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No longer regarded as physiologically inert the endogenous triacylglyceride (TAG) pool within the cardiomyocyte is now recognized to play a dynamic role in metabolic regulation. Beyond static measures of content, the relative rates of interconversion among acyl intermediates are more closely linked to dynamic processes of physiological function in normal and diseased hearts, with the potential for both adaptive and maladaptive contributions. Indeed, multiple inefficiencies in cardiac metabolism have been identified in the decompensated, hypertrophied and failing heart. Among the intracellular responses to physiological, metabolic and pathological stresses, TAG plays a central role in the balance of lipid handling and signaling mechanisms. TAG dynamics are profoundly altered from normal in both diabetic and pathologically stressed hearts. More than just expansion or contraction of the stored lipid pool, the turnover rates of TAG are sensitive to and compete against other enzymatic pathways, anabolic and catabolic, for reactive acyl-CoA units. The rates of TAG synthesis and lipolysis thusly affect multiple components of cardiomyocyte function, including energy metabolism, cell signaling, and enzyme activation, as well as the regulation of gene expression in both normal and diseased states. This review examines the multiple etiologies and metabolic consequences of the failing heart and the central role of lipid storage dynamics in the pathogenic process. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

β-Amyloid (Aβ), a hallmark peptide of Alzheimer's disease, induces both caspase-dependent apoptosis and non-apoptotic cell death. In this study, we examined caspase-independent non-apoptotic cell death preceding caspase activation in Aβ42-treated cells. We first determined the optimal treatment conditions for inducing cell death without caspase activation and selected a double-treatment method involving the incubation of cells with Aβ42 for 4 and 6 h (4+6 h sample). We observed that levels of lamin A (LA) and lamin B (LB) were reduced in the 4+6 h samples. This reduction was decreased by treatment with suc-AAPF-CMK, an inhibitor of nuclear scaffold (NS) protease, but not by treatment with z-VAD-FMK, a pan-caspase inhibitor. In addition, suc-AAPF-CMK decreased the changes in nuclear morphology observed in cells in the 4+6 h samples, which were different from nuclear fragmentation observed in STS-treated cells. Furthermore, suc-AAPF-CMK inhibited cell death in the 4+6 h samples. LA and LB fragmentation occurred in the isolated nuclei and was also inhibited by suc-AAPF-CMK. Together, these data indicated that the fragmentation of LA and LB in the Aβ42-treated cells was induced by an NS protease, whose identity is not clearly determined yet. A correlation between Aβ42 toxicity and the lamin fragmentation by NS protease suggests that inhibition of the protease could be an effective method for controlling the pathological process of AD.

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location.

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π–cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π–cation bridge in 11R-LOX along with Arg106 and Asp173—a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation.

γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its γ-glutamyl group to water (hydrolysis), amino acids or small peptides (transpeptidation). These proteins utilize a conserved Thr residue to process their chains into a large and a small subunit that then form the catalytically competent enzyme. Multiple sequence alignments have shown that some bacterial γ-GTs, including that from Bacillus licheniformis (BlGT), possess an extra sequence at the C-terminal tail of the large subunit, whose role is unknown. Here, autoprocessing, structure, catalytic activity and stability against both temperature and the chemical denaturant guanidinium hydrochloride of six BlGT extra-sequence deletion mutants have been characterized by SDS-PAGE, circular dichroism, intrinsic fluorescence and homology modeling. Data suggest that the extra sequence has a crucial role in enzyme activation and structural stability. Our results assist in the development of a structure-based interpretation of the autoprocessing reaction of γ-GTs and are helpful to unveil the molecular bases of their structural stability.

Plant polyphenol oxidases (PPOs) have a conserved three-domain structure: (i) the N-terminal domain (containing the active site) is connected via (ii) a linker to (iii) the C-terminal domain. The latter covers the active site, thereby maintaining the enzyme in a latent state. Activation can be achieved with SDS but little is known about the mechanism. We prepared domain-swap variants of dandelion PPO-1 and PPO-2 to test the specific functions of individual domains and their impact on enzyme characteristics. Our experiments revealed that the C-terminal domain modulates the pH optimum curve and has a strong influence on the optimal pH value. The linker determines the SDS concentration required for full activation. It also influences the SDS concentration required for half maximal activation (kSDS) and the stability of the enzyme during prolonged incubation in buffers containing SDS, but the N-terminal domain has the strongest effect on these parameters. The N-terminal domain also determines the IC50 of SDS and the stability in buffers containing or lacking SDS. We propose that the linker and C-terminal domain fine-tune the activation of plant PPOs. The C-terminal domain adjusts the pH optimum and the linker probably contains an SDS-binding/interaction site that influences inactivation and determines the SDS concentration required for activation. For the first time, we have determined the influence of the three PPO domains on enzyme activation and stability providing insight into the regulation and activation mechanisms of type-3 copper proteins in general.

