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The osteogenic growth peptide (OGP) was characterized recently in regenerating bone marrow (BM) and normal serum. In vitro, the OGP regulates stromal-cell proliferation and differentiated functions. In vivo, an increase in serum OGP accompanies the osteogenic phase of postablation BM regeneration. The present results in normal mice show that OGP induces a balanced increase in WBC counts and overall BM cellularity. In mice receiving myeloablative irradiation and syngeneic or semiallogeneic BM transplants, OGP stimulates hematopoietic reconstruction and doubles the survival rate; these effects are dependent on initiating the OGP administration before irradiation. Chimerism measurements in semiallogeneic graft recipients suggest no preferential effect of OGP on residual host cells. The data implicate OGP in the acceleration of hematopoiesis secondary to expansion of the stromal microenvironment and/or enhancement of stroma-derived signals to stem cells. The low-dose effectiveness of OGP is explained by the demonstration of an autocrine positive feedback loop that together with the OGP-binding protein sustains high serum levels of the peptide. A potential OGP-based treatment in combination with chemoradiotherapy is attractive because of the OGP-induced balanced multi-lineage enhancement of hematopoiesis and possible replacement of expensive recombinant cytokines by a readily synthesized peptide.

Myosin belongs to the family of motor proteins. Its interaction with actin coupled with hydrolysis of ATP is the molecular basis of muscle contraction. The head segment of myosin, called subfragment 1 (S1), contains the distinct binding sites for ATP and actin and responsible for the ATPase activity. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1-MgADP-Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate (Vi) or beryllium fluoride (BeF(x)), highly stabilizes the complex. We studied the role of the divalent cations in the ATPase activity and in the formation and decomposition of PA-containing stable complexes by substituting Mg2+ with Fe2+, Mn2+, Ni2+, Co2+, and Ca2+. These metal ions supported the actin activation of S1 ATPase and affected the obtained kinetic parameters, Km and V(max). The ATPase activity of S1 in the absence of actin increased with the increasing ionic radius of the metal (Me) ions. These ions also substituted for Mg2+ in the formation of the stable ternary S1-MeADP-PA complexes, which cannot be generated in the absence of divalent cations. Upon formation of stable ternary complexes, S1 reversibly loses its ability to catalyze the hydrolysis of ATP. The formation of the complexes can be followed by monitoring the disappearance of the ATPase activity. The rate of the complex formation depends on the divalent cation present and decreases in the order Mn > Fe > Ni > Co > Mg and Ca > Mn > Fe > Mg > Co in the Vi- and BeF(x)-containing complexes, respectively. The ATPase activity of S1 is recovered upon addition of actin, which causes the decomposition of the complex. The spontaneous decomposition of the complexes was studied in the presence of ethylenediaminetetraacetic acid (EDTA), which chelates the metal divalent cations released from the complex and prevents its reformation. The rate of decomposition was assessed by monitoring the recovery of the ATPase activity of S1 in the presence of EDTA. The rate of decomposition of the Vi- and BeF(x)-containing complexes follows the order Mn > Fe > Co > Mg > Ni and Ca >> Mn > Fe > Co > Mg, respectively. The rate of decomposition increases with the increasing ionic radius of the metal ions, similarly as observed in the case of ionic radius dependence of the ATPase activity. On the basis of this similarity, it is assumed that the decomposition of the complexes consists of two steps, the first step being the very slow release of PA followed by a rapid dissociation of MeADP from S1. The stability of the complexes has been calculated from the formation and decomposition rates. Except in the case of Mg, the stabilities of the BeF(x) complexes are higher than those containing Vi. The results indicate that the metal cations have a significant role in maintaining the proper structure of the transient state complex in the myosin-catalyzed ATP hydrolysis.

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.

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.

Six independent mutations in the Caenorhabditis elegans spe-26 gene cause sterility in males and hermaphrodites by disrupting spermatogenesis. Spermatocytes in mutants with the most severe alleles fail to complete meiosis and do not form haploid spermatids. Instead, these spermatocytes arrest with missegregated chromosomes and mislocalized actin filaments, endoplasmic reticulum and ribosomes. In spite of this arrest some of the nuclei and the organelles that normally transport sperm-specific components to the spermatid mature as if they were in spermatids. The spe-26 gene is expressed throughout the testis in both spermatogonial cells and spermatocytes. It encodes a 570-amino-acid polypeptide, which contains five tandem repeat motifs, each of approximately 50 amino acids. These repeats are similar in sequence to repeats in the Drosophila kelch protein, in the invertebrate sperm protein scruin that cross-links actin filaments, as well as in the mouse and pox virus proteins. The functional importance of these repeat motifs is shown by the fact that five of the spe-26 mutations are in the tandem repeats, and one of the most severe mutations is a substitution in a highly conserved glycine. These results suggest that spe-26 encodes a cytoskeletal protein, perhaps actin binding, which is necessary to segregate the cellular components that form haploid spermatids.

