Raphael Lamed

Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin.

In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002) J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

Covalent coupling of nucleotides to agarose for affinity chromatography

Abstract An improved method for the preparation of agarose hydrazide for coupling of nucleotides and nucleosides is described. Adipic acid dihydrazide is coupled to the agarose by the cyanogen bromide procedure, giving in one step the required agarose hydrazide. Aldehyde of IO 4 − oxidized nucleotides di- and triphosphates are coupled to the resin by hydrazone formation. The application of NADP bound to Sepharose hydrazide for the adsorption of glucose-6-phosphate dehydrogenase is described.

Enzymatic mechanochemistry. I. The interaction of heavy meromyosin with “immobilized adenosine triphosphate”

Abstract ATP was covalently bound to an agarose gel. The insolubilized ATP was found to be capable of specifically binding heavy meromyosin. The adsorbed heavy meromyosin could be eluted by ATP in solution. Both binding and elution by ATP of heavy meromyosin were not much effected by Ca 2+ , Mg 2+ or EDTA. While the water-soluble polyalanine-myosin was also found to be adsorbed, myosin in 0.5 M KCl did not seem to be adsorbed by agarose-ATP. Both Mg 2+ and Ca 2+ appear to activate the splitting of bound ATP by heavy meromyosin to practically the same extent. We prepared water-soluble derivatives of ATP in which ATP underwent the same chemical modification required for its coupling to agarose but in which the agarose component was absent. Their splitting by heavy meromyosin was also activated by Mg 2+ though to a lesser extent but actin did not influence this reaction. Possible relations between our findings and the various stages of the reaction between myosin and ATP, as well as the potential use of columns filled with insolubilized NTPs for the separation and purification of myosin and of its subfragments, are discussed.

A comparative study of muscle contraction and superprecipitation using trinitrophenylated myosin and active myosin subfragments

Summary Myosin and heavy meromyosin (HMM) were trinitrophenylated (TNP-ed) to different tents by 2,4,6-trinitro benzene sulfonate. Protein preparations in which only one active te per molecule has been TNP-ed were isolated by using immobilized ATP affinity chromatography columns. Irrigation of “ghost” myofibrils (i.e. myofibrils from which the myosin has been extracted) with either of these “one-headed” species, followed by the addition of ATP, caused contraction. The myosin derivative exhibited also superprecipitation. On the her hand, fully TNP-ed myosin and HMM could not induce contraction of ghosts and the rst did not give superprecipitation. The relationship between superprecipitation and scle contraction is discussed.

Possible uncoupling of the mechanochemical process in the actomyosin system by covalent crosslinking of F-actin.

It has been speculated that F-actin depolymerizes during muscle contraction. In order to test this speculation, G-actin in F-actins were immobilized by crosslinking. While this did not affect activation of myosin ATPase, superprecipitation could be abolished. Sedimentation, light scattering, electron microscope, electrophoresis and homodyne spectra measurements seem to indicate that the average size of crosslinked segments increases with time of treatment with the crosslinking agent. At the same time, the amounts of dimers and trimers decreased. It appears that crosslinking makes F-actin filaments less flexible. The possible reasons for the loss of the capability to exhibit superprecipitation are discussed.

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Abstract Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca 2+ and Mg 2+ . In the presence of Ca 2+ , the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K + - or Mg 2+ -stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg 2+ -stimulated and a decrease in K + -stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N -ethyl maleimide in the absence of added nucleotides resulted in a loss of K + -ATPase and in an increase in the Ca 2+ -ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K + -ATPase of all preparations whereas the Ca 2+ -ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.

Dynamic affinity chromatography of heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative have been chromatographed on immobilized ATP, ADP and adenosine 5-(geta, gamma-imino) triphosphate affinity chromatography columns, in the presence and in the absence of Ng-2+ or Ca-2+.ma-32-P] ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5 (beta, gamma-imino) triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the Pi-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.

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.