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PubMed1987

19F nuclear magnetic resonance as a probe of structural transitions and cooperative interactions in heavy meromyosin.

An 19F NMR probe has been attached to the reactive sulfhydryl SH1 of the globular heads of rabbit skeletal heavy meromyosin. It serves as a sensitive monitor of the conformational state of the heads of heavy meromyosin in a manner similar to that seen for subfragment-1 (Shriver, J.W., and Sykes, B.D. (1982) Biochemistry 21, 3022-3028; Tollemar, U., Cunningham, K., and Shriver, J.W. (1986) Biochim. Biophys. Acta 873, 243-251). The NMR spectra indicate that there are at least two states for the heads in the SH1 region. The energetics of the interconversion of the two states of heavy meromyosin (HMM) differs significantly from that of S-1. In HMM in the absence of divalent cations, there are two reversible paths between the low temperature and high temperature states with a hysteresis-like behavior. One path is consistent with the head groups behaving independently and similar to S-1 alone. The second path indicates a coupling of the globular head region observed in S-1 with a second region forming a distinctly different cooperative unit. Upon addition of Ca(II) the hysteresis effect is lost and only the second cooperative unit is observed. Two explanations are offered for these results: the globular heads in HMM may couple with the S-2 segment, or the two globular heads of HMM may couple to form a larger cooperative unit. The ability to stabilize the larger cooperative unit with a divalent metal ion implicates a role for the LC2 light chain in coupling regions of the myosin molecule.

Animals↗
PubMed1987

The binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method.

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.

Actins↗
PubMed1984

[Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. VII. The effect of Ca2+ on the nature of the conformational changes in F-actin induced by the binding of heavy meromyosin].

Changes in anisotropy of tryptophan fluorescence and in birefringence of actin filaments induced by the binding to actin of heavy meromyosin (HMM), both containing DTNB light chains and devoid of them, were found in rabbit muscle fibres free of myosin, troponin, and tropomyosin. Ca2+ was shown to affect the pattern of changes in tryptophan fluorescence anisotropy and birefringence of actin filament at the moment of HMM interaction with actin, providing HMM contains DTNB light chains. Anisotropy of tryptophan fluorescence and birefringence of actin filaments rises in the absence of Ca2+ (pCa greater than or equal to 7), while in its presence (pCa less than or equal to 6) these values drop down. Furthermore, these changes become cooperative when Ca2+ concentration increases from pCa = 7 to pCa = 6. It was shown that the binding of HMM devoid of DTNB light chains to F-actin decreases tryptophan fluorescence anisotropy and birefrigence of actin filaments, regardless of Ca2+ concentration. Ca2+-dependent structural changes of F-actin induced by interaction of heads of myosin molecules with actin are assumed to be of great importance in regulation of muscle contraction of vertebrate skeletal muscles.

Actins↗
PubMed1981

Calcium-sensitive binding of heavy meromyosin to regulated actin in the presence of ATP.

Regulated actin, F-actin plus troponin-tropomyosin, activates the myosin ATPase in the presence but not in the absence of calcium. Ultracentrifugation has been used to examine the interaction of regulated actin with two proteolytic fragments of myosin, heavy meromyosin (HMM), a two-headed species, and subfragment 1 (S-1), a single head. In the presence of ATP, the association constant (Ka) for the binding of S-1 to regulated actin is approximately 1.5 X 10(4) M-1, whether or not calcium is present. This is true for S-1 with and without the Mr = 19,000 phosphorylatable light chain. These results confirm the observations of Chalovich et al. (Chalovich, J., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256, 575-578) and support their suggestion that the inhibition of the regulated actin-activated ATPase of S-1 by removal of calcium does not result from preventing S-1 binding. The binding of HMM to regulated actin in the presence of ATP, however, is calcium sensitive. Removal of calcium results in approximately a 4-fold decrease in Ka, from 1.1 X 10(4) M-1 to 2.5 X 10(3) M-1. The results with HMM are more easily reconciled with the low stiffness of relaxed muscle and suggest a functional difference between S-1 and HMM.

Actins↗
PubMed1981

Comparison of DNA damage by methylmelamines and formaldehyde.