We earlier proved the involvement of an autocatalytic step in the oxidation of H 2 by HynSL hydrogenase from Thiocapsa roseopersicina , and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H 2 , we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme–substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein–protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1–K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3–K5CD (K3K4K5CD), K2–K5CD (K2K3K4K5CD) and K1–K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1–2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the ‘addition’ of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the “long-range” enzyme–substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme–substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.

Background Many tyrosinase inhibitors find application in cosmetics and pharmaceutical products for the prevention of the overproduction of melanin in the epidermis. A series of 5-(substituted benzylidene)hydantoin derivatives 2a–2k were prepared, and their inhibitory activities toward tyrosinase and melanin formation were evaluated. Methods The structures of the compounds were established using 1H and 13C NMR spectroscopy and mass spectral analyses. All the synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity. Results The best results were obtained for compound 2e which possessed hydroxyl group at R2 and methoxy group at R3, respectively. We predicted the tertiary structure of tyrosinase, simulated its docking with compound 2e and confirmed that this compound interacts strongly with mushroom tyrosinase residues as a competitive tyrosinase inhibitor. In addition, we found that 2e inhibited melanin production and tyrosinase activity in B16 cells. Conclusions Compound 2e could be considered as a promising candidate for preclinical drug development in skin hyperpigmentation applications. General significance This study will enhance understanding of the mechanism of tyrosinase inhibition and will contribute to the development of effective drugs for use hyperpigmentation.

The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.

During peroxisomal α-oxidation, the CoA-esters of phytanic acid and 2-hydroxylated straight chain fatty acids are cleaved into a (n-1) fatty aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase (HACL1). HACL1 is imported into peroxisomes via the PEX5/PTS1 pathway, and so far, it is the only known peroxisomal TPP-dependent enzyme in mammals. In this study, the effect of mutations in the TPP-binding domain of HACL1 on enzyme activity, subcellular localisation and oligomerisation was investigated. Mutations of the aspartate 455 and serine 456 residues within the TPP binding domain of the human HACL1 did not affect the targeting upon expression in transfected CHO cells, although enzyme activity was abolished. Gel filtration of native and mutated N-His6-fusions, expressed in yeast, revealed that the mutations did not influence the oligomerisation of the (apo)enzyme. Subcellular fractionation of yeast cells expressing HACL1 showed that the lyase activity sedimented at high density in a Nycodenz gradient. In these fractions TPP could be measured, but not when mutated HACL1 was expressed, although the recombinant enzyme was still targeted to peroxisomes. These findings indicate that the binding of TPP is not required for peroxisomal targeting and correct folding of HACL1, in contrast to other TPP-dependent enzymes, and suggest that transport of TPP into peroxisomes is dependent on HACL1 import, without requirement of a specific solute transporter.

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM ( P g-binding group A streptococcal M -like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand–protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients (S°20,w) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence.

SIRT1 is the closest mammalian homologue of enzymes that extend life in lower organisms. Its role in mammals is incompletely understood, but includes modulation of at least 34 distinct targets through its nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase activity. Recent experiments using small molecule activators and genetically engineered mice have provided new insight into the role of this enzyme in mammalian biology and helped to highlight some of the potentially relevant targets. The most widely employed activator is resveratrol, a small polyphenol that improves insulin sensitivity and vascular function, boosts endurance, inhibits tumor formation, and ameliorates the early mortality associated with obesity in mice. Many of these effects are consistent with modulation of SIRT1 targets, such as PGC1alpha and NFkappaB, however, resveratrol can also activate AMPK, inhibit cyclooxygenases, and influence a variety of other enzymes. A novel activator, SRT1720, as well as various methods to manipulate NAD(+) metabolism, are emerging as alternative methods to increase SIRT1 activity, and in many cases recapitulate effects of resveratrol. At present, further studies are needed to more directly test the role of SIRT1 in mediating beneficial effects of resveratrol, to evaluate other strategies for SIRT1 activation, and to confirm the specific targets of SIRT1 that are relevant in vivo. These efforts are especially important in light of the fact that SIRT1 activators are entering clinical trials in humans, and "nutraceutical" formulations containing resveratrol are already widely available.

