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At least 19 recordsLinked to original sources
PubMed1986

Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies.

The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.

Actins↗
PubMedDate not supplied

In vitro cytotoxicity of hexamethylmelamine (HMM) and its derivatives.

The cytotoxicity of hexamethylmelamine (HMM) and its metabolites was investigated in three murine cell lines: one in vitro naturally sensitive to HMM (RC) and two in vivo naturally resistant (P388 and P388D1). The percentage of viable cells was determined both by the in situ reduction of a tetrazolium salt (MTT assay) and by the uptake of labelled thymidine into DNA (3HTdR assay). Short (1h) and long (48h) exposures of cells to drugs were considered. In all experimental conditions used, HMM was found to be inactive, whereas its hydroxylated metabolite hydroxymethylpentamethylmelamine (HMPMM) and one analog N, N 'dihydroxymethyltetramethylmelamine (DHTMM) were found to be cytotoxic. The results further indicated that HMM must be metabolized before it can exert its cytotoxic effect. The activity of HMPMM and DHTMM was found unlikely to be related to extracellular or intracellular release of formaldehyde.

Altretamine↗
PubMed1986

Interaction between actin and HMM.

It is shown that the interaction between actin and HMM results in a rapid precipitation of acto-HMM gel upon addition of MgATP. This is a simple demonstration of the idea that the formation of myosin filaments is not essential for mechanochemical reaction (muscle contraction) to occur and that the soluble myosin heads are competent to interact with actin to produce mechanical effect. Our findings also strongly support earlier suggestion that each head of one HMM molecule is able to bind to a different actin filament.

Actins↗
PubMed1985

Electric birefringence study of rabbit skeletal myosin subfragments HMM, LMM, and rod in solution.

Electric birefringence measurements and depolarized light scattering experiments were performed with HMM, LMM, and rod, the three fragments of myosin, under conditions (0.3 M KCl, 0.02 M PO4, pH 7.3) the medium currently used for biochemical assays of myosin in its native state as well as of its subfragments. The comparison of myosin and rod relaxation times (17.2 and 22.8 microseconds, respectively) suggests that the average bend angle in the tail is sharper in intact myosin (90 degrees) whereas rod, when detached from the heads, is a more elongated species with an average bend angle of 120-135 degrees. The LMM relaxation time (6.4 microseconds) is consistent with a rigid linear stick model of length 78 nm. Flexibility in myosin tail is thus confirmed as located in the HMM-LMM hinge. LMM and rod did not exhibit any significant variation of their apparent relaxation times with concentration and the decay curves were best fitted by a single exponential, evidence that the concentration of parallel staggered dimers was negligible in the concentration range studied here (0-7 g/l). This observation lends support to previous results obtained with myosin. Respective HMM, LMM, and rod molecular weights and homogeneity as evaluated by SDS-PAGE analysis were correlated to the Kerr constants of their solutions. Large variations in LMM Kerr constants could be related to the loss of a COOH-terminal peptide on prolonged chymotryptic digestion. Electric birefringence combined with depolarized light scattering is presented as a potential method for net charge distribution studies.

Animals↗
PubMed1978

Studies of the chemo-mechanical conversion in artificially produced streamings. I. Reconstruction of a chemo-mechanical system from acto-HMM of rabbit skeletal muscle.

Steady and uniform streamings (SUS) of HMM solutions were set up in the presence of Mg-ATP in a circular slit, on both side-walls of which a Millipore filter was fixed; F-actin filaments from rabbit skeletal muscle were bound onto the Millipore filter by cyanogen bromide in the flow. The direction of the SUS was specificially determined by that of the flow during the fixing of F-actin and was independent of the direction of the initial velocity applied externally to the HMM solutions. The SUS continued for about 90 min with a velocity of about 20 mum/s at 20 degrees C. There was a strong correlation between the acto-HMM ATPase activity and the velocity of SUS when the salt concentration was varied. Moreover, this was also the case when the ATPase activity was controlled by Ca2+, when native tropomyosin was bound to F-actin in the circular slit. Careful examination led to the conclusions that F-actin filaments are fixed on the Millipore filter with a specific polarity and that a chemo-mechanical system had been successfully reconstituted in our "stream cells," in which chemical energy from ATP is converted to the mechanical energy of streaming.

