Changes in the mono-and divalentcation-stimulated ATPase activities of myosin progressively labeled with7V-ethyl-[2,3-14 C 2 ]-maleimide were used to classify the readily reacting thiol groups into 3 types. The results show that one thiol-1 and one thiol-2 group are associated with each of the 2 active sites of myosin. Concentrations of KC1 higher than 0.4M and/or temperatures above 10 °C lead to exposure of a variable number of thiol groups of a third class not affecting the enzymic properties. Although modification of thiol groups itself results in changes in structure and function of the protein, the patterns of incorporation of./V-ethyl-[ 14 C 2 ]-maleimide under various conditions of temperature, ionic strength and ligands bound to the protein revealed 9 different conformations of intact myosin. These were distinguished on the basis of the relative reactivity of the 3 different classes of thiol groups. The sequence of blockage of thiol groups reveals that cooperativity between the 2 active sites is induced by binding of a magnesium nucleotide complex to the protein. In the conformation of the long-lived myosin-product intermediate occuring during hydrolysis of Mg-ATP at 25 °C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes. Konformationsunterschiede in Myosin, IV. Radioaktive Markierung spezifischer SH-Gruppen unter dem Einfluß von Liganden-BindungenZusammenfassung: Einbau von7V-Äthyl-[2,3-14 C 2 ]maleinimid und die dadurch bewirkten Veränderungen der mit einwertigen und zweiwertigen Kationen stimulierten ATPase-Aktivitäten erlauben, die leicht reagierenden SH-Gruppen von Myosin in 3 Klassen einzuteilen. Jedem der beiden aktiven Zentren des Myosins kann eine SH-1 und eine SH-2 Gruppe zugeordnet werden. Hohe Salzkonzentrationen (> 0.4M KC1) und/oder Temperaturen über 10 °C bewirken, daß SH-Gruppen der 3. Klasse, die die Enzymeigenschaften des Myosins nicht beeinflussen, leicht mit dem Alkylierungsmittel reagieren. Auch wenn allein durch eine Modifizierung der SH-Gruppen schon Struktur und Funktion des Eiweißes beeinflußt werden, erlaubt das Einbaumuster des radioaktiven JV-Äthylmaleinimids je nach den Bedingungen (lonenstärke, Temperatur, ans Eiweiß gebundene Liganden) neun verschiedene Konformationen des nativen Myosins zu unterscheiden, und zwar aufgrund der relativen Aktivität der drei Arten von SH-Gruppen. Wenn ein Mg-Nucleotid-Kom-

The conformations of the transitory intermediates of the myosin ATPase occurring during the hydrolytic cycle, enzyme without ligand, enzyme-substrate complex and two different forms of enzyme-product complex, have been characterized in terms of numbers and classes of reactive thiol groups based on incorporation of radioactively labeled alkylation reagent. The techniques employed allowed this to be done under steady-state conditions in the presence of high ligand concentrations on intact myosin from rabbit fast skeletal muscles at low ionic strength where the protein is in the gel state as it is in muscle. The binding of a divalent cation (Mg2+ or Ca2+) nucleotide complex exposes thiol-1 as well as thiol-2 groups. The long-lived ATPase intermediate occurring at temperatures above 10 "C adopts the same conformation with Mg2+ and Ca" ions. This intermediate does not protect the thiol-1 and thiol-2 groups but exposes a number of thiol-3 groups which seem to be located distant from the active site. The conformation of the intermediate prevailing in the presence of ATP changes with lowering temperature below 10 "C and is identical with that found in the presence of ADP at 0 "C indicating a change in the rate-limiting step of the hydrolytic cycle. In the absence of divalent cations no such temperature-dependent change in conformation was observed. Evaluation of the activation entropies shows that the structure of the long-lived intermediate occurring above 10 "C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20 "C. In the case of the monovalent-cationstimulated ATPase a constant activation energy of around 70 kJ/mol, typical of many enzyme reactions, was found over the entire temperature range from 0 -35 "C.

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg · ADP and uncomplexed ADP to the active site of myosin subfragment‐1. Additionally, the binding parameters for Mg2+ in the ternary complex protein · Mg · ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol‐1 and thiol‐2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg · ADP just one mole of ligand binds/mole subfragment‐1. The affinity of Mg · ADP at low ionic strength is 2.1 × 106 M‐1 and only five‐times lower in the absence of Mg2+ has a low affinity of 4.1 × 104 M‐1. At high ionic strength the uncomplexed ADP binds with a 43‐times‐lower affinity than Mg · ADP, whose affinity is 6.9 × 105 M−1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 × 105 M−1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.

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The question of a role for water in biochemical and cellular events is ignored by most workers (apart from its obvious role in hydrolysis reactions, which is not under discussion here). But much recent research has pointed to the importance of physical, as well as biochemical, processes of the cell, which focus attention on such straightforward elementary questions as position and relationship in space of cell components. In this communication these questions are examined in terms of a new model of water structure. A radically new feature of this model is that water clusters have long-term rather than flickering existence and are as large as the macromolecular components of the cell. These properties allow the clusters and other components to pack together spatially so giving rise to integrated, large-scale, subcellular structures.