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The present study explored the mechanisms underlying the dopamine releasing effect of phenylbiguanide, a compound commonly used as a 5-HT3 receptor agonist. Phenylbiguanide, and also serotonin and 2-methyl-serotonin, enhanced the outflow of radioactivity from superfused rat striatal slices preloaded with [3H]dopamine. The presence of the dopamine uptake blocker nomifensin prevented the increase in outflow. The effect of phenylbiguanide was not antagonized by 5-HT3 receptor antagonists, did not require the presence of Ca2+ in the superfusion buffer, and also occurred in reserpinized preparations with depleted dopamine stores. Phenylbiguanide caused a greater shift in the distribution of superfusate radioactivity from DOPAC to dopamine than did nomifensin. All these results are in agreement with an exchange mechanism by which phenylbiguanide promotes the efflux of dopamine by operation of the uptake carrier in the reversed direction. In consonance, phenylbiguanide, and also serotonin and 2-methyl-serotonin, inhibited the binding of [3H]CFT to dopamine uptake sites, although the rank order for promoting outflow, serotonin greater than phenylbiguanide greater than 2-methyl-serotonin, differed from that for inhibiting [3H]CFT binding to dopamine uptake sites, 2-methylserotonin approximately serotonin greater than phenylbiguanide. The present results raised the possibility that phenylbiguanide has an additional activity in releasing vesicular dopamine into the cytoplasmic pool.

Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity. The greatest activity was obtained when glutamate or aspartate was incubated with α‐ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids. Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α‐ketoglutarate also did so with pyruvate, although at a lower rate. When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively. Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates. α‐Ketoisocaproate and α‐ketoisovalerate accepted amino groups primarily from the branched‐chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable. A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α‐ketoglutarate, these activities increasing about 10‐fold from birth to adulthood; during this time activities with most other amino acids increased two‐ to threefold. Amino transfers from the branched‐chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood. Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched‐chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

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Myelin basic protein, purified from bovine spinal cord, is cleaved by a purified bovine brain acid proteinase to yield three peptide fragments which were purified by gel filtration procedures combined with preparative gel electrophoresis. The purity of the fragments was established by end group analysis, amino acid analysis, polyacrylamide disc gel electrophoresis, and by their encephalitogenic activity. Chemical and biological characterization shows that one'peptide, (peptide 11), originates from the N-terminal end of the basic protein and contains residues 1-43; a second peptide, (peptide I), contains residues 44-89 and a third peptide (peptide III), originates from the C-terminal region with an N-terminal phenylalanine, residue 90, through arginine, residue 170, of the basic protein. The combined amino acid composition of the three fragments account for the amino acid content of the basic protein. The structure of the three peptides demonstrates that the purified brain acid proteinase selectively cleaves the two phenylalanine-phenylalanine linkages formed between residues 43-44 and 89-90 of the basic protein molecule. This study shows that cathepsin D could be the endogenous enzyme responsible for the breakdown of basic protein to form smaller encephalitogenic fragments, some of which are known to be present in crude tissue extracts.It was established in previous studies that most myelin protein components are subject to turnover [l]. Basic protein of myelin is of particular interest because it is known to induce an autoimmune disease associated with demyelination, experimental allergic encephalomyelitis. Previously, Einstein et af. [2] showed that basic protein can be cleaved by acid proteinase to form smaller encephalitogenic fragments, but the composition of these, and the nature of the susceptible bonds cleaved, have never been established. Indeed, early studies on the isolation and structure of basic protein were accompanied by autolysis during extraction, leading to the fortuitous isolation of polypeptide fragments, some of which were encephalitogenic in a particular species [3-51. Therefore, it is of some interest to establish the exact mechanism of basic protein breakdown using a known intracellular enzyme, since this may have some bearing on demyelinating processes in experimental disease and, in addition, provide some insight into the general mechanism of protein turnover in brain. A limited and specific degradation of basic protein could lead to the production of smaller (diffusible) peptides capable of interaction at the immunological sites related to autoimmune phenomena. The present work was designed to determine the nature of the peptide bonds split by the action of brain cathepsin D, as well as the structure and biological activity of the resultant peptides based on the known structure of bovine basic protein [6]. In other tissues, Coffey and de Duve [7] showed that catheptic activity of lysosomes appear to play a rate-limiting role in the pathways of protein breakdown. Thus, studies on the...

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