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

Karlsson, K., 2004, Substance P Endopeptidase; Purifi cation and Characterization of Enzyme Activity and Evaluation of its Function during Stressful Condition, Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1331, 56 pp. Uppsala. ISBN 91-554-5908-0. The purifi cation 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 purifi ed from rat spinal cord, human spinal cord and cerebrospinal fl uid (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 purifi ed enzymes were characterized with regard to their biochemical and kinetic properties. The typical SPE is neither inhibited by phosphoramidon nor captopril nor phenylmethanesulfonylfl ourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specifi city, inhibition-profi le, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purifi cation of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTATMpurifi er 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 fi ndings 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. Krister Karlsson, Department of Neuroscience, Box 587, Uppsala University, SE-751 24, Uppsala, Sweden © Krister Karlsson 2004 ISSN 0282-7476 ISBN 91-554-5908-0 urn:nbn:se:uu:diva-4130 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4130) Printed in Sweden by University Printers, Uppsala 2004 This thesis is dedicated to: Olle, Solveig, Katarina, Maria, Elsa† and Seok-Jo, Kyung-Ka†, Nam-Sook, Seog-Hwon