Journal zyxwvutsrqpo of Neurochernistry zyxwvutsrqpon 36(3):959-965, March. Raven Press, New zyxwvutsr York zyxwvutsrqpo 0 1981 International Society for Neurochemistry zyxwvutsrq 0022-3042/81/0301-0959/$02.00/0 zyx Newly Synthesized Cholecystokinin in Subcellular Fractions of the Rat Brain N. R. Goltermann, K. Stengaard-Pedersen, J. F. Rehfeld, and "N. J. Christensen Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark and *Department of Endocrinology, KQbenhavns Amtssygehus i Herlev, DK-2730 Herlev, Denmark Abstract: The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [35S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial-synaptosomal fraction, Pz, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH-terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elu- tion constants, K,,, of 1.1 (corresponding to the COOH-terminal octapeptide) and of 1.40 (a component which may correspond to the COOH-terminal tet- rapeptide amide, CCK-4). The findings support the idea that the small molecu- lar forms are the transmitter forms of CCK. Key Words: Biosynthesis- Cholecystokinin-Subcellular fraction-Neuropeptides-Neurotransmitter. Goltermann N. R. et al. Newly synthesized cholecystokinin in subcellular fractions of the rat brain. J. Neurochem. 36, 959-965 (1981). Cholecystokinin (CCK) is located in neurons of most regions of the central nervous system (Pinget et al., 1978; Larsson and Rehfeld, 1979; Innis et al., 1979; Lorkn et al., 1979). In the mammalian cere- bral cortex the concentrations are high, up to 1 nmol equiv. CCK-8/g tissue (Rehfeld, 19786). CCK occurs here in five main forms, of which the small octa- and tetrapeptide-like forms predominate (Dockray, 1976; Rehfeld, 1978a,b; Rehfeld and Goltermann, 1979). By zyxwvutsr in vivo labelling with [35S]methionine we have recently demonstrated a rapid synthesis of CCK in the rat cerebral cortex and a precursor relationship between large molecular forms of CCK and the octapeptide-like form (Rehfeld et al., 1979; Golter- mann et al., 198Oah). The present study examines the subcellular lo- calization of CCK synthesized de novo in different parts of the rat brain. Received June 23, 1980; revised September 5, 1980; accepted Address correspondence and reprint requests to N. R. Gol- September 10, 1980. termann. MATERIALS AND METHODS Reagents [35S]Methionine was purchased from Radiochemical Centre, Amersham, U.K. (specific radioactivity, 300-800 Ci/mmol). Cholecystokinins were generously donated by V. Mutt (Karolinska Institutet, Stockholm [purified porcine CCK-33]), M. Ondetti (The Squibb In- stitute of Medical Research, Princeton, New Jersey [synthetic CCK-81) and J. S. Morley (ICI, Alderley Park, Cheshire, England [synthetic CCK-4 and synthetic human gastrin-171). Sephadex (3-50 superfine and G-25 were purchased from Pharmacia Fine Chemicals (Upp- sala, Sweden), Tris and dithiothreitol from Sigma Chem- ical Company (USA) and Lumagel from Lumac System Ag (Basel, Switzerland). Animals Adult male and female Wistar rats weighing between 200 and 250 g were used, 10 animals in each experiment. Abbreviation used: CCK, Cholecystokinin. 959