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Neurexins are synaptic adhesion proteins encoded by 3 genes (NRXN1, NRXN2, and NRXN3) each transcribed from 2 promoters to yield longer (alpha) and shorter (beta) forms. The primary gene transcripts undergo extensive alternative splicing leading to products that may differ in synaptic coupling properties. Here we show that depolarization of neurons modulates splicing of NRXN2alpha, particularly at splice sites 1 and 3. Furthermore, we demonstrate that exclusion of exon 11 at splice site 3 is calcium-dependent. These data indicate neuronal activity-dependent splicing of NRXN2alpha. This dynamic process may be important for maintenance of mature neuronal circuits.

N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was used to identify melatonin binding proteins in synaptosomes from Syrian hamster brain. Incubation of the synaptosomes with BIM resulted in a concentration dependent, irreversible inhibition of 2-125I-iodomelatonin binding. The radioactive form of BIM, N-Bromoacetyl-2-125I-iodo-5-methoxytryptamine (125I-BIM), became covalently attached to three proteins in the synaptosomes, in a concentration dependent manner. These proteins had apparent molecular weight values of 92, 55 and 45 kilodaltons. The incorporation of 125I-BIM into all three proteins was inhibited by BIM greater than 2-iodomelatonin greater than melatonin whereas the melatonin antagonist N-(1,4 dinitrophenyl)- 5-methoxytryptamine (ML-23) selectively inhibited the labeling of the 45 kDa protein. These results indicate that the 92, 55 and 45 KDa polypeptides are melatonin binding proteins.

The replacement of zinc by a series of other metal ions (Co, Cd, Mn) resulted in enzymatically active carboxypeptidases both as peptidases and esterases. The effect of the metal replacement on the kinetic parameters varies for the various substrates. Cd-CPB, previously known for its lack of peptidase activity, shows enhenced activity as long as the substrate interact with four subsites (including the N-terminal blocking group). Kinetic measurements and chemical modification revealed differences in the nature of the residues necessary for the proper alignment along the active site between the Zn and Cd carboxypeptidases. A simple explanation of the data observed is based on the assumption that replacement of the metal affects the microenvironment of the active site.

Modification of porcine carboxypeptidase B with [14C]-bromoacetyl-p-aminobenzylsuccinic acid results in a marked decrease of the activity toward both peptide and ester substrates. The inactivation correlates with the incorporation of 1 equivalent of inhibitor molecule per carboxypeptidase molecule. Evidence is presented that inactivation is due to alkylation of a single, presumably methionyl residue in the active center of the enzyme.

Microdissected slices of rat hypothalamus were incubated with 3 H -dopamine and then subjected to two successive sets of electrical field stimulation in a superfusion chamber. Neurotransmitter release was found to be calcium dependent and the amount of release was determined by scintillation counting of the effluent buffer. The release obtained following the first train of stimuli served as an internal reference. The samples were exposed to drugs during the interval between the two sets of stimuli. Using this technique, as well as K + -evoked depolarization, we were able to show that subnanomolar concentrations of melatonin, the hormone secreted from the pineal gland, inhibits dopamine release from hypothalamic slices. The possibility that melatonin modulates neurotransmission in the brain is therefore indicated.

Native and zinc reconstituted carboxypeptidase B were nitrated with tetranitromethane. The inactivation of the reconstituted enzyme was faster than that of the native enzyme and was accompanied by the formation of a considerable amount of enzyme dimers. The inactivation and dimerization reflected changes in the reactivity of active site tyrosine residue(s), thus indicating microenvironmental changes which occur during metal substitution. The change in tyrosine reactivity could be correlated with the residence of the enzyme in the metal-free state.

The kinetics of porcine carboxypeptidase B catalyzed hydrolysis of hippurylarginine, hippurylargininic acid, Z·Ala3 and hippurylphenyllactic acid have been examined in order to elucidate the substrate specificity of the enzyme. Activity toward both basic and non-basic (i.e., hydrophobic type) substrates has been confirmed. However, it would appear that these substrates are not all bound in the same manner to the same site. The pH dependencies of the kinetic parameters revealed characteristic differences among the two types of substrates. Furthermore, inhibition studies with L-argininic acid, ɛ-aminocaproic acid, Z- D -Ala- L -Arg and β-phenylpropionic acid support this postulate. These studies provide insite regarding possible pathways for the evolution of the carboxypeptidases.