The isolated 21 kDa N-terminal fragment of myosin binds to actin in an ATP and ionic strength-dependent manner.

Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the 'consensus' ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the 'consensus' ATP site has a role in the ATP-dependent actin binding of the fragment, we isolated a shorter 21 kDa N-terminal fragment, which contains only a part of the 'consensus' site. The 21 kDa fragment was obtained by photocleavage of myosin subfragment-1 in the presence of vanadate (Mocz, G. (1989) Eur. J. Biochem. 179, 373); the cleavage was followed by dissociation of the S-1 heavy chain fragments with guanidine hydrochloride and renaturation. The isolated 21 kDa fragment binds to F-actin, since it cosediments with actin, inhibits the actin-activated ATPase activity of myosin subfragment-1 and shows increase in light scattering upon titration by actin. The affinity of the binding is rather high (Kassoc = 0.83.10(7) M-1). The light scattering increase is reversed, e.g., the 21 kDa-actin complex is dissociated, upon addition of ATP both in the presence and absence of Mg, but less ATP is needed for dissociation when Mg is absent. Other polyphosphates, including inorganic triphosphate, pyrophosphate and ADP, also dissociate both the 21 kDa-actin and 23 kDa-actin complexes but the latter needs a higher concentration of polyphosphates for dissociation. However, these polyphosphates, except ATP, do not dissociate the (subfragment-1)-actin complex in the absence of Mg. The 21 kDa-actin and the 23 kDa-actin complexes are also dissociated by increasing ionic strength or by a low concentration of polyglutamate, which hardly affect the light scattering of the (subfragment-1)-actin complex. The results indicate that the binding of the N-terminal fragments of myosin to actin, unlike that of intact subfragment-1, is essentially of electrostatic nature. The polyanions dissociate the myosin fragment-actin complexes not by reacting with the 'consensus' ATP binding site, but by competing with actin for a positively charged binding site on the 21 kDa fragment. The only positively charged cluster in the amino acid sequence of this fragment is the 143-147 stretch, which may participate in forming the actin binding site.

Studies on the amino groups of myosin ATPase. III. Effect of nucleotides on the fluorescence of beta-naphthoquinone-4-sulfonate bound to amino groups of myosin.

beta-Naphthoquinone-4-sulfonate was used for chemical modification of amino groups of myosin. The reagent was found to affect also the sulfhydryl groups if the reaction was not prevented by previous disulfide exchange with cystamine. When cystamine protection was employed the ATPase (ATP phosphohydrolase, EC3.6.1.3) activity was enhanced in the presence of Mg2+ and decreased in the presence of K+ or Ca2+, a pattern typical of myosin with blocked essential amino groups. On addition of ATP or ADP a blueshift was observed in the fluorescent emission spectrum of beta-naphthoquinone-4-sulfonate bound by myosin, presumably owing to conformational changes in the environment of essential amino groups induced by the binding of nucleotides.

Studies on the amino groups of myosin ATPase. IV. Effects of ATP and its analogs on the spectral properties of trinitrophenylated myosin and its active fragments.

A considerable blue shift was observed in the absorption spectrum of the trinitrophenyl moiety attached to a functional epsilon-lysyl amino group of subfragment-1, heavy meromyosin and myosin on addition of ATP or ATP analogs. The resulting difference spectra showed a maximum at 320 and a minimum at 365 nm. The greatest spectral change was observed with a non-hydrolyzable ATP analog, adenosine 5'-(beta,gamma-imino)triphosphate and it decreased in the order adenosine 5'-(beta,gamma-imino)triphosphate, ATP and ADP. The ATP-induced difference spectrum changed to that of ADP upon the hydrolysis of ATP. The observed spectra were depended on temperature and ionic strength. Difference spectra were produced also by ITP, IDP and pyrophosphate while AMP was practically ineffective. Mg2+ also caused small spectral changes which are not identical with those induced by ATP analogs. On the basis of measurements carried out on a model compound, it is assumed that as a consequence of the reaction of ATP with a myosin head, the environment of the functional lysyl residue becomes less polar, i.e. it becomes buried in the hydrophobic core of the molecule. Changes on addition of ATP or its analogs were observed also in the circular dichroic (CD) spectrum of trinitrophenylated subfragment-1, which also points to conformational changes in the vicinity of the functional lysyl residue.

