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PubMed1992

LC20 and kinetics of gizzard myosin subfragment-1: digestion with papain vs. S. aureus protease.

Previous reports have shown that papain-digested gizzard subfragment-1 (PAP-S1) has a cleaved regulatory light chain (LC20), and Vmax similar to phosphorylated heavy meromyosin (HMM) (Greene et al., Biochemistry 22:530-535, 1983; Sellers et al., J. Biol. Chem. 257:13880-13883, 1982; Umemoto et al., J. Biol. Chem. 264:1431-1436, 1989], while S. aureus protease-digested S-1 (SAP-S1) has intact LC20, but Vmax closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 inhibits ATPase activity for subfragment-1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP-S1 and SAP-S1. SDS-PAGE showed that SAP-S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP-S1 (15 minutes digestion at 20 degrees C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22-24 KDa fragments and LC20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP-S1 and SAP-S1 had Vmax similar to phosphorylated HMM (1.1-1.5 s-1). SAP-S1 had the same KATPase as phosphorylated HMM (38 microM actin), but KATPase for PAP-S1 was 3-fold stronger (11 microM actin). Subsequent digestion of SAP-S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3- to 5-fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding.

Actins↗
PubMed1976

Morphological and biochemical abnormalities in hearts of cardiac mutant salamanders (Ambystoma mexicanum).

The effect of homozygosity for recessive gene c in Ambystoma mexicanum is the absence of a heartbeat even though initially heart development appears normal. Mutant embryos (c/c) are first distinguishable from their normal siblings (+/+;+/c) at stage 34 (7 days after fertilization) when the normals develop contracting hearts. The mutant hearts at this stage, upon gross examination, appear structurally normal but fail to beat. Nevertheless, the mutants survive through stage 41, which is about 20 days beyond the heartbeat stage, and they exhibit normal swimming movements, indicating that gene c does not affect skeletal muscle. Electron microscopic studies of normal hearts show some myofibrils to be present at stage 34; by stage 41, the normal myocardial cells have become highly differentiated muscle cells. Although some mutant heart cells contain a few thin 60 A and thick 150 A filaments, organized myofibrils are absent. Instead, amorphous proteinaceous collections are prominent. Heavy meromyosin (HMM) binding experiments were performed on mutant hearts to determine whether the myocardial cells contain actin. Mutant myocardial cells that are glycerinated but not treated with HMM contain intact amorphous bodies. After incubation in HMM, the amorphous collections are no longer present and large numbers of decorated actin filaments appear. The.results suggest that the amorphous proteinaceous collections contain actin in a nonfilamentous form, and the addition of HMM induces this actin to polymerize into filaments. SDS-polyacrylamide gel electrophoresis of mutant heart tissue supports this conclusion by showing a prominent 43,000 dalton band suggestive of actin. The electrophoresis experiments also demonstrate a significant reduction of myosin heavy chain (200,000 daltons) in mutant hearts when compared to normal, and this latter observation is confirmed by radioimmunoassay experiments. Muscle tropomyosin (34,000 daltons), prominent in normal hearts, is virtually nonexistent in mutants. Thus, it appears that this single gene mutation affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.

Actins↗
PubMed2005

High molecular mass proteome of androgen-independent prostate cancer.

We made a comprehensive study of protein expression of androgen-independent (AI) prostate cancer by means of agarose two-dimensional gel electrophoresis (agarose 2-DE) followed by liquid chromatography-tandem mass spectrometric analysis. The agarose 2-DE method being good at separating high molecular mass (HMM) proteins and alkaline ones, so we were able to successfully reveal differences between the proteomes of androgen-dependent (AD) tumors and those of AI tumors. During the creation of agarose 2-DE protein maps, we successfully identified 295 proteins (91.0%) out of 324 spots excised in total. Excluding redundant and mouse serum proteins, we considered the remaining 225 proteins to be related to the cancer. We divided the 225 cancer-related proteins into HMM and low molecular mass (LMM) groups by their molecular mass being above or below 80 kDa. Functional classification of the proteins in these two groups showed clear differences between the two: more than half (54.8%) of the HMM proteins, but less than one-third (29.1%) of the LMM ones were classified among transcription- or translation-related proteins. Eighteen proteins were regulated when the tumor progressed from an AD to an AI state. Five of these proteins, including antioxidant protein 2, superoxide dismutase 1, thioredoxin peroxidase, GTP-binding protein beta chain homolog, and the ha1225 gene product, had a function to protect cells against oxidant stress-induced apoptosis. We suggest that prevention against oxidative stress is one of the key points for prostate cancer to obtain androgen independency. We also found proteins that had not been previously reported in prostate cancer by use of genomic or conventional 2-DE based proteomic approaches. The proteomic approach, using agarose 2-DE focused on HMM proteins, has the capability to find novel biomarkers of prostate cancer.

