ATPase activity of rabbit skeletal muscles actomyosin complex under ultrasound effect

Influence of continuous and impulsive ultrasound 0.05; 0.2; 0.4; 0.7 and 1.0 W/cm2 on ATPase activity of rabbit skeletal muscle actomyosin has been investigated in this work. It has been shown that most changes of Mg2+, Ca(2+)-ATPase activity are observed under 0.2 and 0.4 W/cm2 continuous ultrasound. K(+)-ATPase activity is inhibited by continuous ultrasound of all intensities studied. Impulsive 2 and 10 ms ultrasound did not change the Mg2+,Ca(2+)-ATPase activity. While K(+)-activity is reliably changed only under impulsive 0.7 and 1.0 W/cm2 ultrasound that can be explained by the thermal effect. It has been determined, when studying the reconstructed actomyosin with sound troponin complex, that troponin complex is the most ultrasound sensitive constituent of actomyosin.     

Tissue transglutaminase has intrinsic kinase activity: identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase

Tissue transglutaminase (TG2) is a ubiquitous enzyme that cross-links glutamine residues with lysine residues, resulting in protein polymerization, cross-linking of dissimilar proteins, and incorporation of diamines and polyamines into proteins. It has not previously been known to have kinase activity. Recently, insulin-like growth factor-binding protein-3 (IGFBP-3) has been reported to be phosphorylated by breast cancer cell membranes. We purified the IGFBP-3 kinase activity from solubilized T47D breast cancer cell membranes using gel filtration, ion-exchange chromatography, and IGFBP-3 affinity chromatography. The fractions containing kinase activity were further purified by high pressure liquid chromatography and analyzed by tandem mass spectroscopy. TG2 was detected in fractions containing kinase activity. Antisera to TG2 and protein A-Sepharose were used to immunoprecipitate TG2 from membrane fractions. The immunoprecipitates retained IGFBP-3 kinase, whereas immunoprecipitation deleted kinase activity in the membrane supernatant. The inhibitors of TG2, cystamine and monodansyl cadaverine, abolished the ability of the T47D cell membrane preparation to phosphorylate IGFBP-3. Both TG2 purified from guinea pig liver and recombinant human TG2 expressed in insect cells were able to phosphorylate IGFBP-3. TG2 kinase activity was inhibited in a concentration-dependent fashion by calcium, which has previously been shown to be important for the cross-linking activity of TG2. These data provide compelling evidence that TG2 has intrinsic kinase activity, a function that has not previously been ascribed to TG2. Furthermore, we provide evidence that TG2 is a major component of the IGFBP-3 kinase activity present on breast cancer cell membranes.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.     

Re-examination of the dimerization state of leucine-rich repeat kinase 2: predominance of the monomeric form

Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene have been identified in PARK8, a major form of autosomal-dominantly inherited familial Parkinson's disease, although the biochemical properties of LRRK2 are not fully understood. It has been proposed that LRRK2 predominantly exists as a homodimer on the basis of the observation that LRRK2, with a theoretical molecular mass of 280 kDa, migrates at 600 kDa (p600 LRRK2) on native polyacrylamide gels. In the present study, we biochemically re-examined the nature of p600 LRRK2 and found that p600 LRRK2 was fractionated with a single peak at ~272 kDa by ultracentrifugation on a glycerol gradient. In addition, p600 LRRK2 behaved similarly to monomeric proteins upon two-dimensional electrophoretic separation. These results suggested a monomeric composition of p600 LRRK2 within cells. The p600 LRRK2 exhibited kinase activity as well as GTP-binding activity, and forced dimerization of LRRK2 neither upregulated its kinase activity nor altered its subcellular localization. Collectively, we conclude that the monomer form of LRRK2 is predominant within cells, and that dimerization is dispensable for its enzymatic activity.     