Comparative studies on amino and thiol groups in myosins from different sources.

Myosins from rabbit white and red skeletal, rabbit heart, fish skeletal and chicken gizzard muscles, as well as from human platelets were subjected to trinitrophenylation by trinitrobenzene sulfonate and alkylation by N-ethylmeleimide which affected their amino and thiol groups, respectively. The blocking of amino groups was carried out in the presence or in the absence of Mg-ADP and was followed both spectrophotometrically and enzymatically. Essential amino groups, whose modification throughly changes the enzymic characteristics of myosin, were found in heart and in all skeletal muscle myosins but were absent in myosins from chicken gizzard muscle and from human platelets. The reaction of these amino groups was highly retarded in the presence of Mg-ADP. Alkylation of thiols led to loss of the K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in all myosins. However, the rate of loss of activity varied from one myosin to another and, for a given myosin, was affected by the presence of nucleotides and by the value of the ionic strength. The change in Ca(2+)-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) on alkylation was influenced by the presence of Mg - ADP during the reaction. In the absence of this nucleotide, the Ca(2+)-ATPase activity increased and reached a plateau as a consequence of modification. The extent of activation largely depended on the origin of the myosin. When alkylation was carried out in the presence of Mg-ADP, the Ca(2+)-ATPase activity as a function of time exhibited a maximum but the descending part of the curve was absent in myosins from heart and gizzard muscles.

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.

Anti-TNP antibody localization of the reactive lysine residues in myosin.

Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.

Characterization of monoclonal antibodies to human platelet myosin that recognize highly conserved epitopes within the 50 kDa fragment of myosin subfragment-1.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.

Polymerization of G-actin by hydrodynamic shear stresses.

In the absence of Ca2+ G-actin can be polymerized by the application of shear stress in low ionic strength buffer. When G-actin in low ionic strength buffer containing EGTA was sheared for predetermined times under different velocity gradients, viscosity attained a maximal value, comparable to that obtained by seeding with F-actin nuclei, at a velocity gradient of 3000 s-1 after about one hour. Such flow-polymerized actin was indistinguishable from KCl-polymerized actin. Under similar conditions, EDTA which can bind both Ca2+ and Mg2+, gave a smaller effect than the Ca2+-chelating agent EGTA which binds Mg2+ weakly. When an Mg2+ salt was added to EDTA- or EGTA-containing buffer to give a free Mg2+ concentration of a few micromoles/liter, flow-induced polymerization was significantly enhanced. It appears that occupancy of only a small fraction of the high affinity binding sites by Ca2+ prevents flow-polymerization while Mg2+ may enhance this type of polymerization by replacing Ca2+. We speculate that the shear stress induces polymerization by promoting nucleation and that Ca2+ bound to the high affinity divalent cation binding site inhibits formation of the nuclei.

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head.

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.

Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge.