Androgens↗
PubMed1997

Cooperative rigor binding of myosin to actin is a function of F-actin structure.

Many aspects of cooperative behavior within pure F-actin filaments have now been described. We have used two myosin fragments, heavy meromyosin (HMM) and Subfragment 1 (S1), to look at the rigor binding to different forms of F-actin. With Ca2+ bound at the high-affinity metal binding site in actin, there is a very large cooperativity in the binding of HMM, but no cooperativity for S1. With Mg2+ bound at the high affinity site, or with conditions that stabilize the conformation of subdomain-2 of actin, there is no cooperativity seen with either HMM or S1. These results show that the two heads of HMM can induce structural changes in F-actin that are not observed with the single head of S1. They also support the notion that the binding of myosin to F-actin induces a conformational change in subdomain-2 of actin, and that under certain conditions this conformational change can be cooperatively propagated through an actin filament.

Actins↗
PubMed2001

Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants.

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.

Amino Acid Substitution↗
PubMed1993

How are myosin fragments bound to nitrocellulose film?

In an attempt to identify the binding sites of myosin fragments to the nitrocellulose film in a flow cell, proteolytic digestion was performed of myosin fragments bound to the substrate. Chymotryptic digestion of papain.Mg-subfragment-1 (S1) showed that a large proportion of S1 were bound near the tail and that the digestion site was protected for some S1. The binding of S1 near the tail was also confirmed by the production of extra long subfragment-2 (S2) by chymotryptic digestion of heavy meromyosin (HMM). Papain digestion of HMM released slightly smaller HMM from the substrate and impaired the motility at the same time. Hence, the motility of HMM is supported largely by those bound to the substrate near the C terminus. These results demonstrate the importance of flexible regions of myosin in generating active movements.

Binding Sites↗
PubMed1998

Skeletal muscle regulatory proteins enhance F-actin in vitro motility.

Using an in vitro motility assay, we have investigated the effects of rabbit skeletal muscle regulatory proteins, troponin and tropomyosin, on the gliding of F-actin filaments or F-actin filaments containing these regulatory proteins. We demonstrate that Ca2+ does not affect the motility of F-actin gliding on HMM, but does in the presence of skeletal muscle tropomyosin and troponin. We conclude that Ca2+ affects motility through troponin because, like F-actin, F-actin-Tm filaments show no Ca(2+)-dependence to their gliding speeds. Furthermore, there is a large enhancement of the gliding speed (about 75%) in the presence of skeletal muscle tropomyosin, troponin + saturating Ca2+ over that seen with F-actin filaments. This enhancement is not due to the action of tropomyosin alone as skeletal muscle tropomyosin without troponin enhances the speed little (about 5%) over that of F-actin. Thus troponin confers Ca2+ sensitivity to the motility and, additionally, potentiates motility greatly along with tropomyosin in the presence of saturating Ca2+. When [HMM] is varied, the decline in speed of F-actin seen at low HMM density is changed little by tropomyosin in the F-actin-Tm filaments. These data show that the skeletal regulatory proteins interact with F-actin to enhance the interaction with HMM particularly in the presence of troponin and saturating Ca2+ and enhance the gliding speed in the in vitro motility assay as they potentiate the ATPase activity in the isolated proteins. This enhancement of speed in the motility assay cannot be ascribed to tropomyosin alone.

Actins↗
PubMed1998

Three-dimensional image analysis of myosin head in function as captured by quick-freeze deep-etch replica electron microscopy.

