Absence Of MgATP Research Articles

Fluorescence properties and contraction characteristics of ANM (N-(1-anilinonaphthyl-4)maleimide)-labeled rabbit psoas muscle fibers.

Fluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (Vo), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though Vo decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM + MgATP or HMM + MgATP + F-actin in comparison with HMM + F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).

Purification and properties of glutamine synthetase from liver of Squalus acanthias

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [ P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456–462 ]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg 2+, MgATP, and l-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and l-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1–7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5–10% ethylene glycol. The apparent K m values for MgATP, l-glutamate, and ammonia (NH 4 +-NH 3) are 0.7, 11.0, and 0.015 m m, respectively. Mg 2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn 2+ cannot replace Mg 2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent K m for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine- N-oxide. Except for activation by halogen anions and the very low apparent K m for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent K m for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.

Discrimination of assembled and disassembled forms of gizzard myosin by papain.

Chicken gizzard myosin in 0.15 M or 0.5 M NaCl was cleaved at two sites of heavy chain with 2-10 micrograms/ml papain. MgATP inhibited these cleavages of myosin in 0.15 M NaCl but not in 0.5 M NaCl. The protective effect of ATP was observed at concentrations as low as 10 microM and increased in proportion to ATP concentration to a maximum at 1 mM. ADP was as effective as ATP, while adenosine 5'-[beta, gamma-imido]triphosphate, an unhydrolyzable ATP analogue, was less effective than ATP or ADP. AMP had no protective effect on the digestion of myosin and GTP inhibited slightly the digestion. When the papain-insensitive myosin in 0.15 M NaCl and 2.5 mM MgATP was phosphorylated by Ca2+/calmodulin-dependent myosin light-chain kinase, the myosin restored the vulnerability to papain. However, the two papain-susceptible forms, nonphosphorylated form in the absence of MgATP and phosphorylated form in the presence of MgATP, yielded very similar but distinct proteolytic fragments upon the digestion. When the extent of myosin assembly was estimated by the turbidimetry of myosin suspension in 0.15 M NaCl, nonphosphorylated myosin in the absence and presence of MgATP was assembled and disassembled, respectively, and phosphorylated myosin in the presence of MgATP was assembled. These results suggest that, at physiological ionic strength, papain as a probe distinguishes disassembled myosin and assembled myosin as papain-insensitive and papain-sensitive forms, respectively.

Binding of MgATP to the nitrogenase proteins from Azotobacter vinelandii.

Binding of MgATP to the MoFe and Fe proteins from Azotobacter vinelandii has been studied. By means of the flow dialysis technique it was demonstrated that one molecule of reduced Fe protein binds one molecule of MgATP, with a dissociation constant of 0.56 +/- 0.11 mM. The oxidized Fe protein binds two molecules of MgATP, with identical intrinsic dissociation constants of 0.29 +/- 0.05 mM. The binding of MgATP to the Fe protein was also studied by equilibrium dialysis. It was found that during dialysis of reduced Fe protein in the presence of MgATP, dithionite was oxidized. Moreover, in the presence of MgATP both reduced and oxidized Fe protein were inactivated during the dialysis. These observations demonstrate that binding of MgATP to the Fe protein can only be measured by a relatively fast method. With the same methods as used for the Fe protein, no binding of MgATP to the MoFe protein of A. vinelandii could be demonstrated. The redox properties of the Fe protein in the presence and absence of MgATP are discussed with respect to the observed binding properties of MgATP for the Fe protein. The implications of these results are discussed with respect to the present models for the interactions between the Fe and MoFe proteins of nitrogenase.

Stimulation of Ca++ binding and ATPase activity of dog cardiac myofibrils by AR-L 115BS, a novel cardiotonic agent.

