Further characterization of brain cholecystokinin-converting enzymes.

Abstract

The brain cholecystokinin-converting enzymes that cleave intact cholecystokinin to its COOH-terminal dodecapeptide and octapeptide also cleave the synthetic dipeptides Arg-Ile (or Arg-Val or Arg-Leu) and Arg-Asp, respectively. Thus, they are not hormone-specific enzymes but are bond-specific. Ultracentrifuge studies demonstrate that there is Arg-Ile hydrolase activity associated with a protein greater in molecular weight than gamma globulin and that both Arg-Ile and Arg-Asp hydrolase activities are associated with one or more proteins between albumin and gamma globulin in molecular weight.We have previously described (1, 2) two partially purified enzymes that are readily solubilized from extracts of mammalian brain and that convert porcine cholecystokinin (pCCK33) to its COOH-terminal fragments, the dodecapeptide (CCK12) and the octapeptide (CCK8). These enzymes were proven to differ from trypsin in size, temperature sensitivity, and substrate specificity (1). Here, we report further characteristics of substrate specificity and other physicochemical properties of these enzymes. MATERIALS AND METHODSPreparation and Partial Purification of Brain Enzymes. Both crude bovine cortical extracts (bCE) and pooled Sephadex G-75 void volume eluates (G75 vv) of bCE were prepared according to published methods (2).In addition, ultracentrifugation was used to fractionate bCE. The extract (0.7 ml), prepared as above, was layered above a discontinuous sucrose gradient (1 ml each of 50%, 25%, 22.5%, and 20% sucrose) in a 5-ml polyethylene tube and centrifuged at 40,000 rpm for 16 hr in a Spinco model L ultracentrifuge fitted with a Beckman swinging bucket rotor (SW 50.1). After the centrifuge came to rest without braking, 0.5-ml samples were removed successively from the bottom of each tube. Each fraction was dialyzed for 1 hr at 40C against the standard diluent (0.1 M barbital buffer pH 8.6, containing 2.5 mg of bovine serum albumin per ml) and then studied for enzymatic activity. As molecular weight markers, 125I-labeled gamma globulin and bromphenol blue-stained albumin were layered in duplicate tubes and centrifuged along with bCE.Substrates and Enzymatic Assay. Studies were performed with both pCCK33 and synthetic dipeptides as substrates.Both pCCK33 (KABI Diagnostica, Studsvik, Sweden) at a concentration of 10 mg/ml and 125I-labeled pCCK33 (pCCK33 used for labeling was a gift of Victor Mutt, Karolinska Institute, Stockholm, and was received through the Gastrointestinal Hormone Research Service of the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, MD) were used as substrates. For enzyme assay, 0.4 ml of bCE or pooled G75 vv or ultracentrifuged sucrose gradient fraction was added to standard diluent containing the substrate. After 6 hr at 370C, loo-M1 aliquots of the incubation mixture were removed, placed in a boiling water bath for 5 min to inactivate the enzyme, and then subjected to starch gel electrophoresis in order to determine the hormonal forms of the reaction products, as des...

Localization of a neutralization site of Theiler's murine encephalomyelitis viruses

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Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: A study by high pressure liquid chromatography

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