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD+ synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD+ synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.

A number of extensive reviews are available discussing the roles of MMPs in various aspects of cancer progression from benign tumor formation to overt cancer present with deadly metastases. This review will focus specifically on the evidence functionally linking the MMPs and tumor-induced angiogenesis in various in vivo models. Emphasis has been placed on the cellular origin of the MMPs in tumor tissue, the requirement of proMMP activation and the resulting proteolytic activity for the induction and progression of tumor angiogenesis, and the pleiotropic roles for some of the MMPs. The functional mechanisms of the angiogenic MMPs are discussed as well as their catalytic detection in complex biological systems. In addition, the contribution of active MMPs to metastatic spread and establishment of secondary metastasis will be discussed in view of the findings indicating that MMPs are involved in the preparation of pre-metastatic niches. Finally, the most recent evidence, indicating the pro-metastatic consequences of anti-angiogenic therapies employing MMP inhibitors will be presented as examples highlighting possible outcomes of interfering with the pleiotropic nature of the MMP functionality.

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme which hydrolyzes bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine. NAAA shows acidic pH optimum in terms of both catalytic activity and maturation by specific proteolysis. However, molecular mechanism involved in this characteristic pH dependency remained unclear. Here we report the important role of Glu-195 of human NAAA by analyzing the mutants E195A and E195Q overexpressed in human embryonic kidney 293 cells. Concanamycin A, raising lysosomal pH, inhibited maturation of the wild-type, but not of the Glu-195 mutants. The purified precursors of the mutants, but not the wild-type, were proteolytically cleaved at pH 7.4 during 24-h incubation. Furthermore, when assayed for N-palmitoylethanolamine-hydrolyzing activity at different pH, the mutants did not exhibit a sharp peak around pH 4.5 in the pH-dependent activity profile. Mutants of other seven glutamic acid residues did not show such an abnormality. These results suggested a unique role of Glu-195 in the pH-dependent activity and proteolytic maturation. Moreover, Arg-142, Asp-145, and Asn-287 as well as previously identified Cys-126 were shown to be essential for the proteolytic activation. Since these residues were predicted to be catalytically important, the results strongly suggested that the proteolysis occurs through an autocatalytic mechanism.

A mathematical model is presented to describe the catalytic mechanism of mammalian NAD-linked isocitrate dehydrogenase (NAD-IDH), a highly regulated enzyme in the tricarboxylic acid cycle, a crucial pathway in energy metabolism and biosynthesis. The mechanism accounts for allosteric regulation by magnesium-bound isocitrate and EGTA and calcium-bound ATP and ADP. The developed model is used to analyze kinetic data for the cardiac enzyme and to estimate kinetic parameter values. Since the kinetic mechanism is expressed in terms of chemical species (rather than biochemical reactants), the model explicitly accounts for the effects of biochemical state (ionic strength, pH, temperature, and metal cation concentration) on the kinetics. Because the substrate isocitrate competes with allosteric activators (ATP and ADP) and an inhibitor (EGTA) for metal ion cofactors (Ca(2+) and Mg(2+)), the observed kinetic relationships between reactants, activator and inhibitor concentrations, and catalytic flux are complex. Our analysis reveals that under physiological conditions, the ADP/ATP ratio plays a more significant role than Ca(2+) concentration in regulating the enzyme's activity. In addition, the enzyme is highly sensitive to Mg(2+) concentration in the physiological range, pointing to a potential regulatory role of [Mg(2+)] in mitochondrial energy metabolism.

The peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor family and plays a central role in the regulation of lipid metabolism, glucose homeostasis and inflammatory processes. In addition to its ligand-induced activation, PPARalpha is regulated by phosphorylation via ERK-MAPK, PKA and PKC. In this study we examined the effect of p38-MAPK on PPARalpha transcriptional activity. In COS-7 cells, anisomycin, a p38 activator, induced a dose-dependent phosphorylation of PPARalpha and a 50% inhibition of its transcriptional activity. In H4IIE hepatoma cells, anisomycin-induced p38 phosphorylation decreased both endogenous and PPARalpha ligand-enhanced L-CPTI and ACO gene expression. Interestingly, PPARalpha/p38 interaction required the molecular adapter ZIP/p62. Reducing ZIP/p62 expression by siRNA, partially reversed the inhibitory effect of anisomycin on L-CPTI gene expression. In conclusion, we showed that p38 activation induced PPARalpha phosphorylation and inhibition of its transcriptional activity through a trimeric interaction between p38-MAPK, ZIP/p62 and PPARalpha.

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.

Positional specificity of NodB-like domain of a multidomain xylanase U from Clostridium thermocellum (CtAxe) was investigated. Of three monoacetates of 4-nitrophenyl beta-d-xylopyranoside the acetylxylan esterase domain showed a clear preference for the 2-acetate. Moreover, the enzyme was significantly activated by Co(2+). Acetylated methyl beta-d-xylopyranosides were deacetylated slightly better at position 3 than at position 2, suggesting that the enzyme binds the substrate with the small methyl aglycone also in the opposite orientation. Nevertheless, both positions 2 and 3 of methyl beta-d-xylopyranoside were deacetylated much faster in the presence of the activating metal ion. In contrast, replacement of the hydroxyl group at either of these positions with fluorine or hydrogen, as well as acetylation of both positions, abolished the enzyme activity, regardless the absence or the presence of Co(2+). Thus, the presence of the free vicinal hydroxyl group seems to be a prerequisite not only for an efficient deacetylation of position 2 or 3, but also for the activation of the enzyme with cobalt ion. The demonstrated involvement of the vicinal hydroxyl groups in the mechanism of deacetylation is in accord with 3-D structures of CtAxe as well as other CE4 metal-dependent deacetylases.

Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A2 (PLA2) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir–Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.

Phosphatidylinositol 4-phosphate 5-kinases [PtdIns4P5Ks] synthesise the majority of cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and phospholipase D1 (PLD1) synthesises large amounts of phosphatidic acid (PtdOH). The activities of PtdIns4P5Ks and PLDs are thought to be coupled during cell signalling in order to support large simultaneous increases in both PtdIns(4,5)P(2) and PtdOH, since PtdOH activates PtdIns4P5Ks and PLD1 requires PtdIns(4,5)P(2) as a cofactor. However, little is known about the control of such a system. Membrane recruitment of ADP-ribosylation factors (Arfs) activates both PtdIns4P5Ks and PLDs, but it is not known if each enzyme is controlled in series by different Arfs or in parallel by a single form. We show through pull-down and vesicle sedimentation interaction assays that PtdIns4P5K activation may be facilitated by Arf-enhanced membrane association. However PtdIns4P5Ks discriminate poorly between near homogeneously myristoylated Arf1 and Arf6 although examples of all three known active isoforms (mouse alpha>beta, gamma) respond to these G-proteins. Conversely PLD1 genuinely prefers Arf1 and so the two lipid metabolising enzymes are differentially controlled. We propose that isoform selective Arf/PLD interaction and not Arf/PtdIns4P5K will be the critical trigger in the formation of distinct, optimal triples of Arf/PLDs/PtdIns4P5Ks and be the principle regulator of any coupled increases in the signalling lipids PtdIns(4,5)P(2) and PtdOH.

The regulation of ATP synthase activity is complex and involves several distinct mechanisms. In bacteria and chloroplasts, subunit epsilon plays an important role in this regulation, (i) affecting the efficiency of coupling, (ii) influencing the catalytic pathway, and (iii) selectively inhibiting ATP hydrolysis activity. Several experimental studies indicate that the regulation is achieved through large conformational transitions of the α-helical C-terminal domain of subunit epsilon that occur in response to membrane energization, change in ATP/ADP ratio or addition of inhibitors. This review summarizes the experimental data obtained on different organisms that clarify some basic features as well as some molecular details of this regulatory mechanism. Multiple functions of subunit epsilon, its role in the difference between the catalytic pathways of ATP synthesis and hydrolysis and its influence on the inhibition of ATP hydrolysis by ADP are also discussed.