Actins↗
PubMedDate not supplied

Studies of the effect of supplementing protected methionine (HMM-Ca) to dairy cows on milk yield and fertility.

A trial with 90 cows was conducted to examine the effect of supplementing 30 g N-hydroxymethyl-DL-methionine-calcium (HMM-Ca) from 14 days pre calving till 100 days in lactation on the performance and some physiological traits. Cows were kept on four energy levels by feeding different amounts of concentrate. At each energy level, half of the cows were given a methionine supplement. Forage consisted of 1/3 corn- and/or beet pulp silage and 2/3 grass silage ad libitum. The amount of daily milk yield differed not significantly from 31.5 kg for the supplement group to 30.6 kg for the control group. No interaction between the methionine supplementation and energy level could be detected. Mean methionine content in blood plasma differed from 22.7 mmol/l for the supplement group to 16.8 mmol/l for the control group. GOT, tau-GT, bilirubin and urea in blood serum and milk urea content did not indicate a positive effect of the methionine supplementation on the metabolism. Conception rate differed slightly from 87% in the supplement group to 73% in the control group.

Animals↗
PubMed1979

HMM-decorated filaments in leukocyte nucleus.

HMM-decorated filaments could be observed in the leukocyte nucleus under an electron microscope. The filaments were distributed in parallel or randomly among chromatin granules and showed "arrowhead" structures similar to those of cytoplasmic filaments. These observations suggest that the leukocyte nucleus may possess contractile filaments containing actins which probably function in the structural transformation of the nucleus.

Actins↗
PubMed1990

[Distribution of F-actin (DACM-HMM staining) in the epidermis in normal subjects and in cases of psoriasis vulgaris].

The epithelium of normal subjects (NE) and that in cases of psoriasis vulgaris, or PV, (PVE) were studied using DACM-HMM staining. In the living cell layer of NE, strong fluorescence was observed in the cell margins, and these bands of fluorescence were narrower in the granular layer than in the basal and squamous layers. In PV, the bands were broad in the lower and middle strata of the squamous layer, and attenuated in the upper strata. Accompanying keratinization, the distribution of F-actin in the keratinocytes was thought to vary. The variations in fluorescence in the lower and middle squamous layer strata of PVE may be attributed to increase of F-actin in the cell margins.

Actins↗
PubMed1988

[Effect of Ca2+ on the motility of myosin head in the F-actin-HMM complex].

Using polarization microfluorimetry, the effect of Ca2+ on the mode of interaction between heavy meromyosin labeled with 1.5-IAEDANS and F-actin was studied. The results obtained suggest that both the orientation and motility of myosin heads in the F-actin-heavy meromyosin complex depend on Ca2+ concentration. The experimental results are suggestive of the existence of a myosin-coupled regulatory system in skeletal muscles.

Actins↗
PubMed1990

The rigor configuration of smooth muscle heavy meromyosin trapped by a zero-length cross-linker.

When chicken gizzard heavy meromyosin (HMM) in its rigor complex with actin was reacted with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), HMM cross-linked with actin but also the two heads of the HMM molecule cross-linked to each other [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry 28, 1898-1904, 1905-1912]. By ultracentrifugal fractionation of the EDC-treated acto-HMM in the presence of Mg-ATP, we obtained a preparation enriched for gizzard HMM with cross-linked heads. When HMM molecules in this preparation were rotary-shadowed and observed in an electron microscope, many head pairs were in contact with each other. The amount of HMM with cross-linked heads determined by electron microscopy was equal to that of the cross-linked NH2-terminal 24K tryptic fragments of HMM heavy chains determined by NaDodSO4 gel electrophoresis, indicating that this cross-linking is primarily responsible for the contact observed between two HMM heads. Most pairs of the contacted heads originated in the same HMM molecule, although a few pairs belonged to different HMM molecules. Cross-linking between the two heads of the same HMM molecule appeared to occur within the distal, more globular half of each head. However, the cross-linking sites were located at different positions within the globular portion. The actin-activated Mg-ATPase activity of the HMM sample treated with EDC in the presence of actin increased in a biphasic manner, depending on the concentration of F-actin, with two apparent association constants: 2.9 x 10(4) M-1 and one much less than 1 x 10(4) M-1. Since the apparent association constant obtained with the HMM control was similar to the latter value, the association constant for HMM molecules with cross-linked heads was identified to be the former value. The binding of HMM to actin was thus strengthened at least by a factor of 3 by the cross-linking between two HMM heads. These results suggest that HMM heads are trapped by treatment with EDC in the rigor complex configuration and that this configuration is retained even after the HMM has been released from actin. The EDC reactivity of rabbit skeletal muscle HMM, however, was different from that of chicken gizzard HMM. The treatment of acto-HMM complexes with EDC did not generate cross-linking between two skeletal muscle HMM heads.