Quick-freeze deep-etch replica electron microscopy combined with mica-flake technique provides high contrast, high time- and spatial-resolution images of protein molecules in solution, whose three-dimensional structure is well preserved. Thus, it might be quite useful to obtain structural information of individual functioning molecules, such as myosin crossbridges under in vitro motility assay conditions. With that method, we could actually show that both heads of heavy meromyosin (HMM) crossbridges are mostly straight and bound to actin filaments with about 45 degree tilt-angle under rigor conditions, whereas they attached to actin through only one head with a wide variety of angles under in vitro sliding conditions. We also demonstrated that free HMM heads are strongly kinked in the presence of ATP or ADP/inorganic vanadate (Vi) in contrast to almost straight configuration in the absence of nucleotide. To examine more detailed structure of individual crossbridges, we tried to reconstruct the three-dimensional architecture of intramolecular subdomains of single HMM molecule. We took a series of tilted images of single HMM-ADP/Vi particle and successfully obtained its 3-D image by filtered back-projection, even with restricted range of tilt-angles. By comparison of the reconstruction with the atomic model of subfragment-1 (S1) without nucleotide, we found some great structural difference, which partly might be attributable to the conformational change by nucleotide binding.

Actins↗
PubMed1977

Actin-like filaments in the myoid cell of the testis.

Microfilaments in the myoid cells of the peritubular tissue in the mouse, swine and human testis bind heavy meromyosin (HMM) and form arrowhead complexes. The periodicity of the arrowhead complexes is about 35 nm. Individual filaments show arrowheads that point in the same direction. Opposing polarity of the HMM-bound filaments is also observed. The microfilaments do not bind HMM in the presence of 10 mM ATP. After treatment with the contraction medium of Hoffmann-Berling, the filaments appear to be undulated. These observations indicate that the microfilaments in the myoid cell are actin-like in nature. A small number of thicker filaments (about 10 nm in diameter) which do not bind HMM is also observed in the cell. Microfibrils which have been reported around the human myoid cell are also found in the swine.

Actins↗
PubMed1975

Intracellular localization of actin with fluorescently labelled heavy meromyosin.

An improved technique for fluorescent labelling of heavy meromyosin has made it possible to detect on a light microscopic level the cellular sites of actin localization. Rabbit heavy meromyosin (HMM) was labelled with fluorescein isothiocyanate so that the actin binding site was protected during the reaction. The specificity of fluorescent HMM binding to cellular actin was tested by using glycerinated myofibrils. Staining was most intense in the I-Bands, and decreased at the edges of the A-Band, where actin filaments overlap with myosin. No staining occurred in the H-Zones or in the Z-Bands. The fluorescent HMM could be removed by washing with a relaxing solution. Similar sarcomeric patterns were obtained when embryonic chick skeletal and cardiac muscle cells were stained with fluorescent HMM. Localized fluorescent staining was also observed in smooth muscle fibers, axons and growth cones of nerves, acrosomal caps of sperm, cleavage furrows of dividing cells and pseudopods of various motile cells, all of which are known to contain actin. In sessile cells, the actin was found predominantly in fibrous bundles. This pattern of actin localization was altered when the cells underwent cleavage or became motile. The relationship between the intracellular distribution of actin and its function in the cell is discussed.

Actins↗
PubMed1988

Pharmacokinetics and metabolism of hexamethylmelamine in mice bearing renal cell tumors.

The pharmacokinetics of hexamethylmelamine (HMM) and its main metabolites hydroxymethylpentamethylmelamine (HMPMM), pentamethylmelamine (PMM), and 2,2,4,6, tetramethylmelamine (2,2,4,6 TetrMM) were studied in renal cell (RC) tumor tissues and plasma of CDF1 mice that had received IP bolus injections of the maximally tolerated dose (200 mg/kg) of HMM. HMM, PMM, and 2,2,4,6 TetrMM concentrations determined in RC tissues were much higher than the plasma values, as indicated by the pharmacokinetic parameters (Cmax and AUC). On the other hand, very low levels of HMPMM, generally considered to be a potentially active antitumor compound, were detected in the target tissues, whereas this hydroxylated metabolite was stable and easily determined in plasma. High HMM concentrations in RC tissues could correlate with the high sensitivity of the tumor to this drug. However, the behavior of HMPMM remains unclear; related hypotheses are presented in this paper.

Altretamine↗
PubMed1986

Superior efficacy of trimelamol to hexamethylmelamine in human ovarian cancer xenografts.

