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Biomedical subjects

M C Schaub

Publications and source records attributed to M C Schaub.

At least 55 records · Page 3Linked to original sources
PubMed1982

Electrophoretic analyses of atrial and ventricular cardiac myosins from foetal and adult rabbits.

1. Rabbit cardiac myosins from atrium and ventricle were found to differ in their native form in pyrophosphate gel electrophoresis as well as in their light chain pattern in one- and two-dimensional electrophoretic systems. 2. The myosin light chain pattern in sodium dodecylsulfate gel electrophoresis differs between atrial and ventricular tissues in the mammalians (rabbit, dog, human, pig, sheep and rat) but not in the chicken. 3. In foetal rabbit ventricle an additional light chain is observed which, in two-dimensional electrophoresis with adult cardiac light chains, was found to be the same as atrial light chain-1.

Animals↗
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↗