An Engineered Version of Human PON2 Opens the Way to Understand the Role of Its Post-Translational Modifications in Modulating Catalytic Activity

The human paraoxonase 2 (PON2) has been described as a highly specific lactonase hydrolysing the quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and having secondary esterase but not phosphotriesterase activity, in contrast with the related enzymes PON1 and PON3. It has been suggested that PON2 enzyme activity is dependent on glycosylation and its N-terminal region has been recently demonstrated to be a transmembrane domain mediating association to membranes. In the present study we describe a mutated form of PON2, lacking the above N-terminal region, which has been further stabilized by the insertion of six amino acidic substitutions. The engineered version, hence forth called rPON2, has been over-expressed in E.coli, refolded from inclusion bodies and purified, yielding an enzyme with the same characteristics as the full length enzyme. Therefore the first conclusion of this work was that the catalytic activity is independent from the N-terminus and protein glycosylation. The kinetic characterization confirmed the primary activity on 3OC12-HSL; accordingly, in vitro experiments of inhibition of the biofilm formed by Pseudomonas aeruginosa (PAO1) have demonstrated that rPON2 is more effective than PON1. In addition, we observed small but significant activity against organophosphorothiotes pesticides, m-parathion, coumaphos and malathion.The availability of fair amount of active protein allowed to pinpoint, by mass-spectrometry, ubiquitination of Lys 168 induced in rPON2 by HeLa extract and to correlate such post-translational modification to the modulation of catalytic activity. A mutational analysis of the modified residue confirmed the result.     

Functional and biochemical features of alcohol dehydrogenase in four species of the obscura group of Drosophila

The biochemical features of ADH of four Drosophila species of the obscura group have been studied. The relationship between ethanol tolerance and ADH activity has been investigated. Propan-2-ol and acetone concentrations have been determined in propan-2-ol treated flies and ADH activity has been followed during 96 h of propan-2-ol treatment. Data on the ADH system confirm constructed phylogenies based on electrophoretic variation and chromosome homologies.     

Natural killers and interleukin-2 in mice of the strain MRL/MpJ-lpr/lpr

The functional activity of natural killers (NK) in the spleen and lymph nodes and interleukin-2 (IL-2) production have been studied in MPL-MpJ-lpr/lpr (H-2k) mice with genetically predetermined autoimmune diseases and CBA (H-2k) mice. In MPL/l mice it has been shown that NK activity in the spleen was markedly depressed already in the first month of life, whereas in lymph nodes there is a substantial NK activity on days 7-10, which reaches its maximum by the second month and by the 6th months is practically intractable. IL-2 production in MPL/l mice was depressed at all stages of investigation.     

The effect of calcium on the translocation of adenine nucleotides in rat liver mitochondria.

The effect of Ca²⁺ on the adenine nucleotide translocase activity of intact rat liver mitochondria has been studied. The results indicate that in mitochondria which have been allowed to accumulate Ca²⁺, the activity of the translocase is strongly diminished; half-maximal inhibition is attained when approximately 40 nmol of Ca²⁺ are accumulated/mg of mitochondrial protein. Inhibition of electron transport or uncoupling prevents the Ca²⁺-induced inhibition of translocase activity; inhibition of Ca²⁺ uptake by ruthenium red also prevents the inhibition of the exchange. These experiments indicate that internal, but not external Ca²⁺ is responsible for the inhibition of adenine nucleotide translocase activity. Inhibition of the exchange activity by Ca²⁺ occurs even in conditions in which external adenine nucleotide concentrations are rate-limiting.     

Effect of recombinant human alpha-interferon (reaferon) on macrophage functional activity in mice

The influence of human recombinant alpha 2-interferon (reaferon) on the parameters of the phagocytic activity of mouse peritoneal macrophages (migration, spreading, adhesion and absorption of corpuscular antigen) has been studied. Reaferon in doses of 5-5 X 10(2) I. U./ml has been found to produce a stimulating effect on all parameters under study. The data obtained in this study suggest that a stimulating effect on the functional activity of macrophages is the same for recombinant (alpha 2) interferon and natural alpha-interferon.     

Histochemical and cytochemical studies of alkaline phosphatase activity in the synapses of rat brain

Summary Although a number of studies have been carried out on alkaline phosphatase (Al-P), this enzyme has not definitely been detected in synapses at the electron-microscopic level. Recently, we have successfully demonstrated, by perfusing specimens with 1% glutaraldehyde for fixation for as short a time as 8–10 min, that Al-P activity is localized on the presynaptic and postsynaptic membranes of the rat central nervous system (CNS). There were four types of presynaptic membrane: (1) those with the activity only on the membrane, (2) those with the activity only on the synaptic vesicle membrane, (3) those with the activity on both the presynaptic membrane and the synaptic vesicle membrane, and (4) those entirely free of the activity. The postsynaptic membranes were classified into two varieties: (1) those with the activity in the postsynaptic membrane and the postsynaptic thickening, and (2) those entirely without the activity. Thus, the occurrence of the enzyme activity assumed various combinations of presynaptic and postsynaptic involvement. The incidence of synapses either with presynaptic or postsynaptic activity varied distinctly from site to site.     

