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Biomedical subjects

J G Watterson

Publications and source records attributed to J G Watterson.

At least 19 recordsLinked to original sources
PubMed1997

The pressure pixel--unit of life?

Life is based on the co-ordinated and efficient function of the molecular nanomachines that biochemists call enzymes. Popular models of these machines are miniature anthropomorphic devices, which function in empty space under conditions bearing little resemblance to the watery subcellular world. The concepts of force and work applicable in our macroscopic world are transposed down to the molecular level where the chaos of thermal energies dominate. Despite four decades of intense research effort, the thermodynamic explanation of water-protein interactions-the first level of living matter is as remote as ever, because the disruptive thermal energies still remain dominant in these theories today. In this work, it is proposed that the important feature of the condensed medium is the formation of clusters, resulting from the bonded state of the molecules. This new view is the basis of the wave model of liquid structure. It is these water clusters, not single molecules, that are responsible for macroscopic pressure. Pressure is exerted on a size scale down to that of a single cluster, the hierarchical level defined by the "pressure pixel'. Below this size, tension between molecules prevails. This tension explains the stability and co-ordinated movement of the subcellular world, where theories based on random collisions fail. It also explains the coherence displayed by the cell in its ability to act as a unit, rather than a collection of independent processes predicted by statistical theories.

Biophysical Phenomena↗
PubMed1990

Protein phosphorylation in intact superior cervical ganglion during regeneration.

The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as microtubule-associated protein-2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85-kDa species and growth-associated protein-43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as alpha- and beta-tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.

Adenosine Triphosphate↗
PubMed1989

Phosphorylation of superior cervical ganglion proteins during regeneration.

The incorporation of radioactive phosphate into proteins of both normal and regenerating ganglia of the sympathetic nervous system of the rat is reported. The incorporation reactions were carried out in vitro by incubating homogenates of excised ganglia with [gamma-32P]ATP under various conditions. It was found that incorporation of phosphate into proteins of regenerating ganglia in the molecular mass range 10,000-100,000 daltons increased up to 40% over incorporation into proteins from control ganglia during the first 3 days following injury and returned to control levels after 14 days. Analysis of the proteins by two-dimensional electrophoresis revealed that only few, i.e., less than 20, became radioactively labelled in homogenates of superior cervical ganglia in the presence of Ca2+, and even fewer in the presence of cyclic AMP. Furthermore, all these proteins fell within a narrow pI range of 4-6. The growth-associated protein, variously designated GAP-43, B-50, F-1, and pp46, has an enhanced level of expression and phosphorylation in regenerating ganglia compared with controls at day 3. Injury also caused consistently higher levels of incorporation into two other proteins with molecular masses at positions 55,000 and 85,000 and pI values of 5.1 and 4.5, respectively; the former protein most probably is beta-tubulin. The fact that both proteins are found in the 15,000 g pellet after the tissue has been solubilized in 0.5% nonionic detergent indicates that they may indeed by components of filament assemblies. Thus, the results suggest that protein phosphorylation is a mechanism involved in cytoskeletal function in regenerating nerve.

Adenosine Triphosphate↗
PubMed1988

The role of water in cell architecture.

The role of water in biochemical and cellular events is ignored by most workers. However, much recent research has pointed to the importance of physical processes of the cell, which focus attention on such straight forward, 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 spacially so giving rise to integrated, large-scale, subcellular structures. The intimate participation of water in these structures would explain the fragility of the cytoplasmic organization.

Cells↗
PubMed1988

A model linking water and protein structures.

This communication presents a new picture of protein and solvent in which they are much more closely related both structurally and functionally than hitherto described. The picture is based on the recently published model of liquid structure, which proposes that clusters in water have long-term rather than flickering existence and are as large as proteins. Their spacial dimensions ensure that the two structures have a mutual tendency to pack together and cooperate in their vibrational motion.

Models, Chemical↗
PubMed1987

A role for water in cell structure.

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.

Body Water↗
PubMed1983

The role of magnesium in binding of the nucleotide polyphosphate chain to the active site of myosin subfragment-1.

The binding of adenosine 5'-[beta, gamma-imido]triphosphate, pyrophosphate and triphosphate to the active site of myosin subfragment-1 was assessed in the presence and absence of Mg2+ by direct and indirect methods. In addition, the affinity and stoichiometry of Mg2+ in the ternary complexes formed by protein, Mg2+ and each of these phosphate compounds have been determined. As direct methods, equilibrium dialysis, sedimentation and quantitative affinity chromatography were used in conjunction with the indirect method of monitoring reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon binding of the ligands at the active site. There was good agreement between the results yielded by the different methods. All three phosphate compounds alone bind just one molecule per isolated myosin head portion with similar affinities lying in the range 1-4 X 10(3) M-1. Again only one molecule/head portion binds when they exist in the form complexed with Mg2+, but now show much higher affinities of between 10(6)-10(7) M-1. In all cases Mg2+ was found to be associated in the ternary complexes with the very high affinity of 10(8)-10(9) M-1. It is postulated that this ion plays a prominent role in fixing the phosphate chain in the myosin active site. In contrast, Mg2+ scarcely affects the affinity of ADP and shows only a low affinity around 4 X 10(4) M-1 in the ternary complex [Watterson, J.G., Foletta, D., Kunz, P.A., and Schaub, M. C. (1983) Eur. J. Biochem. 131, 89-96]. As pyrophosphate displays binding parameters similar to the triphosphate compounds and widely different from ADP, it is argued that it may bind in the beta, gamma-phosphate positions at the active site.

Adenine Nucleotides↗
PubMed1983

Interaction of ADP and magnesium with the active site of myosin subfragment-1 observed by reactivity changes of the critical thiols and by direct binding methods at low and high ionic strength.

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg X ADP and uncomplexed ADP to the active site of myosin subfragment-1. Additionally, the binding parameters for Mg2+ in the ternary complex protein X Mg X 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 X ADP just one mole of ligand binds/mole subfragment-1. The affinity of Mg X ADP at low ionic strength is 2.1 X 10(6) M-1 and only five-times lower in the absence of Mg2+. In the ternary complex Mg2+ has a low affinity of 4.1 X 10(4) M-1. At high ionic strength the uncomplexed ADP binds with a 43-times-lower affinity than Mg X ADP, whose affinity is 6.9 X 10(5) M-1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 X 10(5) M-1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.

Adenosine Diphosphate↗
PubMed1981

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.

Animals↗
PubMed1980

Nucleotide induced head-head interaction in myosin.

In 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.

Adenine Nucleotides↗
PubMed1978

Hydrolytically induced allosteric change in the heavy chain of intact myosin involving nonessential thiol groups.

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.

Adenine Nucleotides↗
PubMed1975

Radioactive labeling 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.

Adenosine Triphosphatases↗
PubMed1975

Temperature-induced transitions in the conformation of intermediates in the hydrolytic cycle of myosin.

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 degrees C adopts the same conformation with Mg2+ and Ca2+ 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 degrees C and is identical with that found in the presence of ADP at 0 degree 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 degrees C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20 degrees C. In the case of the monovalent-cation-stimulated 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 degrees C.

Adenosine Diphosphate↗
PubMed1975

Conformational differences in myosin, IV.[1-3] Radioactive labeling of specific thiol groups as influenced by ligand binding.

Changes in the mono- and divalentcation-stimulated ATPase activities of myosin progressively labeled with N-ethyl-[2,3-14C2]-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 KCl higher than 0.4M and/or temperatures above 10 degrees 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 N-ethyl-[14C2]-malemide 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 degrees C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes.

Adenosine Triphosphatases↗