The regulation of AVP-induced AQP2 expression was investigated in the present study. AVP administration induced AQP2 expression in a dose-dependent manner in association with an increase in intracellular cAMP concentration. PKA activity was stimulated by AVP but PKA inhibitors did not block the upregulation of AQP2 expression. However, AVP also activated both ERK and CREB pathways, and ERK inhibitor attenuated the upregulation of AQP2 expression. These results therefore indicate that the effect of AVP stimulation to upregulate AQP2 expression involves a PKA-independent pathway.

Glucose triggers posttranslational modifications that increase the activity of the Saccharomyces cerevisiae plasma membrane H+-ATPase (Pma1). Glucose activation of yeast H+-ATPase results from the change in two kinetic parameters: an increase in the affinity of the enzyme for ATP, depending on Ser899, and an increase in the Vmax involving Thr912. Our previous studies suggested that Ptk2 mediates the Ser899-dependent part of the activation. In this study we find that Ptk2 localized to the plasma membrane in a Triton X-100 insoluble fraction. In vitro phosphorylation assays using a recombinant GST-fusion protein comprising 30 C-terminal amino acids of Pma1 suggest that Ser899 is phosphorylated by Ptk2. Furthermore, we show that the Ptk2 carboxyl terminus is essential for glucose-dependent Pma1 activation and for the phosphorylation of Ser899.

The principal objective of this study was to investigate the mechanisms regulating the activity of gamma-glutamylcysteine ligase (GCL; EC 6.3.2.2), the rate limiting enzyme in glutathione biosynthesis. Two phylogenetically divergent species, mouse and the fruitfly, Drosophila melanogaster were used to test the hypothesis that reversible protein phosphorylation and pyridine dinucleotide phosphate dependent allostery regulate GCL activity. GCL was almost completely inhibited under phosphorylating conditions, involving preincubations with MgATP and endogenous protein kinases. Maximal GCL inhibitions of 94%, 77%, 85%, 87%, 83%, 95% and 89% occurred, respectively, in mouse cerebellum, hippocampus, brainstem, striatum, cortex and heart, and Drosophila. These changes in GCL activity were detected using saturating levels of substrates, suggesting that V(max) was dramatically affected, whereas K(m) values showed no differences. In vitro activation of GCL, presumably due to dephosphorylation, was blocked by inhibitors of protein phosphatases, suggesting that GCL exists in vivo as a mixture of phosphorylated and dephosphorylated forms. The reversibility of the dephosphorylation-dependent activation was indicated by the time-dependent inactivation of the in vitro activated Drosophila GCL, by preincubation with MgATP. NADPH increased maximal GCL activity by up to 93%, whereas several other nucleotide analogues did not, thereby demonstrating specificity. Kinetic analysis using Hanes-Woolf replots of initial velocity data suggested that the NADPH-dependent stimulation of GCL activity is brought about by a change in the maximal activity, V(max), rather than changes in substrate affinity. Results of this study suggest that mechanisms of modulation of eukaryotic GCL enzymes may include specific binding of ligands such as pyridine dinucleotide phosphates and reversible protein phosphorylation.

Acetate has been found as an endogenous metabolite of beta-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in beta-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.

Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor κB ligand (RANKL), an inducer of osteoclastogenesis via its receptor RANK. We recently demonstrated that OPG also exerts a direct effect in osteoclasts by regulating protease expression. Herein, we showed that OPG-induced pro-matrix metalloproteinase-9 activity was abolished by ras/MAPK inhibitors in purified osteoclasts. OPG induced the phosphorylation of p38 and ERK1/2 in RAW264.7 cells. Only p38 activation was totally abolished by a blocking anti-RANKL antibody or an excess of RANKL. Surface plasmon resonance experiments revealed that RANK, RANKL and OPG are able to form a tertiary complex. These results suggested a potential formation of a tertiary complex RANK–RANKL–OPG on osteoclasts. Thus, OPG is not only a soluble decoy receptor for RANKL but must be also considered as a direct effector of osteoclast functions.

Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug.

Inactivation of PPX1 encoding a major exopolyphosphatase (PPX1) in Saccharomyces cerevisiae results in a change of exopolyphosphatase spectra in the yeast cells. In the PPX1-deficient strain, an elimination of approximately 45 kDa enzyme is observed in cytosol and cell envelopes, and the activity of an exopolyphosphatase with a molecular mass of approximately 830 kDa increases 5-fold in the cytosol. These two enzyme activities differ greatly from each other not only in molecular masses but also in biochemical properties. Inactivation of PPX1 does not result in any changes in the content and properties of vacuolar exopolyphosphatase as compared with the wild strain of S. cerevisiae. In response to PPX1 mutation, exopolyphosphatase properties in the cell as a whole undergo modifications including the ability to hydrolyze polyphosphates with different lengths of the chain.

