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Biomedical subjects

E Reisler

Publications and source records attributed to E Reisler.

At least 109 records · Page 6Linked to original sources
PubMed1984

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges.

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Role of magnesium binding to myosin in controlling the state of cross-bridges in skeletal rabbit muscle.

The effect of Mg2+ on the disposition of myosin cross-bridges was studied on myofibrils and synthetic myosin and rod filaments by employing chymotryptic digestion and chemical cross-linking methods. In the presence of low Mg2+ concentrations (0.1 mM), the proteolytic susceptibility at the heavy meromyosin/light meromyosin (HMM/LMM) junction in these three systems sharply increases over the pH range from 7.0 to 8.2. Such a change has been previously associated with the release of myosin cross-bridges from the filament surface [Ueno, H., & Harrington, W.F. (1981) J. Mol. Biol. 149, 619-640]. Millimolar concentrations of Mg2+ block or reverse this charge-dependent transition. Rod filaments show the same behavior as myosin filaments, indicating that the low-affinity binding sites for Mg2+ are located on the rod portion of myosin. The interpretation of these results in terms of Mg2+-mediated binding of cross-bridges to the filament backbone is supported by cross-linking experiments. The normalized rate of S-2 cross-linking in rod filaments at pH 8.0, kS-2/kLMM, increases upon addition of Mg2+ from 0.30 to 0.65 and approaches the cross-linking rate measured at pH 7.0 (0.75), when the cross-bridges are close to the filament surface. In rod filaments prepared from oxidized rod particles, chymotryptic digestion proceeds both at the S-2/LMM junction and at a new cleavage site located in the N-terminal portion of the molecule. Kinetic analysis of digestion rates at these two sites reveals that binding of Mg2+ to oxidized myosin rods has a similar effect at both sites over the pH range from 7.0 to 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Crossbridge release and alpha-helix-coil transition in myosin and rod minifilaments.

The relationship between crossbridge release and alpha-helix-coil transition in myosin has been investigated by employing synthetic myosin and rod minifilaments prepared in 10 mM-citrate/Tris buffer at pH 7.0 and 8.0. Initial sedimentation velocity and turbidity measurements have established that the minifilament structures obtained at pH 7.0 and 8.0 are relatively similar in size and homogeneity, and can be used in comparative circular dichroism studies. Chemical crosslinkings and proteolytic digestions carried out at pH 7.0 and 8.0 verify that myosin and rod minifilaments undergo the same pH-induced changes as myosin filaments, i.e. a decrease in the rate of subfragment-2 crosslinking to the filament surface, and an increase in proteolytic susceptibility of the light meromyosin-heavy meromyosin hinge at alkaline pH. These results suggest charge-induced release of the S-2 element from the myosin and rod minifilament surface. Circular dichroism measurements reveal a reduced alpha-helical content of myosin (5%) and rod minifilaments (10%) at pH 8.0 compared to the respective pH 7.0 structures. These results establish a direct link between crossbridge release and alpha-helix-coil transition in myosin.

Animals↗
PubMed1983

The actomyosin ATPase of synthetic myosin minifilaments, filaments, and heavy meromyosin.

The actin-activated ATPase activities of myosin minifilaments and heavy meromyosin are similar at high actin concentrations. Under low ionic strength conditions, the minifilaments in Tris citrate buffer yield the same maximal turnover rate (Vmax) and apparent dissociation constant of actin from myosin (Kapp) as heavy meromyosin in standard low salt conditions. The time course of actin-activated ATP hydrolysis of minifilaments is similar to that observed for standard myosin preparations. Depending on the exact protein composition of the assay mixture, either the ATPase activity declines continuously with time, or is accelerated at the onset of superprecipitation. In analogy with myosin filaments, the ATPase of minifilaments shows a biphasic dependence on actin concentration. Super-precipitation of minifilaments follows a well resolved clearing phase during which their structural integrity appears to be fully preserved. These results indicate that minifilaments or similar small assemblies of myosin can fulfill contractile functions.

Actins↗
PubMed1983

Light-chain phosphorylation and cross-bridge conformation in myosin from vertebrate skeletal muscle.