AR-L 115BS, a benzimidazole derivative, is a positive inotropic agent that has been shown to increase active tension development and unloaded shortening velocity of chemically skinned heart muscle preparations at submaximal activating levels of free Ca++. We measured the effect of AR-L on relations between free Ca++, bound Ca++ and ATPase activity of dog cardiac myofibrils. At pCa 6, 100-300 micrometers AR-L increased myofibrillar ATPase activity maximally by about 30%. The concentration of AR-L giving half-maximal activation of myofibrillar ATPase activity was about 10 micrometers, and is similar to plasma concentrations associated with elevated contractility in intact animals. There was no effect of AR-L on myofibrillar ATPase activity at pCa 5 or 8, and the relation between pCa and percent activation of myofibrillar ATPase activity was shifted to the left by 0.4-0.5 pCa units in the presence of 100 micrometers AR-L. Calcium binding by cardiac myofibrils was increased by AR-L in the presence and absence of MgATP by 0.2-0.3 nmol/mg myofibrillar protein over a broad range of free Ca++ concentrations, a result suggesting that AR-L increases the affinity of myofibrillar troponin C for Ca++. The shift in the pCa giving half maximal and myofibrillar ATPase activity induced by raising the free Mg++ from 1.0 to 10 mm was unaffected by AR-L. These results indicate that the positive actions of AR-L 115BS on cardiac contractility may involve direct activation of myofibrils by virtue of an increased affinity of thin filament receptors for Ca++.

Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores

(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg 2+). I 50 values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min −1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg 2+ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg 2+ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane Δ \\ ̃ gm H + . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site ( Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg 2+. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg 2+ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg 2+ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity.

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre-extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat-denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat-denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X-100 extraction of isolated D. discoideum membranes results in a Triton-insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.

A model system for the study of the release of luteinizing hormone-releasing hormone from isolated storage granules.

We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40-160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KCl resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.

A Vesicle-Coated Particle Method for the Determination of Electrophoretic Mobility of Gastric Vesicles

In the presence of both ATP and Mg++, the density of the negative charge on the external surface of hog gastric vesicles had a higher (absolute) value in comparison to values in the presence of ATP only or the absence of both ATP and Mg++ as seen from the electrophoretic mobilities measured with a conventional cytopherometer. Gastric vesicle-coated particles of n-alkanes or n-alky1 halides were used for the study, in which their surfaces were examined under differential interference-contrast microscopy. This showed that the surfaces were coated by the vesicles. On the addition of Mg-ATP, an increase in the density of the surface charge took place irrespective of the oil used. With n-alkyl bromide (<0.1 % v/v), reorganization of the vesicular structure was indicated from the measurements of proton uptake by the vesicles. This uptake was followed by an increase in the proton leakiness of the membrane. This is evidence that the increased density of the negative charge of the vesicle surface is not due to the proton gradient but with most to phosphorylation of the K+-ATPase in the gastric vesicles. The temperature dependence of the viscosity-corrected electrophoretic mobility showed that there was some transition about 23°C in the presence of Mg-ATP, but not in the absence of Mg-ATP.

Circular dichroism and magnetic circular dichroism of nitrogenase proteins

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and Klebsiella pneumoniae (Kp) have been obtained in the near infrared-visible-near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C(3-)) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C(2-) 4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at approximately 8300 cm(-1). The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.

Preparation of a myosin-extracted "ghost" myofibril Sephadex conjugate column and its application to the separation of myosin subfragment-1 giving and not giving the initial burst of phosphate.

A "ghost" myofibril (myosin-extracted myofibril) Sephadex conjugate which specifically binds myosin, HMM and S-1 in the absence of Mg-ATP or Mg-PP can be prepared in a few days by conjugating "ghost" myofibrils to Sephadex beads. Binding ability is retained for over a month. It is used, therefore, for actin-affinity chromatography of myosin and its active fragments. It is under debate whether the two heads of the myosin molecule are functionally identical. Recently several reports have indicated that S-1 could be separated into two kinds of S-1, one giving the initial burst of phosphate and the other not, by assuming a difference in the affinity of the two kinds of S-1 to F-actin. Attempts are reported here to obtain these two components of S-1 separately by using the "ghost" myofibril Sephadex conjugate column. The method of S-1 separation reported by Shibata-Sekiya and Tonomura ((1976) J. Biochem, 80, 1371-1380), which used S-1 treated with CMB, was applied to the "ghost" myofibril Sephadex conjugate column. This resulted in the successful separation of S-1 modified with CMB giving no initial burst of phosphate and unmodified S-1 giving the initial burst of phosphate. A separation method based essentially on the principle employed by Taniguichi and Tawada ((1976) J. Biochem. 80, 853-860) gave an unsuccessful result.