A series of eight human ovarian cancer lines grown in nude mice were used to compare the activity of hexamethylmelamine (HMM) and N2,N4,N6-trihydroxy methyl-N2,N4,N6-trimethylmelamine (trimelamol). The tumor lines differed in histological subtype and growth rate. The drugs were administered i.p. at the maximum tolerated dose at alternate days. Differences in volume of treated and control tumors were endpoints of the study. The tumor lines varied widely in sensitivity to HMM and in four lines a T/C% below 25% was achieved. Trimelamol appeared to be more active than HMM and achieved a T/C below 25% in seven tumor lines. Thus far, the drug has demonstrated significant activity in a phase I trial in ovarian cancer patients. Comparative clinical studies of HMM vs trimelamol have not yet been performed.

Altretamine↗
PubMed1992

Structural changes induced in scallop heavy meromyosin molecules by Ca2+ and ATP.

We have used physicochemical and ultrastructural methods to investigate the effects of Ca2+ and ATP on the structure of purified heavy meromyosin (HMM) from the striated adductor muscle of the scallop, a species with myosin-linked regulation. Using papain as a structural probe, we found that, in the presence of ATP, the head/tail junction was five times more susceptible to digestion at high levels of Ca2+ than at low levels. By HPLC gel filtration, two fractions of scallop HMM with different Stokes radii were detected in the presence of ATP at low Ca2+, while at high Ca2+ a single peak with the larger Stokes radius predominated. Electron microscopy of rotary-shadowed HMM suggested that molecules with the smaller Stokes radius had their heads bent back towards their tails, while those with the larger radius had heads pointing away from the tail. The number of molecules with their heads bent back decreased at high Ca2+ levels. The data also showed that in the absence of ATP or at high salt, HMM molecules behaved similarly to those in the presence of ATP at high Ca2+. These results suggest that scallop myosin heads can exist in two conformations (heads down towards the tail and heads up away from the tail) and that the equilibrium between these two conformations is altered by the concentrations of salt, ATP and Ca2+. However, the equilibrium between the two forms appears to be too slow to be involved in regulating contraction. The 'heads-down' configuration may instead be related to the inactive, folded (10S) form of scallop myosin and possibly involved in filament assembly during development.

Adenosine Triphosphate↗
PubMed1990

The calcium ion dependence of scallop myosin ATPase activity.

The ATPase activity of scallop (Pecten maximus) striated adductor myosin and heavy meromyosin (HMM) have been investigated as a function of [Ca2+] using formycin triphosphate (FTP) as a fluorescent ATP analogue. The FTPase activity of the regulated fraction of these preparations was activated steeply over the range of 0.1 to 1 microM [Ca2+], implying the existence of a form of cooperativity that is intrinsic to the myosin heads. In addition to the previously characterised heterogeneity with respect to an unregulated fraction, the regulated fraction of HMM was resolved into two populations whose activities showed a slightly different dependency on [Ca2+]. This was revealed unambiguously at intermediate levels of activation where, in some experiments, the product release rate constants differed for the two populations by more than fivefold. At maximum relaxation or maximum activation, these rate constants differed by two- to three-fold and were not clearly resolved by the multiexponential fitting procedure. The populations might arise as a consequence of isoenzymes, modification during preparation or slowly interconverting conformers; Ca2+ binding itself being a rapid equilibrium process in both populations. FTP turnover by myosin could not be analysed in such detail because of the technical problems of measuring the fluorescence of a suspension of filaments, but the rates of the elementary steps appeared similar to those of HMM. The fraction of unregulated molecules in myosin preparations was comparable to that of HMM indicating that if it is a consequence of preparative damage, the modification must occur prior to tryptic digestion.

Animals↗
PubMed1999

On identification of Na(+) channel gating schemes using moving-average filtered hidden Markov models.

Transitions between distinct kinetic states of an ion channel are described by a Markov process. Hidden Markov models (HMM) have been successfully applied in the analysis of single ion channel recordings with a small signal-to-noise ratio. However, we have recently shown that the anti-aliasing low-pass filter misleads parameter estimation. Here, we show for the case of a Na(+) channel recording that the standard HMM do neither allow parameter estimation nor a correct identification of the gating scheme. In particular, the number of closed and open states is determined incorrectly, whereas a modified HMM considering the anti-aliasing filter (moving-average filtered HMM) is able to reproduce the characteristic properties of the time series and to perform gating scheme identification.

