Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of the amino-terminal region of myosin alkali 1 light chain (A1) in the interaction between actin and myosin subfragment-1 (S-1) was explored. Papain digestion of skeletal myosin filaments produced S-1 whose A1 was found to lose the basic 13 amino-terminal amino acid residues (A1'). We obtained three types of papain S-1 isoenzymes differing in their alkali light chain content: recombined papain S-1 (A1), papain S-1 (A1'), and papain S-1 (A2). Both the maximum turnover rate (Vmax) and the dissociation constant (Km) for actin-activated papain S-1 (A1') ATPase activity were similar to those for papain S-1 (A2) and remarkably larger than those for recombined papain S-1 (A1). The 13 amino-terminal residue peptide of A1 (N-pep) was isolated and characterized. 1H-NMR spectroscopy suggested that the N-pep was relatively immobilized in the presence of actin filaments. A cross-linking study suggested that N-pep binds to actin. The addition of N-pep to acto-S-1 (A1) made Km and Vmax for the actin-activated ATPase activity close to those for S-1 (A2). Removal of the trimethyl group from the N-pep suppressed the above effect on the actin-S-1 interaction. Our findings suggest that the amino-terminal region of A1 binds to the actin molecule to affect the mechanism of actin-activated S-1 ATPase.
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PMID:Binding of the amino-terminal region of myosin alkali 1 light chain to actin and its effect on actin-myosin interaction. 794 87

The present study was designed to examine the reaction pathway of colloidal bismuth subcitrate (CBS) with thiols. Studies were performed using the monothiol glutathione (GSH), the dithiol dithiothreitol (DTT) and the thiol enzymes papain and H+/K(+)-ATPase. UV-vis spectra showed that CBS forms complexes with GSH and DTT. The GSH/CBS complex but not the DTT/CBS complex was cleared by 5,5'-dithiobis-(2-nitrobenzoic acid). CBS inhibited H+/K(+)-ATPase (IC50: 23 +/- 6.5 mumol/l) but failed to inhibit papain activity. The inhibitory action of CBS on H+/K(+)-ATPase-mediated proton transport was prevented by the dithiol dithioerythritol but not by GSH. These results indicate that CBS forms stable complexes with dithiols and instable complexes with monothiols. We suggest that some of the effects of CBS (i.e., stimulation of prostaglandin production, antibacterial action against Helicobacter pylori) are mediated via the blockade of SH-groups.
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PMID:Studies on the mechanism of action of colloidal bismuth subcitrate. I. Interaction with sulfhydryls. 839 60

We examined whether modification of membrane phospholipids of human erythrocytes by hydrolysis with phospholipase A2 (PLA2 from bee venom) would affect glucose utilization, chosen as a typical model of intracellular metabolism, and, if so, intended to clarify the mechanism of the alteration of glycolysis. Treatment of erythrocytes with PLA2 induced a marked shape change (i.e., crenation) and significantly increased the rate of lactate production from glucose. Available evidence indicated that there is no relevance of this cell-shape change to the alteration of glycolysis. The lack of a detectable effect of papain treatment on glycolysis in PLA2-treated cells suggested that the increase in glycolysis by PLA2 treatment might not be caused by the conformational change of band-3 protein through modulation of membrane phospholipids. The result of the measurement of lactate production in the presence and absence of ouabain did not support the idea that hydrolysis of phospholipids by PLA2 treatment makes plasma membranes leaky to Na+ and consequently enhances glycolysis through activation of Na+/K(+)-ATPase. The action of PLA2 on glycolysis was abolished by extraction of free fatty acids in the cell membrane with bovine serum albumin. Loading erythrocytes with free fatty acid (oleic acid, linoleic acid, or arachidonic acid) caused a significant increase in glycolysis. Analysis of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of 6-phosphofructokinase. The data, thus, indicate that treatment of human erythrocytes with PLA2 significantly accelerates glucose utilization and suggest that the stimulation of glycolysis is caused by activation of 6-phosphofructokinase through liberation of free fatty acids of membrane phospholipids by PLA2.
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PMID:Stimulatory effect of phospholipase A2 treatment on glucose utilization in human erythrocytes. 841 96