Properties of energy-dependent calcium transport by rat liver microsomal fraction as revealed by initial-rate measurements.

Measurements of the initial rate of Ca2+ transport by rat liver microsomal preparations reveal the existence of two phases of transport activity. The first, a phase of rapid transport, is complete by 3-5 min, at which time the second (slower) phase begins; this remains linear for up to at least 40 min. The initial phase is minimal in the absence of MgATP. The initial rate of Ca2+ transport reaches values as high as 25 nmol/min per mg of protein; the Km for Ca2+total is 1-2 micrometer and that for MgATPtotal about 500 micrometer. Ruthenium Red (3-5 nmol/mg of protein) has little effect on the initial rate of transport, whereas tributylin (2 micrometer) inhibits equally in a KC1- or a KNO3-containing medium. Compunds that collapse components of the proton electrochemical gradient in mitochondria (valinomycin and carbonyl cyanide m-chlorophenylhydrazone) each inhibit by 70-80% the initial rate of microsomal Ca2+ transport.

Ligand-dependent tryptic inactivation of the ouabain sensitivity of ADP-ATP exchange catalyzed by canine renal Na +,K +-ATPase

Trypsin treatment of purified canine renal Na +,K +-ATPase preparations causes loss of Na +,K +-ATPase activity with production of tryptic fragments as described previously (Jorgensen, P. L. (1975) Biochim. Biophys. Acta 401, 399–415). With 0.08 – 0.1 M Na + or K + present, the Na +-dependent, ouabain-sensitive ADP-ATP exchange activity is also inactivated by trypsin. Addition of 5 mM ATP to K +-containing digestion mixtures results in protection of ADP-ATP exchange activity with an unexpected loss of ouabain sensitivity. Addition of MgATP to Na +-containing digestion mixtures also protects exchange activity, and in this case ouabain-sensitivity is also lost, regardless of the presence or absence of MgATP. In all cases where ouabain-insensitive exchange activity is generated, the 115,000 dalton α subunit is degraded to an 87,000 dalton fragment. When loss of activity occurs without development of ouabain-insensitive exchange activity the α subunit is cleaved to 52,000 and 67,000 dalton fragments.

Determination of the rate-limiting steps and chemical mechanism of fructokinase by isotope exchange, isotope partitioning, and pH studies.

Isotope exchange studies show that beef liver fructokinase has a random kinetic mechanism in which release of fructose from the enzyme is slower than that catalytic reaction. The stickiness of fructose in the presence of MgATP is confirmed by isotope partition studies, which show it to be released 0.53 times as fast as V1/Et in the presence, and 80--130 times as fast in the absence of MgATP. Fructose-1-P release from it binary complex is not at all rate limiting in the forward direction since no exchange of MgADP back into MgATP could be observed during the forward reaction. Failure to find any isotope effect by the equilibrium perturbation method with [1-18O]fructose (upper limit, 1.003, shows that P--O bond cleavage or formation is not rate limiting. The pH profiles for the forward reaction show a group (probably carboxyl with pK 5.7-6.0 and deltaHion = 0) that must be ionized and a group (perhaps lysine, with pK 9--10, and deltaHion 5-9 kcal/mol) which must be protonated for activity. The profile for the back reaction shows only a group with pK 5.5--6 that must be protonated for activity. A chemical mechanism is proposed in which a carboxyl group on the enzyme accepts a proton from the 1-hydroxyl of fructose during the forward reaction and donates it back during the reverse reaction.

Interaction of filamin with f-actin in solution.

Filamin is a major high-molecular-weight protein in smooth muscle which was recently identified and isolated [Wang, K., Ash, J. F. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4483-4486]. In the present studies, we shown that highly purified chicken gizzard filamin and muscle F-actin react in solution to form aggregates containing both proteins. Occasionally, these aggregates coagulate and contract into a dense gel in the absence of MgATP or CaATP. Immunofluorescence and electron microscopic studies suggest that the F-actin filaments are collected into fiber bundles and a crosslinked fiber meshwork by the binding of filamin molecules. These studies suggest that the function of filamin intact cells may be to regulate the ultrastructural state of F-actin filaments in a variety of dynamic cellular processes.

