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We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein. As a result, 0.9-1.0 mol of fluorescyl group/mol of S-1 was covalently incorporated exclusively into the 95 kDa heavy chain as monitored by spectroscopic measurements. The central 50 kDa segment included the main site of fluorescence attachment as assessed by gel electrophoresis. The extent of S-1--FHS conjugation is strongly sensitive to F-actin binding but not to the interaction of nucleotides. The formation of the rigor F-actin--S-1 complex decreased the level of S-1 labeling to 20% without any competition between actin and S-1 for FHS binding. The derivatization of S-1 did not alter the K(+)-ATPase activity, but it enhanced the Ca(2+)-ATPase and Mg(2+)-ATPase to 150% and 225%, respectively, whereas it lowered the actin-activated ATPase to only 75% of the original activity. A double-reciprocal plot of the ATPase rate against actin concentration indicated a 2-fold decrease of the Vmax value for modified S-1, while the Km for actin was unchanged. Cosedimentation experiments did not reveal disruption of the rigor acto-S-1 interaction by the bound fluorophore. The labeled S-1 heavy chain was isolated, and its total tryptic digest was fractionated by reverse-phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

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The initial steps of the ATPase of covalently cross-linked actomyosin subfragment 1 (acto-SF-1) were studied by the rapid flow quench method, and the results obtained were compared with those with reversible (i.e., non-cross-linked) acto-SF-1 and SF-1 under identical conditions. Cross-linked acto-SF-1 plus [gamma-32P]ATP reaction mixture milliseconds old was quenched either in a large excess of unlabeled ATP (ATP chase) or in acid (Pi burst). The conditions were pH 8 and 15 degrees C at 5 mM or 0.15 M KCl and with or without 40% ethylene glycol. In 40% ethylene glycol (5 mM KCl), as with SF-1 and reversible acto-SF-1, the ATP chase was used to titrate active sites and to study the kinetics of ATP binding. Unlike those with SF-1 or reversible acto-SF-1, saturation kinetics were not obtained. The second-order rate constant for ATP binding was 3.1 X 10(6) M-1 s-1 for cross-linked acto-SF-1, 1.8 X 10(6) M-1 s-1 for reversible acto-SF-1, and 2 X 10(6) M-1 s-1 for SF-1. In Pi burst experiments, a transient phase could not be discerned. Because of a high kcat, cross-linked acto-SF-1 was difficult to study in aqueous solution, but at 5 mM KCl, the ATP chase and Pi burst curves were similar to those obtained in 40% ethylene glycol. At 0.15 M KCl the ATP chase curve was difficult to interpret (small amplitude), and there was a small Pi burst.(ABSTRACT TRUNCATED AT 250 WORDS)

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The reaction of endoproteinase Arg-C on the skeletal myosin head heavy chain was investigated through characterization of peptides and amino acid sequence analysis. The protease splits exclusively the 50 kDa-20 kDa junction at the lysine cluster spanning residues 639~i41 and does not affect any other protease-sensitive region of the entire myosin heavy chain. The sensitivity of the cleavage to actin and nucleotide binding makes this protease a very specific conformational probe of S-1. The nicked S-1 derivative, containing an intact NH2-terminal 75 kDa fragment, may serve as a tool for gaining further insights into the domain structure and function of the myosin head.

The nitration of arginine kinase from lobster muscle (Homarus vulgaris) with tetranitromethane has been studied after reversible blocking of its thiol groups.Experimental procedure for nitration of one essential tyrosyl residue is described. The introduction of one mononitrotyrosine per mole of arginine kinase results in a total loss of activity. Reduction to the aminotyrosine derivative does not restore enzymic activity.The mononitrotyrosyl arginine kinase is unable to bind its nucleotides or guanidine substrates as judged by differential spectrophotometry. An important conformational change occurring on nitration of one reactive tyrosine residue is demonstrated by means of optical rotatory dispersion measurements and immunodiffusion.I n an earlier report [l] from this laboratory it was demonstrated by means of differential spectrophotometry that interaction of arginine with arginine kinase from lobster muscle, Homarus vulgaris, leads to the formation of an enzyme-substrate complex visualized by a blue shift of tyrosine at 280 and 287 nm and a hypochromic phase at 239 nm.This tyrosine shift appears not only with arginine and arginine phosphate but also when the essential thiol group of the enzyme is modified by a specific -SH reagent ; this may suggest a close proximity of these two functional groups in the enzyme active site.I n addition, arginine kinase is the only phosphagen kinase [1,2] which undergoes such structural changes in the presence of its guanidine substrates, as shown by the exposure of a tyrosine to the aqueous medium. I n the present work, we have studied the importance of the chemical modification of tyrosine residues in the biological activity of this phosphagen kinase.Tetranitromethane appears to-day to possess the highest degree of specificity for tyrosine ; however, since -SH groups are also affected by this reagent [3,4], nitration experiments were carried out after reversible blocking of the thiol groups of arginine kinase, the procedure having been used previously to study the function of histidyl and lysyl residues in phosphagen kinases [5,6]. The data presented in this report point out the probable implication of one tyrosyl residue in the conformational maintenance of the active site of arginine kinase. MATERIALSArginine kinase from lobster muscle, Homarus vulgaris, creatine kinase from rabbit muscle, and taurocyamine kinase from Arenicola marina muscle were prepared as previously described [7-91. Before use proteins were dialyzed for 24 hours a t 0" against 0.05 M Tris-acetic acid buffer pH 8, containing 0.1 mM EDTA.Crystallized potassium tetrathionate (Merck product) was dissolved in 0.05M Tris-acetic acid buffer pH 8. Tetranitromethane was obtained from the Aldrich Chemical Co. and diluted in 95O/,, ethanol a t the desired concentration.Dithiothreitol (Nutritional Biochemicals Co.) was used as freshly prepared 0.1 M aqueous solution.1 mM solutions of 5,5'-dithiobis (2-nitrobenzoic acid) (Aldrich Chemicals) in 0.01 M Tris-acetic acid buffer pH 7 were employed. Sodium hydrosulfite w...