1. Binding of MG-ADP to both heart and fast skeletal myosin was found with 3 methods to proceed in 2 steps. One mole of MG-ADP binds with high affinity (K approximately equal to 10(6) M-1) and subsequently a second with lower affinity (K approximately equal to 10(2)-10(4) M-1) per myosin. Only one mole of MG-ADP was found to bind with the high affinity to isolated myosin heads. This implies that binding of MG-ADP to intact myosin exhibits negative cooperativity. 2. When a nucleotide is bound, the 2 heads of a single myosin molecule adopt different conformations since on each head a different type of essential thiol group was found to be the most reactive towards N-ethylmaleimide. In the presence of MG-pyrophosphate a thiol-1 is the most reactive essential group in both heads. Therefore, the nucleoside moiety seems to be required for this latter type of head-head interaction.

Adenosine Diphosphate↗

PubMedDate not supplied

Regenerating neurons. Changes in protein phosphorylation.

We have been studying the phosphorylation of proteins of both normal and regenerating superior cervical ganglia of the rat. Here we report the incorporation of radioactive phosphate into proteins of ganglia homogenates incubated with 32P-labeled ATP under various conditions at day 3 after postganglionic axotomy. The proteins were analyzed by two-dimensional electrophoresis followed by autoradiography. Incubation in the presence of Ca2+ or Ca2+ plus cyclic AMP produced only about 20 spots corresponding to distinctly labeled proteins. This number was reduced to about five under EGTA plus cyclic AMP conditions, whereas the presence of EGTA alone suppressed the phosphorylation reaction almost totally. All these proteins fell within the narrow pI range of 4-6, whereby no qualitative differences between regenerating and control cases were observed. However, the growth-associated protein, variously designated GAP-43, B-50, F-1, and pp-46, had enhanced levels of phosphate incorporation in regenerating ganglia compared to controls. Injury also caused consistently higher levels of phosphorylation of proteins running in the position of alpha- and beta-tubulin. Since these three proteins are major constituents of regenerating axons, these results suggest that the changes in their phosphorylation induced by injury may be involved in the regulation of their transport.

Adenosine Triphosphate↗

PubMedDate not supplied

[The role of water in cell function].

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Binding Sites↗