We have recently reported the discovery of a 14-amino-acid osteogenic growth peptide (OGP). In vivo OGP increases bone formation and trabecular bone density. Physiologically it is found in serum complexed to an OGP binding protein (OGPBP). In vitro OGP has a biphasic effect on osteoblastic MC 3T3 E1 and fibroblastic NIH 3T3 cell proliferation; at low concentrations (0.01-1.0 and 1.0-100.0 pM, respectively) it is highly stimulatory with an inhibition at higher doses. To assess possibilities of labeling synthetic OGP to obtain radio- or fluorescent ligands, OGP analogues were extended at the N- or C-termini with Cys or Cys(S-NEtSucc) or the OGP Tyr-10 replaced by 3-I(Tyr). All analogues with N-terminal modifications, as well as the [Cys15]OGP-NH2 retained the OGP-like dose-dependent effect on proliferation of the MC 3T3 E1 and NIH 3T3 cells, although the magnitude of stimulation was lower, approx. 2/3 that of the native-like synthetic OGP. The [Cys15(S-NEtSucc)]OGP-NH2 and [3-I(Tyr10)]OGP shared only the inhibitory activity of OGP. This suppression is further shared by a number of other positively and negatively net charged, but not net neutral, peptides. Both N-terminal-modified analogues displayed a decreased binding activity to the OGPBP. All analogues except reverse OGP, [3-I(Tyr10)]OGP and [Cys15(S-NEtSucc)]OGP-NH2 reacted with anti-OGP antibodies. These data are not only important for labeling purposes but suggest a respective role for the OGP N-and C-terminal regions in binding to the OGPBP and putative OGP receptor. It appears that the OGP proliferative activity represents the net effect of stimulation specific to the OGP structure and nonspecific inhibition associated with the peptide's high positive net charge.

Regenerating bone marrow produces a potent growth-promoting activity to osteogenic cells.

It is well documented that injury to bone marrow is followed by an osteogenic phase that precedes the complete tissue regeneration. We have recently shown that postablation healing of bone marrow in rat tibiae is associated with a systemic increase in osteogenesis. It was hypothesized that a growth factor(s) with an effect on osteogenic cells is produced in the healing limb, is transferred to the blood circulation, and enhances osteogenesis systemically. To test growth factor production, healing bone marrow-conditioned medium was prepared with tissue separated from rat tibias during the osteogenic phase and assayed for enhancement of mitogenic activity in culture of osteogenic rat osteosarcoma cells (ROS 17/2). Partial purification of healing bone marrow-conditioned medium-derived growth factor(s) consisted of gel filtration on Sephadex G-25, boiling, chromatography on heparin-Sepharose, and gel filtration on Sephadex G-75. Mitogenic activity eluted in the void volume of the Sephadex G-25 column (mol wt greater than 5,000). Potent activity resolved from heparin-Sepharose with PBS, and on filtration by Sephadex G-75 this activity recovered in 3 peaks with mol wt estimates of 35,000, 19,000, and less than 10,000. The partially purified factor also showed considerable stimulatory effect on DNA synthesis in osteoblastic fetal rat calvarial cells and on in vitro elongation of fetal long bone; it had only a small effect on nonosteoblastic ROS and fetal rat calvarial cells. These data indicate that healing bone marrow produces growth factor activity with a preferential effect on osteogenic cells. It is suggested that local growth factors have a role as mediators in the sequence of events whereby bone marrow expresses its osteogenic potential. During postablation healing of bone marrow these factors may also function as systemic promoters to osteogenic cells.

Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals, OGP increases osteogenesis. Immunoreactive OGP (irOGP) in high abundance was demonstrated in normal animal serum mainly as an OGP-OGP-binding protein (OGPBP) complex. Here we show the presence of an OGP-OGPBP system in normal human serum. The total irOGP content, of which the bound peptide comprises at least 80-90%, ranged from 480-4460 mumol/L, several orders of magnitude higher than that of other regulatory polypeptides. The steady state/total irOGP ratio declined between 23 and 49 yr of age. The bound irOGP, purified by boiling, ultrafiltration, and hydrophobic high pressure liquid chromatography, was identical to OGP obtained previously from rat regenerating marrow and mouse stromal cell cultures in terms of its amino acid sequence, immunoreactivity, and mitogenicity. These data demonstrate the usefulness of our immunoassay to measure circulating OGP. More importantly, the identity of the human OGP with that of other species indicates the peptide's evolutionary conservation and, thus, its biological importance. The natural occurrence of OGP in man signifies its potential role in the prevention of bone loss and rescue of bone mass, especially in osteoporosis.

