. Convertingenzyme activity in the whole brain extract is inactivated by exposure to temperatures above 45°C (1). Similar studies were performed with chymotrypsin-free trypsin (Sigma, bovine trypsin, DPCC-treated* type XI) at final concentrations ranging up to 1 mg of trypsin per ml. Additional studies were performed with synthetic sulfated CCK-8 (a gift from Squibb Research Institute, through the courtesy of S. J. Lucania) and synthetic sulfated CCK-12 (prepared by M. A. Ondetti and received through the courtesy of V. Mutt) as substrates. The hormonal form of the final reaction product was determined from starch gel electrophoretic patterns (1). Abbreviations: CCK-33, 33-amino acid cholecystokinin; CCK-12, COOH-terminal cholecystokinin dodecapeptide; CCK-8, COOHterminal cholecystokinin octapeptide; G-34, 34-amino acid "big" gastrin; G-17, heptadecapeptide gastrin. * Diphenylcarbamoyl chloride-treated to inactivate chymotrypsin. 597 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 598 Medical Sciences: Malesci et al.