The cytoxicity and DNA damaging activity of S9-activated hexamethylmelamine (HMM) and pentamethylmelamine (PMM) were compared with suspected active metabolites in mouse leukemia L1210 cells. The presence of semicarbazide hydrochloride did not alter the cytotoxicity of S9-activated HMM and PMM or that of their hydroxylated analogs monomethylolpentamethylmelamine (MPM) and trimethyloltrimethylmelamine (TTM), which have been suggested as active metabolites. Following treatment of L1210 cells with high concentrations of activated HMM and PMM, there were no DNA single-strand breaks or interstrand cross-links observed by DNA alkaline elution and only a low frequency of DNA-protein cross-links. Formaldehyde (FA) at nonlethal concentrations caused far greater DNA-protein cross-linking. In contrast, the polyfunctional TTM produced both DNA-protein cross-linking and DNA interstrand cross-linking. The cytotoxicities of HMM and PMM were found unlikely to be related to extracellular or intracellular release of FA.

Altretamine↗
PubMed1982

Hexamethylmelamine and pentamethylmelamine tissue distribution in M5076/73A ovarian cancer-bearing mice.

The distribution of hexamethylmelamine (HMM), pentamethylmelamine (PMM), and their metabolites N2,N2,N4,N6-tetramethylmelamine (TMM) and N2,N4,N6-trimethylmelamine (TriMM) was investigated in different tissues of M5076/73A ovarian cancer-bearing mice given 100 mg/kg ip of either drug. The area-under-the-curve (AUC) values of HMM and PMM, expressed in microgram/g x min after these drug treatments, were 2432 and 1296 in tumor, 6290 and 8141 in liver, 9779 and 21,294 in spleen, 6840 and 10,800 in kidney, 4003 and 4295 in heart, 1569 and 1327 in brain, 163,689 and 50,809 in adipose tissue, 88,725 and 45,070 in lymph nodes, 15,033 and 18,992 in small intestine, and 393 and 351 in plasma, respectively. The elimination rate of HMM for the different organs was similar, with a half-life of 37-48 mins; PMM disappeared with more variability, the half-life being 19-46 mins. While TMM concentrations were not very different from those of HMM or PMM, TriMM was much higher in all organs evaluated, particularly the brain, where AUC values were 21-24 times those of the administered drug.

Altretamine↗
PubMed1982

[Structural changes in muscle fiber contractile proteins studied by polarization ultraviolet fluorescence microscopy. VI. Conformational restructurings of F-actin induced by the binding of heavy meromyosin].

Decrease in tryptophan fluorescence anisotropy of a single ghost muscle fibre was found at the binding of heavy meromyosin (HMM) to actin. Polarized fluorescence revealed its peak changes at a molar ratio of HMM/actin equal to 0.1. The changes observed in polarized fluorescence at F-actin-HMM interaction were found to depend on the state of HMM. The changes in anisotropy fluorescence under the same conditions were assumed to be independent of tryptophane residues in HMM, reflecting cooperative changes in F-actin conformation. Changes in the conformation of F-actin are of great importance in muscular contraction.

Actins↗
PubMedDate not supplied

A newly developed hexamethylmelamine derivative, SAE9 with both antitumor and aromatase-inhibitory activity.

Hexamethylmelamine (HMM) has previously been shown to be active against ovarian, breast and small cell lung cancer. However HMM dose not have aromatase-inhibitory activity. A newly developed HMM derivative, 2-N,N-dimethylamino-4, 6-bis (1-H-imidazol-1-yl)-1,3,5-triazine (SAE9), was found to have direct antitumor activity as well as aromatase-inhibitory activity. The direct antitumor activity on breast carcinoma cell lines (MCF-7, R-27 and MDA-MB-231) was assessed using the 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) on cells growing in monolayer culture. The 50% inhibitory concentrations (IC50) of SAE9 were found to be approximately 10(-4) M for each cell line, roughly equivalent to those of HMM. When the aromatase-inhibitory effect was assessed using a human placental aromatase-inhibitory assay, the IC50 of SAE9 was 5.5 x 10(-7) M, which was superior to that of aminoglutethimide (AG) (3.8 x 10(-5) M). In a rat uterine growth model treated with androstenedione as the in vivo aromatase inhibition assay, SAE9 had an effect equivalent to that of AG. Since SAE9 has both antitumor and aromatase-inhibitory activity on breast carcinoma cell lines with estrogen dependency, this and similar non-steroidal aromatase inhibitors are thought to be promising for further study.

Altretamine↗
PubMed1996

Simultaneously recorded single units in the frontal cortex go through sequences of discrete and stable states in monkeys performing a delayed localization task.