The effect of phosphorylation of the myosin light chains (LC-2) on cross-bridge conformation in synthetic myosin filaments from vertebrate skeletal muscle was studied by using chemical cross-linking and chymotryptic digestion methods. Phosphorylated and dephosphorylated myosin filaments, which were used in these experiments, had similar sedimentation coefficients, turbidities, and rates of growth from the respective minifilament structures. The proteolytic susceptibility at the heavy meromyosin-light meromyosin (HMM-LMM) junction was somewhat greater in the phosphorylated than in the dephosphorylated filaments at both pH 7.0 and pH 8.0. At the same time, the normalized rate of subfragment 2 (S-2) cross-linking to the filament surface, kS-2/kLMM, was reduced by phosphorylation of myosin. These results are consistent with partial release of cross-bridges from the thick filament surface in phosphorylated myosin filaments.

Animals↗
PubMed1983

Protease-sensitive regions in myosin subfragment 1.

Proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, thermolysin, and Staphylococcus aureus protease reveal that the two trypsin-sensitive regions in S-1 have broad protease susceptibility. The cleavage of S-1 by these enzymes yields products that correspond within 1-2 kilodaltons (kDa) to the 25-, 50-, and 20-kDa fragments produced by trypsin. Papain and thermolysin cut preferentially at the 26-kDa/70-kDa junction, whereas elastase, subtilisin, and S. aureus protease cleave both the 26-kDa/70-kDa and 75-kDa/22-kDa junctions in S-1. Binding of actin to S-1 decreases the rate of all proteolytic reactions in the 95-kDa heavy chain. The protection of the 26-kDa/70-kDa junction by actin is greatest against papain and thermolysin attack. The reaction times of elastase, subtilisin, and S. aureus protease with S-1 increase 2-fold in the presence of actin. However, in contrast to similar reactions with trypsin, they proceed at both junctions and lead to formation of the 50- and 22-kDa fragments. The cleavage of the 22-kDa/50-kDa junction by elastase increases the Km value for the actin-activated ATPase. The presence of the two protease-sensitive regions in S-1 is consistent with a three-domain structure of the myosin head and may have important implications to the mode of intersite communication in this protein.

Actins↗
PubMed1982

Effect of calcium on synthetic myosin minifilaments.

The effect of Ca2+ on the structural properties of myosin filaments has been examined by employing myosin minifilaments as a model system. Paired sedimentation studies of myosin minifilaments at pH 8.0 reveal only a minor increase in their sedimentation coefficient (0.7 +/- 0.3%) upon binding of Ca2+ (pCa = 4.5). This increase and the larger change observed at pH 7.0 (less than 3.7%) are attributed to protein aggregation. Light scattering measurements indicate that both Ca2+ and Mg2+ promote protein association in solutions of myosin minifilaments at pH 7.0 and 8.0 and in myosin filaments at pH 8.0. This association reaction is nonspecific and does not level off with increasing concentrations of divalent cations. Nevertheless, low concentrations of Ca2+ and Mg2+ have a rather limited effect on myosin polymerization. It is suggested that the binding of Ca2+ to the myosin light chains affects the association equilibrium in minifilaments and apparently does not alter other structural properties of these particles.

Calcium↗
PubMed1982

5'-p-Fluorosulfonylbenzoyladenosine. Inactivation of myosin subfragment 1 and a model reaction with cysteine.

