Methods for studying carboxypeptidase E

This chapter discusses the methods for studying carboxypeptidase E (CPE). The most widely utilized CPE assays make use of a change in solubility incurred when substrate is converted into product. The assays differ in their reporter groups; one uses the dansyl fluorescent group, while the other uses a radioactive label. Both assays are rapid and sensitive, but are not specific for CPE. Thus, additional controls must be performed to ensure that the measured activity is because of CPE. These controls include using selective inhibitors and activators at several pH values. In addition to measurements of CPE enzymatic activity, it is often important to quantitate levels of CPE mRNA and protein. Standard methods, such as Northern blots, in situ hybridization, and Western blots, have been used to detect CPE mRNA and protein. Autoradiography with [³H]guandinioethylmercaptosuccinic acid, a selective inhibitor of CPE, has been used to detect the membrane-bound form of CPE in brain and other tissues. In addition to these standard techniques, the rate of CPE biosynthesis has been measured by following the incorporation of radiolabeled amino acids into CPE, which is subsequently isolated by affinity chromatography.