Acetylcholinesterase (AChE) is concentrated at cholinergic synapses, where it is a major factor in controlling the duration of transmitter action. The concentration and localization of AChE within the synaptic cleft are in keeping with the functional requirements of the particular type of synapse. The densities of synaptic AChE at various neuromuscular junctions (NMJs) had been evaluated by quantitative EM-autoradiography using radiolabeled probes. Yet, fundamental issues concerning the precise distribution and location of the enzyme in the cleft remained open: whether and to what extent synaptic AChE is associated with pre- or postsynaptic membranes, or with synaptic basal lamina (BL), and whether it occurs only in the primary cleft (PC) or also in postjunctional folds (PJFs). Nanogold-conjugates of fasciculin, an anticholinesterase polypeptide toxin, were prepared and used to label AChE at NMJs of mouse and frog muscles. Selective intense labeling was obtained at the NMJs, with gold-labeled AChE sites distributed over the BL in the PC and the PJFs. Quantitative analysis demonstrated that AChE sites are almost exclusively located on the BL rather than on pre- or postsynaptic membranes and are distributed in the PC and down the PJFs, with a defined pattern. This localization pattern of AChE is suggested to ensure full hydrolysis of acetylcholine (ACh) bouncing off receptors, thus eliminating its unnecessary detrimental reattachment.

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Buforin is a cationic antimicrobial peptide (AMP) from the stomach of toads. Buforin II is a derivative of this naturally occurring peptide. Buforin IIB is a synthetic analog of buforin II containing a model α-helical sequence (3xRLLR) at the C-terminus. To further increase the antimicrobial activity and decrease toxicity to eukaryotic cells, new derivatives (buforin III analogs) were designed by substituting amino acids in the buforin IIB sequence. In this work, the antimicrobial activity and the actin-and DNA-binding characteristics of buforin IIIB (RVVRQWPIGRVVRRVVRRVVR) and the newly synthetized buforin IIIE (RLLLRQWPIGRLLRRLLRRLLR) were studied. The antimicrobial activity of buforin IIIB (measured against E. coli and E. faecalis) was significantly greater than that of buforin IIIE, while both peptides were nontoxic to macrophages at the minimal concentrations required to inhibit microbial growth. Actin, which inhibited the antimicrobial activity of the two buforin III analogs, was bundled by both peptides; however, less buforin IIIE than buforin IIIB was needed for bundling. Higher levels of NaCl were needed to unbundle actin bundled by buforin IIIE than actin bundled by buforin IIIB, which indicates that buforin IIIE binds more strongly to actin than buforin IIIB. Actin bundled by either peptide was dissociated with the same concentration of DNA; however, buforin IIIE bound more strongly to DNA than buforin IIIB. These results contribute to the understanding of the antimicrobial mechanism of cationic AMPs in general and histone-derived peptides in particular.

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