Spring Chemistry 501 Seminar
UT HOST: Dr. Chris Baker
Speaker: Dr. Stephen Weber
Professor of Chemistry, Professor of Clinicla Translational Science
University of Pittsburgh
Title: "Ectoenzymes: The measurement of their activity in tissue cultures and in vivo, and implications of aminopeptidase activity in selective neuronal damage in the hippocampus in a stroke model"
It is true in every tissue, but especially in the brain, that extracellular chemical events are critical to maintaining homeostasis and for signaling. The fates of signaling molecules like small molecule neurotransmitters in the extracellular space are fairly well understood. However, for the ~ 100-150 neuroactive peptides there is much less understanding of their fate in the extracellular space. Certainly is it known that they are hydrolyzed – but how rapidly? Do rates differ among brain regions or over time? The peptidases thought to be responsible for peptide hydrolysis in the extracellular space are ectoenzymes (ectopeptidases) – integral membrane proteins with the active site facing the extracellular space. Because it is the activity of these enzymes that is important, it is necessary to have a method for their determination that has good spatial resolution.
For a method to be useful in this pursuit, it must be able to (1) perfuse tissue and collect the perfusate for quantitative analysis of the solutes introduced and reaction products produced, (2) control the average residence time of the active solutes, and (3) have the appropriate spatial resolution for the process of interest. We developed a perfusion technique based on electroosmosis (EO), called EO push−pull perfusion (EOPPP), that supplies peptide substrate (and an internal standard). We collect and measure product and unreacted substrate (capillary liquid chromatography). It turns out to be somewhat complicated to infer activity, in this case Vmax, from the data, but with the use of simulations we are able to do so.
It has been known for over a century that the hippocampus, the center for learning and memory in the brain, is selectively vulnerable to ischemic damage, with the CA1 being more vulnerable than the CA3. It is also known that leucine enkephalin, or YGGFL, is neuroprotective. We hypothesized that the extracellular hydrolysis of YGGFL is greater in the CA1 than the CA3, which would lead to the observed difference in susceptibility to ischemia. In rat organotypic hippocampal slice cultures, we estimated the Michaelis constant, Km, and Vmax. in the CA1 and CA3 regions. We found nearly three-fold higher ecto-aminopeptidase activity in the CA1 than the CA3. The aminopeptidase inhibitor bestatin significantly reduced hydrolysis of YGGFL in both regions by increasing apparent Km. Based on propidium iodide cell death measurements 24 h after oxygen−glucose deprivation, we demonstrate that inhibition of aminopeptidase activity using bestatin selectively protected CA1 against delayed cell death due to oxygen−glucose deprivation and that this neuroprotection occurs through enkephalin-dependent pathways.
Steve received his BA (biology and chemistry) at Case Western Reserve University in 1970 and then spent four years in the Navy, mostly in clinical chemistry and drug analysis labs. He received his PhD (chemistry) from McGill University in 1979 for the development of electrochemical immunoassay, and started at the University of Pittsburgh the same year. His interests are in electrochemistry and separations, especially how aspects of mass transport phenomena and non-covalent interactions can control chemical behavior in them. Following a sabbatical in Sweden he became interested in so-called reperfusion injury that occurs in stroke. Fifty-nine PhD or MS graduates have come from his lab and 31 postdoctoral fellows and visiting scientists/professors have spent time there.
Thursday, February 7 at 3:45pm to 5:00pm
Buehler Hall, 555
1420 Circle Drive, Knoxville, TN 37996