SUBCELLULAR-DISTRIBUTION OF 2 ENZYMES WITH NEUROPEPTIDE CONVERTING ACTIVITY IN RAT SPINAL-CORD

The subcellular distribution of a dynorphin converting enzyme (DYNase) and a substance P endopeptidase (SPase) has been examined in rat spinal cord using differential and density gradient centrifugation. The DYNase activity was highest in a 10,000g supernatant after differential centrifugation. In the step gradient, the highest activity was found in synaptosomal fractions. In the continuous density gradient using a synaptosomal lysate as starting material, peak activities were found in the top fractions and at 0.8-1.05 M sucrose. This distribution is compatible with a localization in a soluble compartment and to synaptosomes. The SPase activity was highest in the P1 (nuclear) and P2 (mitochondrial) fractions, suggesting it is mainly membrane bound during these conditions. In a step gradient centrifugation (0.32/0.8/1.4 M sucrose), most of the activity was found in synaptosomal (0.8 M) fractions. After partial osmotic lysis of the synaptosomal fraction and centrifugation on a continuous density gradient (0.1-1.4 M sucrose), peaks of SPase activity were found around 0.55 M, possibly representing material associated with neuronal or plasma membrane vesicles, and at 1.0-1.25 M, representing synaptosomes, and/or mitochondria.