John G. Watterson

Radioactive labelling and location of specific thiol groups in myosin from fast, slow and cardiac muscles

1. Based on incorporation of radioactively labeled N-ethylmaleimide, the readily reactive thiol groups of isolated myosin (EC 3.6.1.3) from fast, slow and cardiac muscles could be classified into 3 types. All 3 myosins contain 2 thiol-1, 2 thiol-2 and a variable number of thiol-3 groups per molecule. Both thiol-1 and thiol-2 groups which are essential for functioning of the K+-stimulated ATPase, are located in the heavy chains in all 3 myosin types. 2. The variation in the incorporation pattern of N-ethylmaleimide over the 3 thiol group classes under steady-state conditions of Mg(2+) - ATP hydrolysis allowed different conformations of some reaction intermediates to be characterized. In all 3 types of myosin the hydrolytic cycle of Mg(2+) - ATP was found to be controlled by the same step at 25 degrees C. In all three cases, this rate-limiting step is changed in the same way by lowereing temperature. 3. Using the chemically determined molecular weights for myosin light chains, their stoichiometry was found on the basis of sodium dodecyl sulfate electrophoresis to be 1.2 : 2.1 : 0.8 for light chain-1: light chain-2:light chain-3 per molecule of fast myosin, 2.0 : 1.9 for light chain-1:light chain-2 per molecule of slow myosin and 1.9 : 1.9 for light chain-1:light chain-2 per molecule of cardiac myosin. This qualitative difference in light subunit composition between the fast and the two types of slow myosin is not reflected in the small variations of the characteristics exhibited by the isolated myosins, but rather seems to be connected with their respective myofibrillar ATPase activities.

ISOLATION OF CYANOGEN BROMIDE AND TRYPTIC PEPTIDES CONTAINING THE ESSENTIAL THIOL GROUPS FROM ISOLATED MYOSIN HEADS

Received 6 September 1977 1. Introduction The two classes of essential thiol groups of myosin have been defined according to the effect of their blockage on the enzymic properties of the protein [ 1 ]. According to this functional definition the so-called thiol-1 and thiol-2 groups are essential for the K÷-depen - dent ATPase. Blockage of thiol-I has a variety of effects on the divalent cation-dependent ATPases depending on the conditions of the ATPase tests [2]. The second group, the thiol-2 group, can be differen- tiated since after blockage of thiol-1, it is essential for the functioning of the divalent cation-dependent ATPases. With the use of radioactive reagents it has been established that there is per heavy meromyosin subfragment-1, i.e., per myosin head, just one of each group and that both reside in the uncleaved head heavy chain of mol. wt 90 000 [3], obtained by chymotryptic digestion of myosin. The results of experiments employing a thiol cross- linking reagent have shown that these two thiol groups are in close spatial proximity in the native myosin molecule [4], which raises the question of their positions relative to one another in the heavy chain. We prepared isolated myosin heads in which, in one case thiol-1, and in another thiol-1 and thiol-2, were specifically labelled according to their effect on the enzymic function. From these the labelled CNBr-

SHEAR-INDUCED PROTEIN-PROTEIN INTERACTION AT THE AIR- WATER INTERFACE

Abstract Increases in the viscosity of solutions of different proteins varying over a wide range of molecular shape and size have been studied in a cone and plate viscometer. The protein concentrations used were lower than those needed to produce viscosities of the solutions significantly greater than the solvent medium. The high values of shear stress finally reached are attributed to a shear-induced formation of an intermolecular structure resulting from protein-protein interactions at the air water interface. The rate of formation of this structure was found to increase with ionic strength, indicating that the rate of adsorption at the interface may influence the development of high shear stress values. However, this effect also shows that hydrophobic intermolecular interactions and the conformation of the protein molecules in solution are important factors in the development of the structure. The presence of small amounts of detergents was found to be able to prevent increases in shear stress. The fact that the double-chained lipid lecithin does not produce this effect, even in high concentrations, may be due to its ability to take part in protein-lipid-protein interactions.

Symmetry and asymmetry in the contractile protein myosin

The subunit composition of the myosin molecule which is built up from 3 pairs of identical polypeptide chains (2 heavy chains and 2 pairs of light chains), gives it the appearance of having symmetric structure. This homodimeric arrangement in the molecule is in fact asymmetric in its construction as a result of the natural folding of the chains. There are also heterodimers which result from combinations of pairs of heavy chains and/or light chains which are not identical in their amino acid sequence. Enzyme kinetics and ligand binding are characterised by homogeneous processes in studies on isolated myosin heads. With the double-headed molecular species, myosin and its water-soluble fragment heavy meromyosin, the enzyme kinetics, nucleotide and metal ion binding exhibit negative cooperativity. Binding of Mg-ADP to active centres induces site-site and therefore head-head interaction, thus intact myosin is designed to be able to function asymmetrically. It is suggested that the ligand-induced asymmetry between the heads plays a central role in crossbridge function. The two heads, even in rest, adopt non-equivalent conformations and it is argued that this built-in constraint complements the asymmetric mode of interaction they subsequently undergo with their reaction partners on the actin filament. It is concluded that the enzyme is so constructed that during contraction the heads can perform their function in an alternating cooperative way.