Actins↗
PubMed1972

Heavy meromyosin: evidence for a refractory state unable to bind to actin in the presence of ATP.

The binding of actin to heavy meromyosin (HMM) in the presence of ATP was studied by analytical ultracentrifuge and ATPase studies. At 0 degrees C, at very low ionic strength, the double-reciprocal plot of HMM ATPase against actin concentration is linear. If one assumes that all of the HMM is bound to actin when the ATPase activity equals V(max), then, at an actin concentration where the actin-HMM ATPase is 85% of V(max), all but 15% of the HMM should be complexed with actin. However, when the binding of HMM to actin in the presence of ATP was measured with the analytical ultracentrifuge, more than 60% of the HMM was not bound to actin. From experiments with EDTA- and Ca-ATPases it seemed unlikely that the unbound HMM was denatured. It is thus possible that during the steady-state hydrolysis of ATP, HMM spends more than 50% of its cycle of interaction with actin and ATP in a "refractory state," unable to bind to actin, i.e., while an HMM molecule goes through one cycle of interaction with actin and ATP, an actin monomer could bind and release several HMM molecules so that the turnover rate per mole of added actin would be considerably greater than that per mole of added HMM. Comparison of the rate of ATPase activity at very high actin concentration with that at very high HMM concentration shows that this is indeed so. Therefore, both kinetic and ultracentrifuge studies suggest that the HMM exists in a refractory state during a large part of its cycle of interaction with actin and ATP.

Actins↗
PubMed1987

Conformational changes in myosin and heavy meromyosin from chicken gizzard associated with phosphorylation.

Heavy meromyosin (HMM) undergoes a conformational transition between a rapidly and a slowly sedimenting form, during which it sediments as a single peak in the ultracentrifuge with sedimentation coefficients between 7.5 and 9S. Changes in sedimentation velocity and ATPase activity produced by changes in ionic strength, phosphorylation of HMM or addition of MgATP are interpreted in terms of equilibria between the rapidly and slowly sedimenting forms, the observed values of activity and sedimentation velocity being determined by the fraction of HMM in each form. Phosphorylation of the 20 kDa light chain or raising the ionic strength decrease the sedimentation velocity, by decreasing the fraction of HMM in the rapidly sedimenting form, while addition of ATP increases sedimentation velocity upon forming a 9S HMM-ADP-Pi complex. Electron microscopic studies support this interpretation showing the presence of two distinct conformations of HMM--extended and flexed, which correspond to the 7.5S and 9S forms, respectively (Suzuki et al., 1985). In samples prepared at high ionic strengths, the heads extend away from the tail in a more or less random orientation, while at low ionic strength, the molecule is flexed at the head-tail junction assuming a more compact structure, that appears to account for its more rapid sedimentation rate. The degradation rates of the heavy chain and the 20 kDa light chain of HMM on digestion with papain indicate the presence of three forms of HMM differing in their susceptibility to papain. At 25 mM NaCl, HMM is rapidly digested in the absence of ATP, while addition of ATP decreases digestibility by a factor of ten, upon formation of a complex of HMM with the products of ATP hydrolysis. Above 0.4 M NaCl, HMM is degraded at an intermediate rate that is not affected by ATP. When the ionic strength is varied, the rate of disappearance of the heavy chain depends linearly on the sedimentation velocity in both the phosphorylated and dephosphorylated states, indicating that the rate of proteolysis is determined primarily by the fraction of HMM in the rapidly and slowly sedimenting forms. The same pattern is seen in the disappearance of the 20 kDa light chain of dephosphorylated HMM on cleavage at a site 4 kDa from the N-terminus, indicating that the cleavage of the light chain also depends on the fraction of HMM in the rapidly and slowly sedimenting forms.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