The first step in the decay of several yeast mRNAs is the shortening of the poly(A) tail, which for the MFA2 transcript triggers decapping and 5'-to-3' degradation. To understand the basis for differences in mRNA decay rates, it is important to determine if deadenylation-dependent decapping is specific to the unstable MFA2 transcript or is a general mechanism of mRNA degradation. To this end, we analyzed the turnover of the stable PGK1 mRNA by monitoring the decay of a pulse of newly synthesized transcripts while using two strategies to trap decay intermediates. First, we used strains deleted for the XRN1 gene, which encodes a major 5'-to-3' exonuclease in Saccharomyces cerevisiae. In xrn1 delta cells, PGK1 transcripts lacking the 5' cap structure and a few nucleotides at the 5' end were detected after deadenylation. Second, we inserted into the PGK1 5' untranslated region strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates. These secondary structures led to the accumulation of PGK1 mRNA fragments, following deadenylation, trimmed from the 5' end to the site of the secondary structure. The insertion of strong secondary structures into the 5' untranslated region also inhibited translation of the mRNA and greatly stimulated the decay of the PGK1 transcripts, suggesting that translation of the PGK1 mRNA is required for its normally slow rate of decay. These results suggest that one mechanism of degradation of the PGK1 transcript is deadenylation followed by decapping and subsequent 5'-to-3' exonucleolytic degradation. In addition, by blocking the 5'-to-3' degradation process, we observed PGK1 mRNA fragments that are consistent with a 3'-to-5' pathway of mRNA turnover that is slightly slower than the decapping/5'-to-3' decay pathway. These observations indicate that there are multiple mechanisms by which an individual transcript can be degraded following deadenylation.

The atomic structure of myosin subfragment-1 (S1) has been recently solved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and near-ultraviolet CD spectra, the methylation does not affect the secondary structure of S1 but causes limited changes in its tertiary structure. The methylation significantly decreases the affinity of S1 for actin in rigor and, to a lesser degree, that of S1 to actin in the presence of MgATP gamma S. This modification, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on S1 either by phosphate analogs or by cross-linking of the SH1 and SH2 thiols. Methylation strongly impairs the coupling between the actin- and nucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a complete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication between other sites on S1 including that between the nucleotide-binding site and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent photocleavage experiments, respectively. The increased rate of thermal denaturation of S1 and the loss of S1 protection by ADP and actin from this process also indicate flawed communications in methylated S1. It is concluded that these relatively mild but numerous and important changes impair the function of methylated S1.

Beryllium fluoride (BeFx) has been widely used as a phosphate analogue in nucleotide-binding proteins. It was found to bind tightly to F- but not G-actin (Combeau C., and Carlier M. F. (1988) J. Biol. Chem. 263, 17429-17436) and to affect the three-dimensional structure of filaments by stabilizing the subdomain 2 region of the actin promoter (Orlova, A., and Egelman, E. H. (1992) J. Mol. Biol. 227, 1043-1053). In this work we examined the BeFx-induced structural and functional changes in G- and F-actin by using proteolysis, chemical modifications, ATPase, and in vitro motility assays. The results of proteolysis studies show that BeFx binds also to MgADP-G-actin and renders its subdomain 2 region more similar to that in MgATP-G-actin. This is manifested in enhanced subtilisin and decreased tryptic digestions in subdomain 2 of G-actin. BeFx had a strong effect on the proteolysis of MgADP-F-actin: both the tryptic and subtilisin digestions in subdomain 2 were completely inhibited. Significant protection against proteolysis in this region was observed even at 1:14 molar ratios of BeFx to actin indicating cooperative effects on the structure of the actin filament. A similar although milder effect of phosphate on the proteolysis of F-actin suggests that BeFx acts as a phosphate analogue in this system. BeFx also induces changes in the subdomain 1 region of F-actin. This is revealed via reduced rates of Cys-374 alkylation with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and an increased subtilisin cleavage near the C terminus of actin in the presence of BeFx. The BeFx-induced structural changes in actin have little effect on its interactions with myosin. BeFx inhibits only slightly the actin-activated ATPase activity of S1 by decreasing Vmax without affecting KM. Additionally, the binding of BeFx to actin does not change the sliding velocity of actin filaments in the in vitro motility assays. The BeFx-induced specific and distinct changes in G- and F-actin point to the dynamic nature of actin structure and the local differences between monomeric and polymeric forms of actin.

