An endogenous aminoenkephalinase inhibitor: purification and characterization of Arg0-Met5-enkephalin from bovine striatum.

Arg0-Met5-enkephalin (Arg0-MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroacetic acid, followed by column chromatography successively on Bio-Sil C8, semipreparative HPLC Radial-Pak C18, fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C18, Lichromsorb C18, and mu Bondapak C18. The peptide content was followed by radioimmunoassay with an antibody against synthetic Met-enkephalin. For each of the six HPLC and FPLC systems, the elution time of the immunoreactive fractions coincided exactly with that of synthetic Arg0-MEK. The purified peptide showed a highly homogeneous profile in three different analytical HPLC systems. Its retention time and three-dimensional UV spectrum were identical to those of the synthetic Arg0-MEK. The structure of the purified material was identified by microsequencing as the hexapeptide Arg-Tyr-Gly-Gly-Phe-Met. Ninety percent of the purified peptide was in oxidized form containing equimolar amounts of Met-(R)- and Met-(S)-sulfoxide. The reduced Arg0-MEK inhibited aminoenkephalinase with a Ki of 2.2 microM, and its sulfoxide analogue inhibited it with a Ki of 8.9 microM. The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED50 of 5 microM, and the effect could be reversed by equimolar naloxone. Our data indicate that Arg0-MEK is an immediate Met-enkephalin precursor and an endogenous inhibitor.

A new soluble brain-specific protein: identification and partial purification.

A new soluble 170-kDa protein (BP170) was found to be present exclusively in the brain of all the vertebrates that we studied by Western immunoblotting. It was not detected in peripheral rat tissues, including heart, kidney, liver, spleen, lung, muscle, adrenal, intestinal mucosa, sciatic nerve, or pituitary. In rat brain, its regional distribution was found to be heterogeneous, with its highest concentration in the cerebrum and its lowest in the hypothalamus, and 89% of it was in the post-microsomal fraction. BP170 constitutes at least 0.05% of the total brain cytosol proteins. Its level increases during development, being the lowest at 5 days and the highest at 90 days postnatal. BP170 is a single-chain polypeptide. It could be partially purified by precipitation with polyethylene glycol followed by column chromatography on Q Sepharose. Although BP170 was identified by an antiserum against puromycin-sensitive aminopeptidase (PSA), the two proteins differ in molecular weight, chromatographic properties, regional and subcellular distribution, developmental changes, immunoreactivity, and enzyme activity. Self-incubation or trypsin treatment of the partially purified BP170 generates no PSA activity, indicating that BP170 is not a PSA precursor. Furthermore, BP170 is neither an inhibitor nor an activator of PSA. Our data suggest that BP170 is a novel brain-specific protein not previously described.

Major rat brain membrane-associated and cytosolic enkephalin-degrading aminopeptidases: comparison studies.

The major cytosolic and membrane-associated enkephalin-degrading aminopeptidases were purified in parallel by column chromatography successively on DEAE-cellulose, AH-Sepharose, hydroxylapatite, Sephadex G-200, Affigel Blue, AH-Sepharose, and hydroxylapatite. With the final hydroxylapatite column, the cytosol (S) and the membrane (M) enzymes could each be resolved into two peaks, one eluted with 0.05 M phosphate (SI, MI) and the other with 0.25 M phosphate (SII, MII). The overall purification, with Arg BNA as substrate, for the SI and MI was about 450-fold; for SII and MII, 1,200-fold. The yield for each enzyme was about 2%. the major protein integral units of the four enzymes are similar; they are single polypeptide chains with a molecular weight of 100,000 daltons. Their pH optimum, substrate specificity, and sensitivity to puromycin show that they are similar to lysosomal aminopeptidase (EC 3.4.11.-). The two forms of the cytosol and the membrane enzymes have slightly different kinetic constants. With the inhibitors, SII is more sensitive to proctolin, whereas MII is more sensitive to bestatin and Arg-Phe-Ala. Mn2+ activates SI on Met-enkephalin degradation, but inhibits SII, MI, and MII.

Proctolin: a potent inhibitor of aminoenkephalinase.

Proctolin is a potent selective inhibitor of aminoenkephalinase. The specificity of its inhibition of various aminopeptidases is similar to that of puromycin; it inhibits aminoenkephalinase, but not leucine aminopeptidase or aminopeptidase M. Enkephalin breakdown by synaptic plasma membrane, but not by brain slices, is sensitive to proctolin. The inhibition by proctolin is partially caused by its resistance to enzymatic breakdown. The inhibition is of mixed type and is concentration dependent, and the two amino acids at the N-terminal are important for its action. The minimal structure for inhibition is a dipeptide with a basic amino acid at the N-terminal and a basic or an aromatic amino acid at the C-terminal.