Myosin subfragment 1 ((S-1) is inactivated upon incubation with the nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo). The rate of inactivation is increased in the presence of MgADP and MgPPi and decreased in the presence of MgATP. Complete loss of ATPase activity correlates with the loss of two sulfhydryl groups on S-1, and for reactions carried out in the presence of MgADP, this is accompanied by noncovalent trapping of the nucleotide (0.75 ADP/S-1). Treatment of the inactivated S-1 with dithiothreitol leads to the recovery of the lost sulfhydryl groups, release of the trapped MgADP, and recovery of the protein's activity. Importantly, all of these changes are achieved without any significant release of the incorporated radioactive nucleotide analogue. Remarkably similar results were obtained for the reaction of 5,5'-dithiobis-(2-nitrobenzoic acid) with S-1 (Wells, J. A., and Yount, R. G. (1980) Biochemistry 19, 1711-1717) and were interpreted in terms of disulfide bond formation between the reactive SH1 and SH2 thiols. It is proposed that the inactivation of S-1 by 5'-FSO2BzAdo is caused by a two-step reaction involving the initial formation of a thiosulfonate, with the SH1 group, followed by a displacement of the analogue by the SH2 residue to yield a disulfide. The stable and measurable incorporation of the analogue into S-1 proceeds in a parallel reaction at site(s) which did not affect the ATPase activity. The general feasibility of the proposed role of 5'-FSO2BzAdo in forming a disulfide bond in proteins is demonstrated by monitoring its reaction with free cysteine. As detected by pH stat titrations, 5,5'-dithiobis-(2-nitrobenzoic acid) titrations, and amino acid analysis, 5'-FSO2BzAdo rapidly converts cysteine into cystine.

Adenosine↗
PubMed1982

Growth of synthetic myosin filaments from myosin minifilaments.

Addition of KCl to a solution of synthetic myosin minifilaments in 10 mM citrate-Tris buffer (pH 8.0) induces the growth of filaments. These filaments, at pH 8.0, resemble in their morphological and hydrodynamic properties the synthetic filaments described by Josephs and Harrington [Josephs, R., & Harrington. W. F. (1966) Biochemistry 5, 3474--3487]. The rate of filament growth depends critically on the KCl concentration in the solution. Low rates of filament formation are noted in the presence of both low (below 80 mM KCl) and high (above 0.15 M KCl) salt concentrations, whereas at the intermediate KCl concentrations the filaments are formed at a fast rate. The formation of filaments from minifilaments is a reversible process, and under moderate salt concentrations, these two polymeric systems appear to exist in a dynamic equilibrium. Small amounts of minifilaments can induce rapid polymerization of dissociated myosin; i.e., they can act as a seeding material. These and other observations are discussed in terms of a direct route for filament formation from myosin minifilaments.

Animals↗
PubMed1981

Interaction of myosin subfragment 1 with Cibacron Blue F3GA.

Cibacron Blue F3GA and its immobilized derivatives have been shown before to bind and inhibit nucleotide-dependent enzymes and, among them, myosin subfragment 1. Experiments have been carried out to examine the mechanism of the subfragment 1--dye interaction. Binding of subfragment 1 to immobilized dye (Affi-Gel Blue) does not involve the ATP binding site on myosin. Subfragment 1 hydrolyzes MgATP and CaATP while bound to the Affi-Gel Blue column. Inactivated subfragment 1, which contains [3H]ADP noncovalently trapped at the active site, binds and elutes from the Affi-Gel Blue column in the same manner as unmodified, active protein. Free Cibacron Blue inhibits the ATPase activity of subfragment 1. The inhibition is pH, salt, and time dependent. Complete inhibition correlates with the noncovalent binding of four to five dye molecules per mole of subfragment 1. Three to four of these dye molecules can be preferentially removed from subfragment 1 in the presence of 1 M KCl without relieving the inhibition. This inhibition, which can be traced to one dye molecule per subfragment 1, is reversible and is facilitated in the presence of MgADP and MgATP, suggesting that the dye does not bind at the active site of subfragment 1. Our observations are explained in terms of hydrophobic and electrostatic protein--dye interactions.

Adenosine Diphosphate↗
PubMed1981

Structural changes in synthetic myosin minifilaments and their dissociation by adenosine triphosphate and pyrophosphate.