Nucleotide induced head-head interaction in myosin

SummaryIn isolated myosin the reaction sequence of essential thiol groups with N-ethylmaleimide was studied using the following five approaches: kinetics of the modification reaction, effects of modification on enzyme properties, affinity chromatography of isolated subfragment-1 stemming from modified myosin, isolation of cyanogen bromide peptides and identification of the tryptic thiol peptides thereof. All techniques involved revealed differences whether the modification was performed in the presence or absence of pyrophosphate on the one hand and in the presence of ADP or ATP on the other. In the former cases the two thiol-1 groups per myosin, one per active site, reacted at an equal rate indicating an equivalent microenvironment of these groups and hence a symmetric site-site relationship. In contrast, the nucleotides induce the sequential modification of thiol-1 on one head followed by the thiol-2 on the other head. This indicates non-equivalence in microenvironment of the essential thiols connected with each active site and hence that a form of asymmetric head-head interaction is operative.

Evidence for head-head interactions in myosin from cardiac and skeletal muscles@@@Wechselwirkungen zwischen den beiden Köpfen in Myosin aus Herz- und Skelettmuskeln

Summary1.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∼106M−1) and subsequently a second with lower affinity (K∼102–104 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.Zusammenfassung1.In 3 verschiedenen Methoden zeigte sich, daß Mg-ADP in 2 Schritten an Myosin aus Herz- und schnell kontrahierenden Skelettmuskeln bindet. 1 Mol Mg-ADP bindet mit hoher Affinität (K∼106 M−1) und darauf ein zweites mit niederer Affinität (K∼102–104 M−1) pro Myosin. Nur 1 Mol Mg-ADP bindet mit hoher Affinität an isolierte Myosinköpfe. Dies deutet auf negative Kooperativität zwischen den beiden Bindungsstellen in intaktem Myosin.2.In Gegenwart von Nukleotiden zeigen die beiden Köpfe eines Myosin-moleküls verschiedene Konformationen, da an jedem Kopf eine andere essentielle Thiolgruppe leicht mit N-Aethylmaleinimid reagiert. In Gegenwart von Mg-Pyrophosphat reagiert an beiden Myosinköpfen eine Thiol-1-Gruppe besonders leicht. Der Nukleosidanteil scheint für diese zweite Art von Wechselwirkung zwischen den beiden Myosinköpfen verantwortlich zu sein.

Evidence for head-head interactions in myosin from cardiac and skeletal muscles

Summary1.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∼106M−1) and subsequently a second with lower affinity (K∼102–104 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.Zusammenfassung1.In 3 verschiedenen Methoden zeigte sich, daß Mg-ADP in 2 Schritten an Myosin aus Herz- und schnell kontrahierenden Skelettmuskeln bindet. 1 Mol Mg-ADP bindet mit hoher Affinität (K∼106 M−1) und darauf ein zweites mit niederer Affinität (K∼102–104 M−1) pro Myosin. Nur 1 Mol Mg-ADP bindet mit hoher Affinität an isolierte Myosinköpfe. Dies deutet auf negative Kooperativität zwischen den beiden Bindungsstellen in intaktem Myosin.2.In Gegenwart von Nukleotiden zeigen die beiden Köpfe eines Myosin-moleküls verschiedene Konformationen, da an jedem Kopf eine andere essentielle Thiolgruppe leicht mit N-Aethylmaleinimid reagiert. In Gegenwart von Mg-Pyrophosphat reagiert an beiden Myosinköpfen eine Thiol-1-Gruppe besonders leicht. Der Nukleosidanteil scheint für diese zweite Art von Wechselwirkung zwischen den beiden Myosinköpfen verantwortlich zu sein.

Shear induced structure formation at the air-water interface of aqueous, synthetic polymer solutions

Abstract The occurrence of anomalous viscosity behavior of dilute polymer solutions, indicated by increases in shear stress with time at constant shear rate, have been investigated in a cone and plate viscometer. The ionic strengths of the solutions influence the kinetics of increases whereas the presence of small amounts of detergent abolishes the effect. The results are interpreted by assuming the shear induced development of a structural network of the polymer solute at the air-water interface.

Cooperation between the two myosin heads interacting with actin in presence of ADP in myofibrils

Alkylierung von 2 Thiolgruppen pro Myosin mit NEM bei 0°C in Gegenwart einer Diophosphatkette inaktiviert die K-ATPase vollständig.In Myofibrillen werden diese Thiolgruppen durch Rigor-≪interaction≫ beider Myosinköpfchen mit Actin vor Alkylierung geschützt. In Gegenwart von Mg-ADP tritt eine spezifische vom Rigor verschiedene ≪interaction≫ zwischen Myosin und Actin auf. Man muss annehemen, dass dabei nur 1 Myosinköpfchen ans Actin bindet und dass das andere Köpfchen ein ADP gebunden hat.

Interactions between contractile and regulatory proteins of the myofibril

Die Regulationseiweisse (Tropomyo sin und Troponinkomplex), welche die Wechselwirkung der kontraktilen Eiweisse in der Myofibrille steuern, zeigen eine spezifische und unspezifische Bindung an den Actomyosinkomplex. Die spezifisch gebundene Menge der Regulationseiweisse beträgt nur etwa 1/100 derjenigen von Actomyosin, genügt aber trotzdem, um die Steuerung der Mg-ATPase und damit der Muskelkontraktion durch Ca2+ zu gewährleisten.