Cell Line↗
PubMed2005

Revealing molecular targets for enterovirus type 71 detection by profile hidden Markov models.

The enterovirus infection in 1998 claimed 78 deaths in Taiwan, with an average of 40 fatalities each year after. Traditional serum-based diagnostic methods often fail to detect enteroviruses due to antigenic changes. As a result, many isolates remain untyped and are absent from the enterovirus surveillance and epidemiological investigations. We present a profile hidden Markov model (HMM) method for molecular typing of enterovirus 71 (EV71). Based on the enteroviral sequences retrieved from GenBank, we build a nucleotide-based and an amino acid-based profile HMM for each EV71 gene using the package HMMER. HMMER bit score-based Z-scores for EV71 and non-EV71 sequences are calculated for each of these profile HMMs. In a genome-wide analysis, we find that the distribution of the EV71 Z-scores and that of the non-EV71 Z-scores have disjoint support for nucleotide-based VP1 profile HMM if the sequence is longer than 150 bases; a VP1-based molecular typing method for EV71 is thus proposed. We also report VP4 an alternative molecular target for detecting EV71, while the two UTRs and all the genes coding the internal proteins cannot be used for such purpose. To demonstrate the performance of the nucleotide-based EV71 VP1 profile HMM, 330 enterovirus VP1 nucleotide sequences newly reported to GenBank are typed with this method. All the EV71 sequences are detected with no error.

Amino Acid Sequence↗
PubMed1992

Demonstration of similar calcium dependencies by mammalian high and low molecular mass phospholipase A2.

The in vitro Ca2+ dependencies of arachidonyl (AA)-selective high molecular mass phospholipase A2 (HMM, 85 kDa-PLA2) and human low molecular mass (LMM-Type II, 14 kDa)-PLA2 were compared. When the LMM-PLA2 and HMM-PLA2 enzymes were examined for hydrolysis against [3H]AA Escherichia coli in an ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA)-free buffer system, neither enzyme demonstrated activity below 10 microM free Ca2+. Beyond 11 microM Ca2+ both enzyme activities increased steadily exhibiting 50% of maximal activity at 0.1 and 1.0 mM, respectively. Using EGTA-regulated free Ca2+ buffers, both enzymes responded in a biphasic manner, achieving 50% of the maximum response by 0.5 microM Ca2+, stabilizing up to 0.1 mM, then further increasing with exposure to millimolar Ca2+ concentrations. Replacement of [3H]AA-labeled phosphatidylethanolamine vesicles for [3H]AA E. coli or using Tris-HCl buffer instead of HEPES buffer did not alter these findings significantly. The presence of EGTA had a pronounced concentration-dependent effect on the activity of both the HMM- and LMM-PLA2 enzymes but only in the range of 0 to 100 microM free Ca2+. EGTA (EC50 approximately 200 microM) reduced the concentration of Ca2+ required by PLA2 to achieve 50% of maximal acylhydrolysis. In contrast, the Type I bovine pancreatic PLA2 required millimolar Ca2+ concentrations to elicit 50% of the maximal response in both EGTA-free or EGTA-containing systems, which is concordant with its extracellular role as a digestive enzyme. These data suggest that the LMM-Type II PLA2 and HMM-PLA2 are both activated at submicromolar, intracellularly relevant, Ca2+ concentrations and therefore have the ability to contribute to cellular lipid metabolism.

Animals↗
PubMed1980

In vitro cytotoxicity of the methylmelamines.

Hexamethylmelamine (HMM) and a number of its derivatives are toxic to PC6 plasmacytoma cells in vitro. However, there is no correlation between in vitro cytotoxicity and activity against this tumour in vivo. N-Methylolmelamines are significantly more toxic than HMM itself. These compounds break down to release formaldehyde which is itself a highly cytotoxic agent. Pentamethylmonomethylolmelamine rapidly inhibits the growth of PC6 cells in culture, whereas HMM and pentamethylmelamine (PMM) require prolonged contact with the cells in order to exert a cytotoxic effect. HMM and its metabolites are also toxic to a number of other cell lines. The toxicity of the N-methylols to cultured L1210 leukaemia and Walker 256 ascites cells appears to be due entirely to formaldehyde release, whereas in PC6 cells the methylols appear to be acting more selectively.

Altretamine↗