Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age‐related changes in calpain specificity and substrate susceptibility were measured. Several age‐related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).
Cocaine and its metabolites were measured in plasma and brain from mice injected i.p. with cocaine and monitored for spontaneous locomotor behavior. Cocaine concentrations in the brain reached peak values within 5 min after administration of cocaine. At all time points between 5 and 60 min the concentrations of cocaine in the brain were 7-fold higher, on the average, than those in plasma. The opposite was true for the concentrations of benzoylecgonine; brain to plasma ratios of benzoylecgonine were approximately 0.1 from 5 to 30 min after i.p. cocaine injection. After i.p. injection of either 10 or 25 mg/kg of cocaine, cocaine disappeared from plasma and brain with a half-life of 16 min and benzoylecgonine disappeared from plasma with a half-life of 62 min. There was good correspondence between locomotor stimulation and concentration of cocaine in the brain measured at 12, 22 and 32 min after i.p. administration of 25 mg/kg of cocaine. Among individual animals there was a significant correlation between their locomotor stimulation and their brain cocaine concentration, indicating that differences in cocaine levels in the brain between animals contribute to their different behavioral response; however, the correlation analysis also indicated the role of other factors determining the locomotor response to cocaine.