Synthesis and anti-HIV properties of new water-soluble bis-functionalized[60]fullerene derivatives

A new series of bis-functionalized fullerene C₆₀ derivatives bearing two or more solubilizing chains have been evaluated for their activity against HIV-1 and HIV-2 strains. Some of the compounds show activity against HIV-1 type in the low micromolar range. The effect of the positions of the addends on the C₆₀ nucleus has been investigated, indicating that only trans-2 isomers possess promising activity. The presence of a quaternary pyrrolidinium nitrogen is essential to increase solubility.     

Effect of ATP, ADP and magnesium ions on the activity of phosphorylase kinase from rabbit skeletal muscles

The dependence of enzymatic activity of phosphorylase kinase on ATP and magnesium concentrations has been studied. The enzyme activity has been shown to be inhibited by the substrate surplus (Mg-ATP) but free Mg2+ stimulates the enzyme. At saturating concentrations of ATP the activating effect of Mg2+ is maximum at the Mg/ATP ratio of 6-10. The ADP inhibition action is characterized by an incompetitive type towards ATP. The apparent Ki value is equal to 0.2 mM. It is suggested that the specific ADP-binding site spatially removed from the active site has an importance for the phosphorylase kinase activity regulation.     

Leucine aminopeptidase in extracts of swine muscle

1. Leucine aminopeptidase (EC 3.4.1.1) has been demonstrated in swine muscle at a level of activity one-fifth that of the swine kidney. 2. The enzyme has been purified 110-fold by precipitation with ammonium sulphate, heat treatment and chromatography on Sephadex G-100. 3. The enzyme is heat-stable, but is rapidly inactivated below pH7. It requires Mg(2+) or Mn(2+) for activity. The Michaelis constant for leucine amide with Mg(2+)-activated enzyme is 5.0x10(-3)m. 4. Muscle leucine aminopeptidase is very similar to the kidney enzyme.     

The role of zinc binding in the biological activity of botulinum toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.     

Inhibitory kinetics of mercuric ion on the activity of beta-N-acetyl-D-glucosaminidase from green crab (Scylla serrata)

Chemical modification of p-chloromercuribenzoate (PCMB) on beta-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) from green crab (Scylla serrata) has been studied. The results show that sulfhydryl group is essential for the activity of the enzyme. Inhibitory kinetics of the enzyme by mercuric chloride (HgCl2) has been studied using the kinetic method of the substrate reaction during inhibitor of enzyme. The kinetic results show that the inhibition of the enzyme by mercuric ion (Hg2+) at lower than 1.0 microM is a reversible reaction with residual activity and the inhibition belongs to be competitive. The inhibition kinetics model of Hg2+ on the enzyme was set up and the microscopic rate constants were determined and the data obtained were well fitted with the model. It was also turned out that only one molecule of HgCl2 binds to the enzyme molecule to lead the enzyme lose its activity. The above results suggest that the cysteine residue is essential for activity and is situated at the active site of the enzyme.     

Partial Purification and Characterization of Detergent-Solubilized N-Sulfotransferase Activity Associated with Calf Brain Microsomes

Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.     

Effect of ethanol and yeast on cellulase activity and hydrolysis of crystalline cellulose

The process of bioconverting lignocellulosic materials into ethanol in the simultaneous saccharification and fermentation system depends upon the activity of Penicillium decumbens cellulase. The influence of both ethanol and the yeast on this cellulase activity has been studied and it has been found that ethanol in concentrations between 1% and 7% inhibits the enzymatic hydrolysis of crystalline cellulose but the inhibition is reversible. At ethanol concentrations between 1% and 9%, the activity of β-glucosidase increases with increasing ethanol concentration. Yeast has no effect on the enzymatic activity.     

Too much of a good thing: the role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2

CK2 is a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, constitutively active and has been implicated in multiple cellular functions, as well as in tumorigenesis and transformation. Elevated CK2 activity has been associated with the malignant transformation of several tissues and is associated with aggressive tumor behaviour. While the precise roles of CK2 in tumorigenesis remain incompletely understood, mounting evidence suggests a role for CK2 in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity. Consequently, CK2 has emerged as a potential therapeutic target, and strategies to inhibit CK2 have been ongoing in pre-clinical trials. This review will focus on published evidence highlighting the molecular mechanisms by which CK2 functions in the promotion of tumorigenesis, as well as review current strategies being used to inhibit CK2.