Faculty Bibliography

Proctolin is a potent selective inhibitor of aminoenkephalinase. The specificity of its inhibition of various aminopeptidases is similar to that of puromycin; it inhibits aminoenkephalinase, but not leucine aminopeptidase or aminopeptidase M. Enkephalin breakdown by synaptic plasma membrane, but not by brain slices, is sensitive to proctolin. The inhibition by proctolin is partially caused by its resistance to enzymatic breakdown. The inhibition is of mixed type and is concentration dependent, and the two amino acids at the N-terminal are important for its action. The minimal structure for inhibition is a dipeptide with a basic amino acid at the N-terminal and a basic or an aromatic amino acid at the C-terminal

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 2 X 8 mg/kg retro-orbital) to BALB/cBy mice reduced [3H]mazindol binding to striatal membranes by 50%. Reactive oxygen derivatives have been suggested to be involved in MPTP neurotoxicity; therefore we examined the effects of ascorbic acid (an antioxidant). Ascorbic acid (100 mg/kg) given 20 min prior to MPTP administration appreciably prevented the reduction of [3H]mazindol binding. The involvement of oxidative processes in the mechanism of MPTP neurotoxicity may suggest a relationship to the etiology of Parkinson's disease, and the possible benefit of treatment with ascorbic acid

The action of three previously isolated electrophoretically homogeneous brain proteinases--cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and high-molecular-weight aspartic proteinase (Mr = 90K; EC 3.4.23.-)--on human angiotensins I and II has been investigated. The products of enzymatic hydrolysis have been identified by thin-layer chromatography on Silufol plates using authentic standards and by N-terminal amino acid residue analysis using a dansyl chloride method. Cathepsin D and high-molecular-weight aspartic proteinase did not split angiotensin I or angiotensin II. Cathepsin B hydrolyzed angiotensin I via a dipeptidyl carboxypeptidase mechanism removing His-Leu to form angiotensin II, and it degraded angiotensin II as an endopeptidase at the Val3-Tyr4 bond. Cathepsin B did not split off His-Leu from Z-Phe-His-Leu. Brain cathepsin B may have a role in the generation and degradation of angiotensin II in physiological conditions

Spontaneous locomotor activity of mice was stimulated by IP administration of cocaine and its closely related phenyltropane analogs. In contrast, locomotion was inhibited by IP administration of cocaine congeners such as norcocaine, (+)-pseudococaine, and tropacocaine, and of isomers of phenyltropane analogs. Also inhibitory were the local anesthetics procaine, tetracaine, benzocaine, lidocaine, and prilocaine. The locomotor inhibition induced by IP norcocaine or tetracaine could be reversed by subsequent treatment with cocaine. Both cocaine and norcocaine were centrally stimulatory when injected intracerebroventricularly. The rank order of potencies of cocaine congeners and local anesthetics in depressing locomotion was similar to that of their potencies in interacting with sodium channels. From these results we infer that the locomotor depression induced by systemic administration of cocaine congeners results from a local anesthetic action involving inhibition of the ion conductance of sodium channels

There was a highly significant correlation between IC50 values of various drugs in inhibiting the Na+-independent [3H]cocaine binding in the mouse striatum and their values in inhibiting the synaptosomal uptake of [3H]serotonin. In contrast, there was no correlation between the inhibition of binding in the absence of Na+ and the inhibition of [3H]dopamine uptake. Lesioning of serotonergic nerve terminals with 5,7-dihydroxytryptamine reduced the Na+-independent [3H]cocaine binding, without affecting the Na+-dependent binding. These results indicate that the bulk of the Na+-independent [3H]cocaine binding in the mouse striatum is associated with serotonergic nerve terminals

In a continuing study of nicotine binding sites, we determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both [3H]nicotine and [3H]acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of [3H]nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-[3H]nicotine and that of [3H]acetylcholine

Aluminum injection in rabbits leads to neurofibrillary changes which are at light microscopic level similar to those found in Alzheimer's disease. We used this animal model to see whether changes in proteolytic activity occur that may affect protein degradation in the altered neurofibrillary structure. Rabbits were injected via the cisterna magna with aluminum chloride, and after ten days tissue was excised from the spinal cord, hippocampus, occipital lobe, and cerebellum. Sections from the hippocampus and spinal cord were examined for neurofibrillary changes; enzyme activity was measured in all four areas. The enzymes studied were cathepsins A, B, and D, and the angiotensin-converting enzyme. No significant differences could be established in enzymatic activity in aluminum-injected animals compared to controls. However, a significant decrease in Triton-soluble proteins was observed in the treated animals, which correlated with changes in neurofibrillary structure. This decrease was most noticeable in the spinal cord (from 16.6 to 12.5 mg/g)

In a continuing study of control processes of cerebral protein catabolism we compared the activity of cathepsin D from three sources (rat brain, bovine brain, and bovine spleen) on purified CNS proteins (tubulin, actin, calmodulin, S-100 and glial fibrillary acidic protein). The pH optimum was 5 for hydrolysis with tubulin as substrate for all three enzyme preparations, and it was pH 4 with the other substrates. The pH dependence curve was somewhat variable, with S-100 breakdown relatively more active at an acidic pH range. The formation of initial breakdown products and the further catabolism of the breakdown products was dependent on pH; hence the pattern of peptides formed from glial fibrillary acidic protein was different in incubations at different pH's. The relative activity of the enzyme preparations differed, depending on the substrate: with tubulin and S-100 as substrates, rat brain cathepsin D was the most active and the bovine spleen enzyme was the least active. With calmodulin and glial fibrillary acidic protein as substrates, rat brain and spleen cathepsin D activities were similar, and bovine brain cathepsin D showed the lowest activity. Actin breakdown fell between these two patterns. The rates of breakdown of the substrates were different; expressed as ?g of substrate split per unit enzyme per h, with rat brain cathepsin D activity was 8-9 with calmodulin and S-100, 4 with glial fibrillary acidic protein, 1.8 with actin, and 0.9 with tubulin. The results show that there are differences in the properties of a protease like cathepsin D, depending on its source; furthermore, the rate of breakdown and the characteristics of breakdown are also dependent on the substrate. We recently measured the breakdown of brain tubulin by cerebral cathepsin D in a continuing study of the mechanisms and controls of cerebral protein catabolism (Bracco et al., 1982a). We found that tubulin breakdown is heterogeneous, that membrane-bound tubulin is resistant to cathepsin D but susceptible to thrombin (Bracco et al., 1982b), and that cytoplasmic tubulin was in at least two pools, one with a higher, another with a lower, rate of breakdown. The pH optimum of tubulin breakdown by cerebral cathepsin D differed significantly from the pH optimum of hemoglobin breakdown by the same enzyme. These findings showed that the properties of breakdown by a cerebral protease depend on the substrate. To further examine this dependence of properties of breakdown on the substrate, we now report measurements of pH dependence of breakdown of several purified proteins (tubulin, actin, calmodulin, S-100, glial fibrillary acidic protein [GFA]) from brain by cathepsin D preparations from three sources, rat brain, bovine brain, and bovine spleen. We also compare the rate of breakdown of the various proteins with the rate of hemoglobin breakdown