Biochemical and Pharmacological Mechanisms Underlying by P.B. Bradley and R.W. Brimblecombe (Eds.)

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By P.B. Bradley and R.W. Brimblecombe (Eds.)

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The second question depends on whether AChE is present on the inside or the outside of the nerve terminals. Most people working with subcellular fractionation have obtained evidence to show that the main part of the AChE must be on the outside (Fonnum, Rodriguez de Lores Arnaiz and Marchbanks, independent studies, unpublished observations). This is mainly based on two kinds of study. , the enzyme, unlike ChAc, is not protected behind the membrane. (2) If the synaptosome is loaded with labelled ACh, the latter is not hydrolysed; but if the synaptosomes are broken, ACh diffuses out and is immediately hydrolysed by the AChE.

It was then centrifuged at 9,500 rpm in a Servall centrifuge S S 34 for 20 min. The supernatant was placed on a gradient and spun for 2 h at 25,000 rpm in an SW 40 rotor. The vesicle-containing fraction was used for further experiments with phospholipase A. Fractions mainly containing membrane fragments were saved and later analysed for intrinsic phospholipase A activity. Effect of phospholipase A on cholinergic vesicles Suspensions of isolated vesicles were tested for ACh activity before and after acid boiling, the latter method releasing the vesicle-bound ACh.

6): M’, outer two-thirds of stratummoleculare (theinner one-third, containing terminals of commissural fibres, was discarded); G, stratum granulare plus 20-30 pm of each of the adjacent AChE-rich zones; H, hilus fasciae dentatae. For assay of ChAc (Fig. 3) and AChE, M was subdivided into Mo and Mm and the underlying parts of area dentata were subdivided into 4 zones with limits differing from the ones in this figure, but with widths as indicated in Fig. 3 and with the center of zone G coinciding in the two figures.

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