The C-terminal 24-kDa fragment of myosin subfragment-1 (S-1) heavy chain was isolated by papain digestion of porcine aorta myosin followed by ethanol fractionation. The isolated 24-kDa fragment was digested by lysylendopeptidase. When the digest was ultracentrifuged with F-actin, 7- and 2-kDa peptides coprecipitated with the actin. These two peptides were isolated by high performance liquid chromatography, and their amino acid sequences were determined. The 7- and 2-kDa peptides correspond to residues 692-744 and 835-846, respectively, of the chicken gizzard myosin heavy chain (Yanagisawa, M., Hamada, Y., Katsuragawa, Y., Imamura, M., Mikawa, T., and Masaki, T. (1987) J. Mol. Biol. 198, 143-157). The 7-kDa peptide contains the reactive cysteine residues, SH1 and SH2. The isolated 7- and 2-kDa peptides also bound to F-actin with dissociation constants of 0.6 and 12 microM, respectively. The 2-kDa peptide was found to compete with rabbit skeletal S-1 for the binding to F-actin by examining the binding of S-1 to actin in the presence of various concentrations of the 2-kDa peptide. The 2-kDa peptide was also shown to interact with the regulatory light chain of aorta myosin by difference UV absorption spectroscopy. The 2-kDa peptide inhibited the actin-activated ATPase activity of skeletal S-1, and the inhibition was canceled by the addition of the isolated regulatory light chain. These results suggest that the newly found 2-kDa peptide region may be related to the regulation of smooth muscle actomyosin ATPase activity by phosphorylation of the regulatory light chain.
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PMID:Actin-binding peptides obtained from the C-terminal 24-kDa fragment of porcine aorta smooth muscle myosin subfragment-1 heavy chain. 842 12

The susceptibility of papain cleavage sites on the cardiac myosin essential light chain (LC1) was studied at low and high Ca2+ concentration in cardiac myosin filaments alone and complexed with pure skeletal actin or cardiac regulated actin in the absence or presence of ATP. Enzymatic properties of cardiac myosin containing papain cleaved LC1 were compared to those of intact myosin. It was found that the kind of divalent cations (Mg2+, Ca2+) saturating the regulatory light chains influences the susceptibility of essential light chains to papain cleavage. The cardiac myosin having shortened essential light chains showed increased affinity for skeletal pure actin and a significant decrease of Ca2+ sensitivity of Mg2+ activated ATPase activity. This was observed both in the case of cardiac myosin complexed with cardiac regulated actin and skeletal actin complexed with cardiac regulated proteins.
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PMID:The influence of regulatory light chains on structural organization of cardiac myosin heads interacting with actin and ATP. 858 50

Single-headed scallop myosin (shM) was prepared by papain digestion of filamentous scallop myosin and purified by hydrophobic interaction chromatography. The shM preparation consisted of equimolar amounts of polypeptides corresponding to an intact heavy chain, rod chain, essential light chain, and regulatory light chain. In electron micrographs the shape of shM showed the presence of a single head domain to which a normal looking rod was attached. Myosin and shM bound Ca2+ with association constants of 5 x 10(6) and 11 x 10(6) M-1, respectively. The ATPase activity of shM was activated about 3-fold by Ca2+. Both heads of myosin and shM had comparable ATPase activities in the presence of Ca2+. The activation of the ATPase activity of single-headed scallop myosin by Ca2+ paralleled closely the Ca2+ binding, in sharp contrast to the activation of intact myosin by Ca2+, which is highly cooperative. Single turnover experiments of myosin with radioactive ATP gave a half-life for the ATPase cycle of approximately 3 min in the presence of EGTA, whereas that of single-headed myosin was shorter than approximately 30 s, which was the resolution time of these measurements. The results suggest that the presence of two heads, as well as the attachment of the head to the coiled coil rod, contribute to the regulation of scallop myosin by Ca2+.
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PMID:Single-headed scallop myosin and regulation. 890 Jan 58

The effects resulting from the removal of the N-terminus of myosin A1 by limited papain cleavage are investigated. The myosin and heavy meromyosin K+-ATPase and Ca2+-ATPase activities, and actin-activated ATPase activity of heavy meromyosin (HMM) and subfragment-1, are studied. Myosin and HMM preparations devoid of the A1 N-terminus exhibits lower Ca2+-ATPase activities at low ionic strength whereas no differences in K+- or Ca2+-ATPase activities are observed at high ionic strength. Direct binding of actin to monomeric myosin under K+-activated ATPase conditions is much more effective for myosin containing a shortened A1 light chain. The kinetic constants K(app) for actin and V(max) are calculated from actin-activation curves for HMM and subfragment-1. The kinetic constants for HMM are determined under conditions assuring saturation of regulatory light chains (RLC) either with Mg2+ or Ca2+. The removal of the A1 N-terminus influences the actin-myosin interaction in a Ca2+- and phosphorylation-dependent manner; in most cases, this leads to an increase in affinity. In the case of subfragment-1, the removal of the N-terminus of A1 led to a decrease in affinity. It is reasonable to assume that the intact A1 light chain may cause weakening of the actin-myosin interaction under certain conditions. This weakening may be regulated by RLC phosphorylation and RLC-bound calcium-for-magnesium exchange. Such an effect requires a structural minimum that is present in HMM but not in subfragment-1. Implications of such a role for the A1 N-terminus in the myosin-actin interaction are discussed.
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PMID:The possible role of myosin A1 light chain in the weakening of actin-myosin interaction. 921 20

