Properties of a Leu‐Phe‐Cleaving Endopeptidase Activity Putatively Involved in β‐Endorphin Metabolism in Rat Brain

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Abstract
Incubation of β‐endorphin with cytosolic and particulate fractions of rat brain resulted in the formation of several peptides, including γ‐endorphin [β‐endorphin‐(1–17)] and β‐endorphin‐(18–31), indicating the presence of enzyme activity cleaving the Leu17‐Phe18 bond of β‐endorphin. An assay for this Leu‐Phe cleaving activity, based on the cleavage of the 14C‐labeled substrate acetyl‐Val‐Thr‐Leu‐Phe‐[ε ([14C]CH3)2]Lys‐NHCH3, was used to examine the properties of this enzyme activity. β‐Endorphin‐(1–31) competitively inhibited the Leu‐Phe‐cleaving enzyme activity on the pentapeptide substrate. Over 90% of activity was recovered in the cytosolic fraction. Leu‐Phe‐cleaving activity behaved like a thiol endopeptidase because it was inhibited by low concentrations of N‐ethylmaleimide, p‐chloromercuribenzoate, p‐chloromercuribenzoyl sulfate, and low concentrations of Hg2+. Low concentrations of sulfhydryl compounds stimulated Leu‐Phe‐cleaving activity. The activity was optimal between pH 8.5 and 9.0. The Km of Leu‐Phe‐cleaving activity in the cytosolic fraction was 35 μM and in the particulate fraction 88 μM with Vmax values of 193 and 15 nmol mg protein−1 h−1, respectively. The apparent molecular mass of the Leu‐Phe‐cleaving enzyme was estimated by gel filtration to be ∼200 kilodaltons. These properties of Leu‐Phe‐cleaving activity indicate that the Leu‐Phe‐cleaving enzyme is distinct from any known brain endopeptidase.