Characterization of a nontrypsin cholecystokinin converting enzyme in mammalian brain

Abstract

An enzyme has been partially purified from canine and porcine cerebral cortical extracts that differs from trypsin in that it manifests some degree of hormone specificity since it converts porcine cholecystokinin to smaller immunoreactive forms, i.e., the COOH-terminal dodecapeptide and octapeptide fragments, but fails to convert big gastrin (34 amino acids) to heptadecapeptide gastrin. This enzyme is distinguishable from trypsin not only in substrate specificity, but also in several physicochemical proprties. It is not inhibited in the presence of concentrations of lima bean trypsin inhibitor sufficient to inhibit 1 mg of trypsin r ml of incubation mixture. Over the past decade it has become evident that many, if not all, peptide hormones are found in their tissues of origin in more than one form (see ref. 1 for review). Following the discovery that proinsulin is the precursor of insulin (2) it has generally been assumed that, if the larger hormonal form contains a peptide with amino acid sequence identical to or if it is convertible by enzymatic conversion to a smaller, well-characterized, and more biologically active form, the larger molecule is a prohormone, whether or not the biosynthetic precursor relationship has been established. Thus, a precursor relationship has been definitely established for several peptide hormones, including proinsulin (2) and proparathyroid hormone (3-5), but it has only been assumed for others, such as big (34-amino-acid) gastrin (gastrin-34) (6, 7) and the 39-amino-acid cholecystokinin variant (CCK-39) (8). Similarly, cholecystokinin (CCK-33) can be considered to be a precursor for the cholecystokinin dodecapeptide and octapeptide (CCK-8), the COOH-terminal fragments which have biologic potencies greater than that of the presumed parent CCK-33 molecule.Although several groups have reported on the existence in the tissues of origin of tryptic-like enzymes involved in the conversion of proinsulin (9) and proparathyroid hormone (10, 11), precursors to insulin and parathyroid hormone, respectively, no evidence has as yet been presented to determine whether the same enzyme may be involved in processing more than one hormonal family. In this report we characterize and partially purify an enzyme, obtained from extracts of mammalian brain, which converts porcine cholecystokinin (pCCK-33) to smaller immunoreactive forms but which fails to convert gastrin-34 to heptadecapeptide gastrin (gastrin-17 (pH 7.5). The extracts were centrifuged at 10,000 X g for 15 min. The supernatants were tested for ability to convert CCK-33 and gastrin-34 to smaller immunoreactive forms as described below. They were also chromatographed on 1 X 50 cm columns containing Sephadex G-50 or G-75. The columns were calibrated by application of radioactive markers to establish the positions of the void volume and the radioiodide peak. After application of brain extract, the columns were eluted with 0.9% NaCl and 1-ml fractions were collected and tested for enzymatic activity.The active fractions from the Sep...

Extraction and immunochemical characterization of cholecystokinin-like peptides from pig and rat brain.

“…Portions of tissue extracts were also subjected to starch gel electrophoresis as described (17). This electrophoretic system Abbreviations: CCK, cholecystokinin; iCCK, cholecystokinin-like immunoreactive peptides; pCCK33, 33-amino acid porcine cholecystokinin; CCK3 through CCK12, COOH-terminal peptide fragments of CCK33 containing the indicated numbers of residues; pGI, porcine heptadecapeptide gastrin (unsulfated); QUSO, microfine precipitated silica; RIA, radioimmunoassay.…”

“…iCCK contained in tissue extracts was fractionated in three systems: adsorption to microfine precipitated silica (QUSO), which binds big iCCK but not small iCCK (16); Sephadex G-50 fine column chromatography (6, 7); and starch gel electrophoresis (17).…”

Characterization of Cholecystokinin Binding Sites in Rat Cerebral Cortex Using a 125I‐CCK‐8 Probe Resistant to Degradation

“…Innis and Snyder (1980) found excessively high levels of nonspecific binding in these membranes and Used guinea pig cortical membranes instead; whereas Saito et al (1981a) defined binding parameters in mouse cerebral cortical membranes after finding extensive degradation and large amounts of nonspecific binding in the same membranes from rat. Rapid processing of CCK-33 to smaller peptides has been demonstrated in brain tissue extracts frorn various species including rat (Straus et al, 1978. Goltermann et al, 1980), and could account for t h i rapid degradation of the radioligand observed.…”

“…The improved resistance of our 12511E-CCK-8 to degradation in contrast to 1251-BH-CCK-33 may reflect the location of the 1251. CCK-33 is rapidly processed in rat brain in vivo to smaller peptides including CCK-8 (Straus et al, 1978;Goltermann et al, 1980). Malesci et al (1980) have identified and partially isolated two enzymes from cow brain that specifically cleaved CCK-33 to its C-terminal dodecapeptide and octapeptide.…”

Further characterization of brain cholecystokinin-converting enzymes.

“…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.…”