Morphologically similar short myosin and rod filaments (minifilaments) have been prepared in 10 mM Tris--citrate buffer, pH 8.0, in the absence of other myosin or rod forms. Both minifilament systems are dissociated in the same manner in the presence of ATP or pyrophosphate. Identical binding of these ligands to myosin and rod minifilaments suggests that myosin heads play no role in substrate-induced destabilization of the minifilaments. The effects of ATP and pyrophosphate on minifilaments are similar to their dissociating effect on synthetic filaments [Harrington, W. F., & Himmelfarb, S. (1972) Biochemistry 11, 2945--2952], thus justifying their use in conformational studies in lieu of filaments. In view of their small size and homogeneity, the minifilaments constitute an appropriate material for such studies. The binding of pyrophosphate to myosin and rod minifilaments decreases their alpha-helical content, as measured by circular dichroism. No change in the secondary structure of subfragment 1 and light meromyosin is observed upon binding of pyrophosphate, but substantial changes (10%) are detected in subfragment 2. The structural changes in myosin, possibly relevant to contraction, are localized in the subfragment 2 region of the molecule. These results emphasize the importance of charge interactions in the functional behavior of thick filaments.

Adenosine Triphosphate↗
PubMed1981

On the alkali light chains of vertebrate skeletal myosin. Nucleotide binding and salt-induced conformational changes.

The interaction of alkali light chains of vertebrate skeletal myosin with nucleotides and KCl has been examined by chemical modifications of these proteins, by direct binding measurements, and in circular dichroism studies. The reactivities of the single thiol groups in the isolated alkali light chains A1 and A2 have been studied by reacting these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). MgATP and MgADP reduced the reactivities of thiol groups while high concentrations of KCl increased them. Subsequent equilibrium dialysis experiments verified the presence of a low-affinity nucleotide binding site per each alkali subunit. Circular dichroism measurements revealed that KCl induced local (around phenylalanines) and overall (alpha-helical content) conformational changes of equal magnitude in the two alkali light chains. However, salt induced different conformational changes in the subfragment 1 isoenzymes, S-1(A1) and S-1(A2). This differential salt effect on the S-1 isoenzymes was confirmed by comparing their thermal stability in different salt conditions. AT low KCl concentrations (5 mM), S-1(A1) was found to be considerably more heat labile than S-1(A2); at higher salt levels (50 mM KCl) the stability of S-1(A1) approached that of S-1(A2). These experiments are discussed in terms of the relationship between the alkali subunits and the ATP and the actin-binding sites of myosin.

Adenosine Diphosphate↗
PubMed1980

Effects of actin and calcium ion on chymotryptic digestion of skeletal myosin and their implications to the function of light chains.

Experiments have been carried out to assess the involvement of the myosin light chains [obtained by treatment of myosin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2)] in the control of cross-bridge movement and actomyosin interactions. Chymotryptic digestions of myosin, actomyosin, and myofibrils do not detect any Ca2+-induced change in the subfragment 2 region of myosin. Actin, like Ca2+, protects the in situ Nbs2 light chains from proteolysis and causes a partial switch in the digestion product of myosin from subfragment 1 to heavy meromyosin. This effect is independent of the state of aggregation of myosin, and it persists in acto heavy meromyosin and in actinomyosin in 0.6 M NaCl. Digestions and sedimentation studies indicate that there is no direct acto light chain interaction. Proteolysis of myosin shows a gradual transition from production of heavy meromyosin to subfragment 1 with lowering of the salt level. In the presence of Ca2+ heavy meromyosin is generated both in digestions of polymeric and of monomeric myosin. These results are explained in terms of localized changes within the Nbs2 light chains and subfragment 1. Subunit interactions in the myosin head lead to a Ca2+-induced reduction in the affinity of heavy meromyosin for actin in the presence of MgATP. The resulting Ca2+ inhibition of the actin-activated ATPase of myosin can be detected at high salt concentrations(75 mM KCl).

Actins↗
PubMed1980

Kinetic studies with synthetic myosin minifilaments show the equivalence of actomyosin and acto-HMM ATPases.

The kinetic properties of actomyosin have been examined using complexes of actin with the recently described (Reisler, E., Smith, C., and Seegan, G. (1980) J. Mol. Biol., in press) short, bipolar synthetic myosin filaments (minifilaments). It is shown, in contrast to previous observations with aggregated and insoluble myosin, that the kinetic behavior of actomyosin is similar to that of acto-heavy meromyosin. Owing to their size, solubility, and stability under conditions of the actin-activated ATPase measurements, the minifilaments provide a well defined experimental system. Thus, they constitute a convenient and appropriate material for studying actomyosin interactions.

Actins↗