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Biomedical subjects

E Reisler

Publications and source records attributed to E Reisler.

At least 127 records · Page 7Linked to original sources
PubMed1980

The interaction of Cibacron Blue F3GA with troponin and its subunits.

Binding of troponin to Cibacron Blue F3GA-agarose column and its selective release from the gel in the presence of 0.5 M KCl provides the basis for a new purification method. The two-step procedure consists of isoelectric precipitation of tropomyosin and chromatography of the resultant crude troponin supernatant on Affi-Gel Blue column. Adsorption of troponin to the immobilized dye appears to occur through the troponin-T subunit. Troponin-I and troponin-C do not bind to the blue agarose column, whereas troponin-T binds to it very tightly. Binding of the dye to troponin-T prevents formation of troponin-T-troponin-C complex, but does not interfere with direct interaction of troponin-T with troponin-I. The activity of troponin in conferring calcium sensitivity on actomyosin ATPase is not affected by Cibacron Blue. Circular dichroism and difference absorption measurements of complexes of the blue dye with troponin and its subunits reveal the presence of a tight binding site on whole troponin and on troponin-T (KA greater than or equal to 10(6) M). The existence of weak binding sites for the dye on troponin and all of its subunits is deduced from difference absorption studies. Cibacron Blue appears to be a sensitive probe for subunit interactions in troponin.

Actins↗
PubMed1979

Salt-induced conformational changes in skeletal myosin light chains, troponin-C, and parvalbumin.

Evidence for salt-induced changes in myosin light chains [dissociated by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)], troponin-C (TnC), and parvalbumin was obtained from chymotryptic digestion, circular dichroism, fluorescence, and difference absorption studies. High salt (0.6 M NaCl) protects the DTNB light chain from proteolysis, increases its alpha-helical content, and quenches the tryptophan fluorescence. These effects are similar to the changes induced by Ca2+ but smaller in magnitude. TnC is affected by monovalent cations in a similar manner. Changes in the alpha-helical content resemble the effect of Ca2+. The enhancement of tyrosine fluorescence reflects conformational changes in the Ca2+-Mg2+ binding sites. The increase in the fluorescence of dansylaziridine-labeled TnC suggests perturbation of Ca2+-specific sites by salt. Cancellation of this effect by Mg2+ binding to the high-affinity sites is indicative of site-site interactions. In Whiting parvalbumin, salt-induced a perturbation of tryptophan absorption similar in nature to the Ca2+ effect.

Animals↗
PubMed1977

Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin. Chemical probing of movement of residues during conformational transitions.

The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.

Adenosine Diphosphate↗
PubMed1977

Reactivity of essential thiols of myosin. Chemical probes of the activated state.

14C-Labeled fluorodinitrobenzene and N-ethylmaleimide have been used as chemical probes of the conformational states of myosin induced by the binding of MgADP and MgATP. The results indicate that in the high-energy conformation, MMgADP-Pi, the essential thiols are protected from modification but their diminished reactivity does not result from depletion of the reagent by reaction at nonessential thiols. The binding of MgADP to myosin exposes the essential thiols as reflected by an increased rate of their modification. The influence of the divalent cations Mg2+ and Ca2+ on the conformation of the M species has also been investigated. By monitoring the incorporation of fluorodinitrobenzene, the conformations of the M state in the presence of these cations can be clearly discerned.

Adenosine Diphosphate↗
PubMed1977

Bovine serum albumin and aqueous guanidine hydrochloride solutions. Preferential and absolute interactions and comparison with other systems.

The partial specific volume, upsilon20, of bovine serum albumin at 25 degrees C was found to be 0.728 +/- 0.001 ml/g in solutions of guanidine hydrochloride (GuHC1), 0.01 M dithioerythritol (DTE), independent of GuHC1 concentration (3-6 M). The volume decrease upon denaturation is about 400 ml/mol (upsilon20 in water at the same temperature was found to be 0.734). From the reduced density increments at constant chemical potential of diffusible solutes, The apparent volumes, phi, were found to increase from 0.693 ml/g at 3 M GuHC1 to about 0.725 ml/g at 7 M GuHC1. The phenomenological interaction parameter, xi3 (grams of GuHC1 "bound" per gram of protein), was found to decrease from about 0.2 at 3 M GuHC1 to about 0.07 at 6.4 M GuHC1. The phenomenological interaction parameter, xi1 (grams of water "bound" per gram of protein), is negative and become less negative with increase in GuHC1 concentration. The relation between xi3 and xi1 and physical binding and exclusion of low-molecular-weight components are discussed in terms of simple model consideration. It is concluded that over the range of GuHC1 concentrations studied about 0.2 g of water as well as 0.28 g of GuHC1 are bound per gram of protein. This corresponds on the average to 1.3 molecules of water and 0.35 molecule of GuHC1 per amino acid residue. Similar results were found by recalculating some previous results for aldolase. These results on proteins in GuHC1 solution are in marked contrast to the behavior of DNA at high concentrations of NaCl and CsCl, which is analyzed on the basis of earlier work.

DNA↗
PubMed1976

Affinity chromatography of heavy meromyosin subfragment-1 reacted with thiol reagents.

Separation of heavy meromyosin subfragment-1 treated with N-ethyl maleimide (MalNEt) into native -SH1- and -(SH1, SH2)-blocked protein populations could be achieved by affinity chromatography on agarose-ATP columns in the presence of Mg2+ or Ca2+. Covalent bridging of the two -SH groups by p-phenylenedimaleimide gave a product which has the same affinity of binding to ATP columns as the doubly blocked MalNEt preparation. Treatment with p-phenylenedimaleimide abolished binding to immobilized F-actin columns, whereas modifications by MalNEt did not affect adsorption by this chromatographic medium. Affinity chromatography on immobilized nucleotide and actin columns is suggested as an analytical tool in the study of the involvement of thiol groups in the myosin active site and its conformation.

Actins↗
PubMed1975

An activation mechanism for ATP cleavage in muscle.

Evidence for a proposed activation mechanism is summarized. The low rate of ATP cleavage in the resting state of muscle is considered to result from the formation of a stable ring structure involving the two essential sulfhydryl groups on each myosin head and MgATP. Activation is thought to occur by interaction of actin in the vicinity of one of the essential sulfhydryl groups. Thus opening the stable ring leading to rapid dissociation of split products. This idea is consistent with the kinetic scheme of ATP cleavage developed recently by other workers and allows a prediction of the shift in population of intermediate states with changes in solvent conditions. It is also supported by our recent studies on the spatial geometry of the ring. The possibility that other nucleophilic groups may replace the sulfhydryl groups in other contractile systems is considered. The relevance of the ring structure to the tension generating event is discussed on the basis of recent measurements of the rate of contraction of modified (SH1-blocked) actomyosin threads. Results indicate the ability to form the ring structure is an essential requirement of the contractile process in these systems, and, moreover, that single, modified heads of myosin can act independently to produce the same rate of contraction as native myosin. This latter finding suggests that the myosin duplex exhibits some type of negative cooperativity in the contractile process.

Actins↗