Substance P Endopeptidase Purification and Characterizataion of Enzyme Activity and Evaluation of its Function during Stressful Condition

Sammanfattning: The purification and biochemical characterization of the substance P (SP) hydrolyzing enzyme, substance P endopeptidase (SPE), have been carried out; with subsequent orientation in neurobiological fundamental processes involved in opioid dependence, withdrawal, and heat-stress.SPE was purified from rat spinal cord, human spinal cord and cerebrospinal fluid (CSF), rat ventral tegemental area (VTA), and rat hippocampus. The enzyme activity was found to release the biologically active fragments SP(1-7) and SP(1-8) as major products. The purified enzymes were characterized with regard to their biochemical and kinetic properties. The typical SPE is neither inhibited by phosphoramidon nor captopril nor phenylmethanesulfonylflourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specificity, inhibition-profile, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purification of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTA™-purifier system. Studies revealed a crucial role of SPE in a series of clinically important neuropathological conditions, such as opioid tolerance, and withdrawal (SPE, increased); and heat-stress (SPE, increased). These findings emerged from assessment of enzyme activity in hypothalamus, nucleus accumbens (NAc) periaqueductal gray (PAG), pituitary, striatum, substantia nigra (SN), VTA, spinal cord. Viewing the role of SPE in morphine tolerance, it was possible to note regional differences with a decrease in PAG, and striatum, whereas an increase was seen in SN, and VTA. After heat-stress treatment, SPE was raised in several regions (cerebral cortex, hippocampus, diencephalon, cerebellum, spinal cord), and the most precise observation of this was located to the hippocampus structure.