Effect of the integrity of the myofibrillar structure on the tryptic accessibility of a hinge region of the myosin rod.

Limited proteolysis has been used to study the influence of actin, in the absence or presence of regulatory proteins of the thin filament (tropomyosin and troponin), as well as that of the myofibrillar structure on the tryptic cleavage of the heavy meromyosin (HMM)/light meromyosin (LMM) hinge region in myosin heavy chain. Cleavage at the HMM/LMM hinge is almost absent in myofibrils, whereas this hinge is accessible to tryptic digestion in actomyosin, in native thin filaments attached to myosin and in myosin heavy chain alone. This observation indicates that it is the myofibrillar structure which profoundly affects the tryptic accessibility of this specific hinge region of myosin. This provides a good example of the manner by which a highly organized supramolecular structure might affect the chemical properties of a specific site in a macromolecule.

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1.

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.

A novel 27/16 kDa form of subtilisin cleaved actin: structural and functional consequences of cleavage between Ser234 and Ser235.

A new 27/16 kDa form of cleaved actin was prepared by subtilisin cleavage between Ser234 and Ser235 of F(MgADP)-actin complexed with BeFx. The cleavage had little effect on actin-actin interactions as probed in polymerization measurements and by electron microscopy. In circular dichroism melting experiments the thermostability of F-actin was reduced by about 10 degrees C by this cleavage. The in vitro motility and Vmax, but not Km, of actomyosin ATPase were decreased by about 20% upon 27/16 kDa cleavage of F-actin. The binding of tropomyosin to actin was unchanged by this modification.

The effect of antibodies to human platelet membrane and cytoplasmic myosins on myosin ATPase activity and platelet aggregation.

Myosins were purified from the membrane fraction and the cytoplasm of human platelets. Polyclonal antibodies to the purified myosins were induced in rabbits. Their effects on the ATPase activity of the purified myosins as well as on the process of platelet aggregation were studied. A strong cross reactivity was found between the two myosins and their respective antibodies by the ELISA technique. It was found that the antibodies preferentially bind to the "head" segment of the myosins, since purified myosin "rod" reacted only weakly with the two kinds of antibodies. The two antimyosin antibodies strongly inhibited the K+(EDTA) ATPase activity of both myosins, as well as the activity of the isolated myosin "heads". The amount of antimembrane myosin antibody required to inhibit the above enzymatic activity was smaller than that of the anticytoplasmatic myosin antibody. Similar results were observed with F(ab)2 fragments of the two kinds of antibodies. No effect of these antibodies or their F(ab)2 fragments was observed on platelet aggregation induced by various agonists, although their inhibitory effect on the platelet myosin ATPase activity was strong.

Distribution of actin, myosin and actin binding protein in platelets of patients with hyperlipoproteinemia.

Significant anomalies in the quantity and relative distribution of the contractile proteins actin, myosin and actin binding protein (ABP) were observed in platelets obtained from patients with hyperbetalipoproteinemia (type IIa) and in patients with hypertriglyceridemia (type IV). Changes were observed in unfractionated platelets, (increased ABP in type IIa patients and increased actin in both type IIa and type IV) in isolated platelet membranes (increased ABP and actin in type IV, and increased myosin in type IIa) and in the KCl extract of platelets (increased actin in type IV and increased myosin in type IIa). The myosin ATPase specific activity was increased in platelets of type IV patients. No changes were observed in the concentrations and distribution of membrane glycoproteins in the platelets of these patients. The above anomalies in the contractile proteins might be relevant to the known functional anomalies of the platelets of patients with hyperlipoproteinemias.

Structural and immunological properties of myosin from human platelet external and internal membranes.