The first step in the decay of some eukaryotic mRNAs is the shortening of the poly(A) tail. To examine how the transcript body was degraded after deadenylation, we followed the decay of a pulse of newly synthesized MFA2 transcripts while utilizing two strategies to trap intermediates in the degradation pathway. First, we inserted strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the MFA2 5' UTR. Following deadenylation, fragments of the MFA2 mRNA trimmed from the 5' end to the site of secondary structure accumulated as full-length mRNA levels decreased. In addition, in cells deleted for the XRN1 gene, which encodes a major 5' to 3' exonuclease in yeast, the MFA2 transcript is deadenylated normally but persists as a full-length mRNA lacking the 5' cap structure. These results define a mRNA decay pathway in which deadenylation leads to decapping of the mRNA followed by 5'-->3' exonucleolytic degradation of the transcript body. Because the poly(A) tail and the cap structure are found on essentially all mRNAs, this pathway could be a general mechanism for the decay of many eukaryotic transcripts.

Decay rates of individual mRNAs in the yeast Saccharomyces cerevisiae can vary by 10- to 20-fold. To determine the basis for the rapid degradation of the mRNA encoded by the yeast MFA2 gene we have used a genetic screen to isolate mutations that increase the stability of this transcript. Analysis of point mutations obtained from this screen, and of additional lesions constructed in vitro, indicated that the MFA2 3'-untranslated region (UTR) contains sequences that specify rapid mRNA decay. Moreover, the lesions that affected mRNA decay rate also affected the process of mRNA deadenylation. Mutations in one region of the 3' UTR both decreased the rate of poly(A) shortening and increased the stability of an intermediate form in the decay pathway with an oligo(A) tail (approximately 10 nucleotides). Mutations in a second region primarily increased the stability of the oligo(A) form. These results suggest that the decay of the MFA2 mRNA initiates with the shortening of the poly(A) tail and there are specific sequences within the 3' UTR that stimulate poly(A) tail shortening as well as subsequent steps in the decay pathway. Given the similarity of this decay pathway to that seen for some mammalian mRNAs, these results suggest that mRNA deadenylation may be a common mechanism of mRNA turnover.

We have incorporated Fe2+ into the high-affinity metal-ion-binding site of actin. By supplying the system with oxygen from air and a reductant (dithiothreitol or ascorbate), we have induced freeradical generation, with the intent of causing peptide cleavage at the metal-ion-binding site. By analysis of the resulting fragments from actin in the F-form, we have deduced that cuts occurred at positions 159–160 and 301–302 (at the latter location we could not be sure if more than one cut occurred). We considered that these two cuts occurred in the chain strand coursing from the outer to the inner domain and vice-versa. Our results harmonize very well with the recently reported atomic structure of actin [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37–44] and remove ambiguities that had remained in the structure. The results partly bear out the homology-based prediction of Strzelecka-Golaszewska et al. [Strzelecka-Golaszewska, H., Boguta, G., Zmorzynshi, S. & Moraczcwska, J. (1989) Eur. J. Biochem. 182, 299–305].

Recently we reported that the isolated 23 kDa N-terminal fragment of myosin heavy chain, which contains the ‘consencus’ ATP binding site, binds to actin in an ATP-sensitive manner (Muhlrad, A. (1989) Biochemistry 28, 4002). In order to determine whether the ‘consencus’ 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 ‘consencus’ 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 ( K assoc = 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 ‘consencus’ 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.

Near-UV irradiation in the presence of vanadate cleaves the heavy chain of myosin subfragment 1 at three specific sites located at 23, 31, and 74 kDa from the N-terminus. Increasing the pH from 6.0 to 8.5, gradually, reduces the efficiency of the cleavage and completely eliminates the 31-kDa cut. Actin specifically inhibits the photocleavage at the sites located 31 and 74 kDa from the N-terminus. ATP strongly protects from cleavage at the 23- and 31-kDa sites and less strongly from the cut at the 74-kDa site. ADP and pyrophosphate have similar, but less pronounced, effects as ATP. Orthophosphate inhibits the photocleavage at the 23- and 74-kDa sites with a similar efficiency. In the ternary actin-S-1-ATP complex, the photocleavage is inhibited at all sites, and the effects of actin and ATP are additive. Photocleavages affect the K + (EDTA)-, Ca 2+ -, and actin-activated ATPase activity of subfragment 1. Loss of all three ATPases is caused by cleavage at the 23-kDa site, while the cut at the 74-kDa site only leads to the loss of acting-activated ATPase activity. It is concluded that subfragment 1 contains at least two distinct phosphate binding sites, the first being part of the «consensus» ATP binding site wherein the 23-kDa photocleavage site is located. This site is responsible for the bindings and hydrolysis of ATP. It is possible that the 31-kDa cleavage site is also associated with the «consensus» site through a loop. The 74-kDa cleavage site a part of another phosphate binding site which may play a role in the regulation of the myosin-actin interaction

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.

Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin--27 kDa, 50 kDa and 20 kDa--aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the 'anti-27 K' and 'anti-50 K' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the 'anti-50 K' to S-1 decreased upon actin binding, while that of the 'anti-27 K' was not affected by binding of S-1 to F-actin. The 'anti-50 K' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the 'anti-27 K' had no effect. The results indicate that either the epitope of the 'anti-50 K' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.

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.

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.