To test whether spiking activity of six to eight simultaneously recorded neurons in the frontal cortex of a monkey can be characterized by a sequence of discrete and stable states, neuronal activity is analyzed by a hidden Markov model (HMM). Using the HMM method, we are able to detect distinct states of neuronal activity within which firing rates are approximately stationary. Transitions between states, as expressed by concomitant changes in the firing rates of several units, occur quite abruptly. The significance and consistency of the states are confirmed by comparison with simulated data. The detected states are specific to a monkey's response in a delayed localization task, allowing correct prediction of the response in approximately 90% of the trials. Similar predictive power is achieved by a model based simply on the response histograms (PSTH) of the units. The two models reach this predictive ability with different time courses: the PSTH model gains predictive power with a higher rate in the first second of the delay, and the HMM gains predictive power with higher rate in the next 3 sec. In this later period, conventional methods such as the PSTH cannot detect any firing rate modulations, but the HMM successfully captures transitions between distinct states that are specific to the monkey's behavioral response and occur at highly variable times from trial to trial. Our results suggest that neuronal activity in this later period is described best as transitions among distinct states that may reflect discrete steps in the monkey's mental processes.

Animals↗
PubMed1997

Enantioselective determination of the hydroxylated metabolites of mexiletine in human plasma.

Pre-column derivatization with o-phthaldialdehyde and N-acetyl-l-cysteine was used for liquid-chromatographic diastereomeric resolution of p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM), metabolites of mexiletine formed by aromatic and aliphatic hydroxylation, respectively. The resulting diastereomeric derivatives were resolved on a C18 column and monitored by fluorescence detection. The diastereomeric elution order for both metabolites was determined on the basis of the circular dichroism spectra of each eluted fraction. Plasma samples (500 microliters) showed recoveries greater than 75% for both the metabolites. Calibration curves in plasma samples were linear over the concentration ranges 10-500 and 20-1,000 ng/ml for each enantiomer of PHM and HMM, respectively. The limits of quantitation were found to be 10.0 and 5.0 ng/ml for both enantiomers of PHM and HMM. The within-day and between-day coefficients of variation were less than 10%. The assay was shown to be suitable for a pharmacokinetic study performed in a patient with ventricular arrhythmias following the short-term oral treatment of 200 mg t.i.d. of racemic mexiletine hydrochloride.

Anti-Arrhythmia Agents↗
PubMed1976

Hexamethylmelamine. An evaluation of its role in the therapy of cancer.

Hexamethylmelamine (HMM) has been undergoing clinical trials for about a decade under the sponsorship of the National Cancer Institute. It has been tested in Phase II and III cooperative group studies and has a wide spectrum of activity in solid tumors. Its activity is most marked in ovarian cancer, lymphomas, and carcinoma of the cervix; the drug is also active in bronchogenic carcinoma and carcinoma of the breast. Considerable clinical evidence suggests a lack of cross-resistance between HMM and alkylating agents. the currently popular dose is 300 mg/m2/day p.o. for indefinite periods if tolerated. The dose-limiting toxicity is neurologic, but gastrointestinal side effects and a moderate degree of myelosuppression are also observed. Combination chemotherapy using HMM is underway in ovarian and lung cancer. Futher exploration of its activity as a single agent in tumors such as those of the bladder, prostate, and uterus, and in combination chemotherapy in lymphomas, and mammary, cervical, and pulmonary tumors is warranted.

Altretamine↗
PubMed1994

Characterization of high-molecular-mass heat shock proteins and 42 degrees C-specific heat shock proteins of murine cells.

There are two isoforms of high-molecular-mass heat shock protein (HMM-HSP), hsp105A and hsp105B, in murine FM3A cells. To characterize the HMM-HSPs, we here purified hsp105A and hsp105B, as well as 42 degrees C-specific HSPs that are specifically induced by continuous heating at 42 degrees C, from the cytoplasmic extracts of the FM3A cells heat-shocked at 42 degrees C for 8 h. Digestion of the hsp105A, hsp105B, and 42 degrees C-specific HSPs with lysyl endopeptidase generated 17,000-Da polypeptide fragments in common, and the N-terminal amino acid sequences of the fragments revealed a homology with those of the adenosine binding domain of hsp70 family proteins and actin. Thus, the two isoforms of hsp105 and the 42 degrees C-specific HSPs seemed to be very similar proteins having a ATP binding domain in common, and these HSPs may constitute a HMM-HSP family in murine cells.

Amino Acid Sequence↗
PubMed1996

Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A2B2 heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50-70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B4 forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15-35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.

Amino Acid Sequence↗
PubMed1994

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine kappa-casein.