We have developed a mass spectrometry based method for the identification of linker regions and domain borders in multidomain proteins. This approach combines limited proteolysis and in-gel proteolytic digestions and was applied to the determination of linkers in the transcription factor NtrC from Escherichia coli. Limited proteolysis of NtrC with thermolysin and papain revealed that initial digestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domain and the still covalently linked OC-domains. Subsequent steps in limited proteolysis afforded further cleavage of the OC-fragment into the O- and the C-domain at accessible amino acid residues. Mass spectrometric identification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker between R- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement with papain and subtilisin digestion. The region between amino acid residues Thr-389 and Gln-396 marked the hitherto unknown linker sequence that connects the O- with the C-domain. High abundances of proline-, alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of related NtrC response regulator proteins. While R- and C-domains remained stable under the applied limited proteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised the sequence from Ile-140 to Arg-320. ATPase activity was lost after separation of the R-domain from the OC-fragment. However, binding of OC- and C- fragments to specific DNA was observed by characteristic band-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain. The outlined strategy proved to be highly efficient and afforded lead information of tertiary structural features necessary for protein design and engineering and for structure-function studies.
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PMID:Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry. 1046 Jan 56

Cells of Lactococcus lactis MG1363 growing in batch culture in TYG (tryptone, yeast extract, glucose) medium at constant pH 7.0 became gradually more acid sensitive shortly after inoculation until a point of maximum sensitivity was reached in early log-phase. The acid tolerance then gradually increased in the mid- and late-log phase until maximum tolerance was reached at the onset of stationary phase. This pattern has been termed the growth-phase acid tolerance. The variation in acid tolerance seen in pH 7.0 grown cells of L. lactis MG1363 did not result from changes in internal pH or membrane H+ ATPase activity levels. Neither the amount of glucose present during mid-log phase nor the amount of lactate produced by the cells correlated with the pattern of the log-phase acid tolerance. Cells grown in partially spent TYG medium showed a reduced growth rate and increased acid tolerance compared to cells grown in fresh TYG medium. Supplementing the spent medium with tryptone or yeast extract or both restored the growth rate and cells became more sensitive to acid. Fractionation of tryptone yielded a fraction which stimulated the growth of MG1363 in partially spent medium and delayed the acquisition of acid tolerance. The active compound(s) has a putative molecular weight of about 1 kDa and was partially degraded by papain and trypsin.
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PMID:Changes in acid tolerance of Lactococcus lactis during growth at constant pH. 1079 46

In order to compare the ability of different isoforms of myosin essential light chain to interact with actin, the effect of the latter protein on the proteolytic susceptibility of myosin light chains (MLC-1S and MLC-1V - slow specific and same as ventricular isoform) from slow skeletal muscle was examined. Actin protects both slow muscle essential light chain isoforms from papain digestion, similarly as observed for fast skeletal muscle myosin (Nieznanska et al., 1998, Biochim. Biophys. Acta 1383: 71). The effect of actin decreases as ionic strength rises above physiological values for both fast and slow skeletal myosin, confirming the ionic character of the actin-essential light chain interaction. To better understand the role of this interaction, we examined the effect of synthetic peptides spanning the 10-amino-acid N-terminal sequences of myosin light chain 1 from fast skeletal muscle (MLC-1F) (MLCFpep: KKDVKKPAAA), MLC-1S (MLCSpep: KKDVPVKKPA) and MLC-1V (MLCVpep: KPEPKKDDAK) on the myofibrillar ATPase of fast and slow skeletal muscle. In the presence of MLCFpep, we observed an about 19% increase, and in the presence of MLCSpep about 36% increase, in the myofibrillar ATPase activity of fast muscle. On the other hand, in myofibrillar preparations from slow skeletal muscle, MLCSpep as well as MLCVpep caused a lowering of the ATPase activity by about 36%. The above results suggest that MLCSpep induces opposite effects on ATPase activity, depending on the type of myofibrils, but not through its specific N-terminal sequence - which differs from other MLC N-terminal peptides. Our observations lead to the conclusion that the action of different isoforms of long essential light chain is similar in slow and fast skeletal muscle. However the interaction of essential light chains with actin leads to different physiological effects probably depending on the isoforms of other myofibrillar proteins.
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PMID:The effects of the interaction of myosin essential light chain isoforms with actin in skeletal muscles. 1242 41


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