The two globular head portions, each bearing an active site, contain an uncleaved heavy chain when isolated by chymotrypsin from intact myosin. By specific labeling with radioactive N-ethylmaleimide the essential thiol 1 and thiol 2 groups were found to reside in this heavy chain. In intact myosin nonessential thiol 3 groups become the most reactive during ATP hydrolysis above 15 degrees C. These thiol 3 groups are located in a portion of the myosin heavy chain which appears as a fragment with an apparent molecular weight of 11 000 during proteolysis. The facts that this fragment is produced in an almost 1: 1 molar ratio with the head heavy chain and that it bears unblocked N-terminal amino groups whereas the heavy chain does not and is not contained in the rod portion of the myosin molecule indicate that it may orginate from the heavy chains in the neck region where the heads are joined to the rod. Since this fragment is removed by ion-exchange chromatography, it is not part of the functioning head and hence not involved in the active site. As its nonessential thiol 3 groups are rendered the most reactive of all thiol groups in the enzyme-product complex M**ADP.Pi, the hydrolytic step induces an allosteric conformational change in the neck region of intact myosin.

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Electrophysiological experiments were done to investigate the effect o p-nitrophenyl diazonium fluoroborate (p-NPD) on motor endplates of the frog's m. cutaneus pectoris. The compound has no direct depolarizing effect on the postsynaptic membrane and stabilizes it irreversibly when added to the bath. Longtime iontophoretical applications of p-NPD produce a biphasic effect: initially a potentiation of the depolarizations due to acetylcholine (ACh) (both iontophoretically applied and presynaptically liberated), and subsequently an inhibition of the response to ACh. When the acetylcholinesterase (AChE) is inactivated previously, only the inhibiting effect of the compound is demonstrable. The association constant of p-NPD to purified AChE and to membrane fragments of electroplax was determined by biochemical methods. The compound's affinity to the AChE was found to be about 20 times greater than to the acetylcholine receptor (AChR). Iontophoretical application of p-NPD to cholinergic neurons in the hippocampal cortex of the cat also produced the characteristic biphasic effect on ACh-induced activity of these investigated neurons. The results suggest that the biphasic effect depends on the capacity of p-NPD to combine with both the AChE and the AChR. The AChE is first inhibited with low concentrations thereby potentiating the ACh response. At higher concentrations the AChR's are progressively inhibited too, thereby diminishing the excitability of the postsynaptic membrane up to a complete block.