The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine kappa-casein was studied. A 4-h digest (pH 6.2, 15 degrees C) of kappa-casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1-160, 1-151, 1-95 and 1-79 of kappa-casein, whereas the main LMM products found were the 161-169 and 152-160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160-161 and 151-152 peptide bonds. Two minor LMM products were identified as the fragments 96-104 and 103-106, indicating additional cleavage at positions 102-103, 104-105 and 106-107 of the sequence. Also several peptide bonds within the 161-169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococcal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79-80 and 95-96.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Pharmacokinetics and metabolism of hexamethylmelamine in mice after IP administration.

The pharmacokinetics of hexamethylmelamine (HMM) and its first metabolite (hydroxymethylpentamethylmelamine: HMPMM) following IP bolus dose of 200 mg/kg were studied in mice. The drug concentrations were determined by a sensitive reversed-phase HPLC assay. Thus, for the first time, HMM major hydroxylated and demethylated metabolite plasma levels canbedetermined at the same time. Pharmacokinetic data were analyzed by an original method using a nonlinear cost function minimized by a simplex algorithm. An important property of this computer program is that convergence is ensured in contrast to linear or nonlinear least-square regression analysis, which leads to lack of convergence or to false convergence. Both HMM and HMPMM data fit a one-compartment open model. The parameters obtained indicate that the parent drug would probably be rapidly and completely transformed by the human body into HMPMM.

Algorithms↗
PubMed1985

Structure of myosin decorated actin filaments and natural thin filaments.

Negatively stained paracrystals of reconstituted thin filaments decorated with myosin subfragment 1 (S1), at high calcium concentrations (greater than or equal to 10(-5) M), exhibit pgg plane group symmetry with component filaments having 28 subunits in 13 turns of the actin genetic helix. Isolated S1 decorated F-actin filaments trapped in a stain film were also observed to form spontaneously paracrystals with pgg plane group symmetry. We conclude that a favourable S1-S1 interaction must exist in order to stabilize these structures. Three-dimensional helical reconstructions, calculated from these paracrystals show S1 to be curved, 12 to 14 nm long and tilted with respect to the helical axis, in broad agreement with previous reconstructions calculated from isolated particles. Reconstructions of S1 and HMM decorated filaments that resolve actin show a principal myosin binding site located on the side of the actin subunit reported by Taylor & Amos [J. molec. Biol. 147, 297-324 (1981)] and a possible small interaction on the opposite side. The appearance, symmetry and helical reconstructions of isolated F-actin filaments decorated with heavy meromyosin (HMM) were similar to those of S1 decorated filaments, except at high radii where extra mass was observed. This probably arose from the connection between the two heads of HMM bound to the same long-pitch strand of actin. In contrast to most studies on thin filaments, which use reconstituted filaments, we present data on natural I-segments of muscle homogenates. Individual filaments exhibited actin helical symmetry which on reconstruction gave a two-domain motif oriented consistently with its long axis approximately perpendicular to the helical axis, but inclined towards the 5.9 nm genetic helix. Our original interpretation of these maps [Seymour & O'Brien, Nature, Lond. 283, 680-2 (1980)] depended upon reconstructions from F-actin paracrystals, which suggested actin was rather symmetrical in shape. New data from two- and three-dimensional crystal studies and reconstructions of actin-tropomyosin filaments show that actin is rather elongated and consists of two domains. These results indicate that actin contributes towards both domains of our I-segment motif and are consistent with the monomer long axis lying approximately perpendicular to the helical axis. Although tropomyosin is not resolved, comparison of the actin-tropomyosin and I-segment reconstructions suggests that tropomyosin is strongly merged with the actin domain at a lower radius from the helical axis and that the domain at higher radius arises solely from actin.

Actins↗
PubMed1976

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K+- and Ca2+-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.

Adenosine Triphosphatases↗
PubMed1982

Isolation of a cationic fragment of high molecular mass kininogen and effect of this fragment on human plasma kallikrein activity.

The effect of a cationic fragment of high molecular mass (HMM) kininogen on kallikrein activity and the autocatalytic process of prekallikrein-to-kallikrein conversion were studied. Prekallikrein and kallikrein were obtained by ion exchange and affinity chromatography; the purification of HMM kininogen was achieved using QAE- and DEAE-Sephadex, and separation of the cationic fragment was carried out by gel filtration. It was shown that the fragment of HMM kininogen (mol. mass. 7000, high lysine content) suppressed the activity of a labile form of kallikrein and prevented activation of prekallikrein. A model of the prekallikrein-kallikrein system is proposed. The model is based on the recently established fact of occurrence of two kallikreins and two kallikrein precursors. It involves formation from kininogen, besides kinins, of a kallikrein inhibitor.

Cations↗