Our previous studies indicate that platelets contain two myosin isoforms, one of them localized in the membrane while the other in the cytoplasmic compartment. Structural and functional differences of these myosins have been characterized. In this study two platelet membrane subfractions, the external and the internal membranes, were isolated simultaneously from a crude membrane fraction and their purity was characterized using specific marker enzymes. Myosin was shown to be present in both membrane fractions and its structural and immunological properties were investigated. The electrophoretic mobilities of myosin in both membrane preparations were identical to the mobility of its cytoplasmic counterpart. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the maps of the myosins from the external and internal membranes as compared to their soluble counterpart. Our data suggest that myosin is located in three distinct platelet compartments: cytosol, external and internal membranes. The same myosin isoform is located in the two membrane compartments, while the isoform found in the cytosol is different. The observed variations in the structure of the two isoforms may reflect differences in their respective physiological functions.

Altered structural and functional properties of myosins, from platelets of idiopathic scoliosis patients.

Platelets of patients with idiopathic scoliosis (IS) have been shown to have decreased capacity to aggregate and secrete in response to certain agonists. Similarities between the contractile protein system of platelets and muscle have made the platelets a popular model for muscle disease. We attempted to characterize the function and structure of myosin in platelets of IS patients. Blood was obtained from seven IS patients and seven matched non-scoliotic healthy controls. The mean Cobb angle measurement of the IS patients was 35.4 degrees with a mean Risser sign of 2.2. Washed platelets were isolated from the blood, and the contractile proteins from the membrane and the cytosol compartments were isolated and analyzed by two-dimensional peptide mapping. As previously reported (J Biol Chem 258:9290, 1983), peptide maps of normal platelets revealed that the heavy chain of myosin located in the platelet membrane lacks one major spot relative to the cytoplasmic myosin. In IS patients the cytoplasmic myosin lacks the same peptide that is missing in the membrane myosin of normal individuals. In addition, the ATPase specific activity of the cytoplasmic myosin from IS platelets was significantly lower compared with the activity of the cytoplasmic myosin from normal platelets. These results suggest the presence of a fundamental abnormality of IS platelet contractile proteins.

Near UV circular dichroism from biomimetic model compounds define the coordination geometry of vanadate centers in MeVi- and MeADPVi-rabbit myosin subfragment 1 complexes in solution.

The circular dichroism (CD) spectrum was measured from vanadate (Vi) cyclic esters of chiral vicinal diols, hydroxycarboxylates, and cyclodextrines as a function of Vi concentration ([Vi]) and at the lowest energy transitions of the vanadium. At low [Vi] and in the presence of excess vicinal diols, hydroxycarboxylates, or cyclodextrines the CD signal intensity scales linearly with [Vi] indicating the predominance of a monomeric cyclic ester. At higher [Vi], the signal intensity in the presence of the vicinal diols and hydroxycarboxylates become nonlinear in [Vi], indicating formation of a dimeric cyclic ester. Vanadium-51 NMR (51V-NMR) indicates the coordination geometry of several of these model Vi centers in solution and identifies the CD signals characteristic to Vi trigonal bipyramidal (tbp) and octahedral (Oh) coordination geometries from monomeric and dimeric species. The CD spectra from monomeric and dimeric forms of the tbp-coordinated model compounds have two apparent transitions with amplitudes of opposite sign at wavelengths > or = 240 nm. Spectra from the monomeric and dimeric Oh coordinated species are distinct from the tbp-type spectra over the same wavelength domain because of the presence of two additional transitions with opposite sign amplitudes. These model spectra were compared to the vanadate CD spectra from Vi bound to rabbit myosin subfragment 1 (S1) in solution, in the presence of divalent metal cations (MeVi-S1) or trapped with MeADP (MeADPVi-S1). Polymeric MeVi binds to the active site of S1 and the vanadate centers in MnVi-S1 or CoVi-S1 produce a CD signal resembling that from the tbp model. The trapped ATPase transition state analog MeADPVi produces a different CD signal resembling that from the Oh model.