Adenosine cyclic 3′,5′-monophosphate-dependent protein kinase from human platelets

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent K m for ATP in the presence of 10 μM Mg 2+ was 4 μM (plus cyclic AMP) and 4.3 μM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 μM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 μM (presence of MgATP) were exhibited by the “protein kinase-cyclic AMP complex”. The enzyme required Mg 2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28 000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.

An active site-directed, irreversible inactivation of yeast hexokinase by ( r, s)2′,3′-epoxypropyl β d-glucopyranoside

(1) Only ( R, S)2′,3′-epoxypropyl β- d-glucopyranoside of the complete series of mono ( R, S)2′.3′-epoxypropyl ethers and glycosides of d-glucopyranose significantly inactivated yeast hexokinase. (2) ( R, S)2′,3′-Epoxypropyl β- d-glucopyranoside inactivates yeast hexokinase in the absence of MgATP 2−, The rate of inactivation is unaffected by MgATP 2−. (3) The rate of inactivation of hexokinase with ( R, S)2′,3′-epoxypropyl β- d-ilucopyranoside was much greater when hexokinase was present in a monomeric form than when it was present in a dimeric form. (4) ( R, S)2′,3′-Epoxypropyl β- d-glucopyranoside has a high K t (0.38 M) and at a saturating concentrarion, the first order rate constant for the inactivation of monomeric hexokinase is 8.3 · 10 −4 sec. (5) d-Glucose protects against this inactivation and this was used to derive a dissocistion constant of 0.21 mM for d-glucose in the absence of MgATP 2−. (6) The alkylation of yeast hexokinase by ( R, S)2′,3′-epoxypropyl β- d-gluco-pyranoside was not specific to the active site. When the concentration of ( R, S)2′,3′-epoxypropyl β- d-glucopyranoside was 50 mM two thiol groups outside the active site were also alkylated. (7) The reaction between 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and yeast hexokinase was examined in detail. Two thiol groups per monomer (mol. wt. 50000) reacted with a second order rate constant of 27 1 mole −1 sec −1. A third thiol group reacted more slowly with a second-order rate constant of 1.6 1 mole −1 sec −1 and a fourth thiol group reacted very slowly with inactivation of the enzyme. Tue second-order rate constant in this case was 0.1 1 mole −1 sec −1.

The active site of myosin A-adenosine triphosphatase. 8. Modification of the enzyme with diazonium-1H-tetrazole.

Myosin was treated with DHT (diazonium-1H-tetrazole) in the presence or absence of Mg-ATP. Results indicated that the SH-groups of myosin were modified by DHT. To prevent reaction of the SH-groups of myosin with DHT, the groups were blocked reversibly by DGM through their conversion to disulfide bonds. The Ca2+.ATPase [EC 3.6.1.3] activity of myosin disappeared on treatment with DGM (dithiodiglycolic acid dimethyl ester) for 50 hr and the amount of free SH-groups decreased to 2 moles per mole of myosin. The Ca2+ATPase activity was restored to the original level by reversing the blocking reaction with excess βME or DTT. DGM-treated myosin was treated with DHT, and then with β-ME or DTT. The Ca2+-ATPase activity decreased linearly with increase in the amount of monoazotyrosine in myosin, and complete inactivation occurred when approximately one mole of monoazo-tyrosine was formed per 4.0×105g of myosin. During treatment of DGM-treated myosin with DHT, the Ca2+-ATPase activity was protected to some extent by the presence of Mg-ATP. H-Meromyosin prepared by tryptic digestion of azo-myosin had the same monoazo-tyrosine content as the parent myosin. All the monoazo-tyrosine was present in the f-subunit. When a few moles of tyrosine residues per mole of myosin were modified with DHT, myosin became soluble even in water. One mole of tyrosine residue per mole of L-meromyosin fraction 1 reacted specifieally with DHT, and filament formation in L-meromyosin fraction 1 was inhibited by this modification with DHT.