Lysozyme from hen egg white was identified as an immunoglobulin production stimulating factor (IPSF) that enhances immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of lysozyme was facilitated by heat treatment. The heat treatment of lysozyme at 83 degrees C for 30 min activated its specific IPSF effect 30.0-fold compared with that of native lysozyme. The IPSF activity of lysozyme heat-treated at 83 degrees C in 4 M urea solution was enhanced 8.4-fold than that of native lysozyme. However, lysozyme that was not heated in 4 M urea solution completely lost its IPSF activity. This means that the IPSF activity of this enzyme in 4 M urea was reactivated by thermal treatment. Moreover, coexistence of 0.5 mM 2-mercaptoethanol (2-ME) during heating in 4 M urea solution extremely enhanced the IPSF activity up to 77.8-fold. The uptake of lysozyme by hybridoma cells was enhanced by heat denaturation in 4 M urea. The hydrophobicity of lysozyme was extremely increased by heat-treatment in 2-ME containing urea solution. It is expected from these findings that the increase in the hydrophobicity caused the enhancement of incorporation of lysozyme into target cells, and resulted in the acceleration of IgM production.

Ibuprofen and its major metabolites were incubated with catalase and fumarase, in the presence of protein-modifying biomolecules, to explore the mode of action of ibuprofen in protection against cataract. Both 2 and 10 mM ibuprofen/metabolites protected catalase against fructose-, cyanate- and prednisolone-induced inactivation; the carboxy-metabolite gave the highest protection (31%). The 2 mM ibuprofen/metabolites protected fumarase against fructose- and cyanate-induced inactivation by up to 26%, but had no effect on prednisolone-induced inactivation. Ibuprofen/metabolites did not bind to catalase or fumarase. They penetrated into the lens in vitro. When in the lens, the metabolites may reduce the risk of cataract by protecting lenticular enzymes.

Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1).

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.

Liberation of arachidonic acid by cytosolic phospholipase A(2) (cPLA(2)) upon cell activation is often the initial and rate-limiting step in leukotriene and prostaglandin biosynthesis. This review discusses the essential features of cPLA(2) isoforms and addresses intriguing insights into the catalytic and regulatory mechanisms. Gene expression, posttranslational modification and subcellular localization can regulate these isoforms. Translocation of cPLA(2)alpha from the cytosol to the perinuclear region in response to calcium transients is critical for the immediate arachidonic acid release. Therefore, particular emphasis is placed on the mechanism of the translocation and the role of the proteins and lipids implicated in this process. The regional distribution and cellular localization of cPLA(2) may help to better understand its function as an arachidonic acid supplier to downstream enzymes and as a regulator of specific cellular processes.

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.

Protein disulfide isomerase (PDI), a member of the thioredoxin (Trx) superfamily, consists of five consecutive domains, a-b-b'-a'-c. Domain combinations, AB, A'C, B'A'C and AB-C, and hybrids of PDI domains with Trx, Trx-C and Trx-B'A'C, have been constructed and expressed in Escherichia coli to examine the contributions of PDI domains to its enzyme and chaperone activities. All the combination and hybrid products are considerably less active than intact PDI in their enzyme activities. Recombinant products containing C, at low concentrations, inhibit the reactivation of lysozyme in HEPES buffer, while those without C do not. Only the intact PDI molecule and the hybrid molecule, Trx-B'A'C, but to a much lower level, show general chaperone activity in assisting the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase. It is suggested that all domains of PDI contribute to the binding of target protein for its chaperone activity.

Nitric oxide (NO) and its derivatives peroxynitrite and S-nitrosothiols inhibit mitochondrial respiration by various means, but the mechanisms and/or the reversibility of such inhibitions are not clear. We find that the NO-induced inhibition of respiration in isolated mitochondria due to inhibition of cytochrome oxidase is acutely reversible by light. Light also acutely reversed the inhibition of respiration within iNOS-expressing macrophages, and this reversal was partly due to light-induced breakdown of NO, and partly due to reversal of the NO-induced inhibition of cytochrome oxidase. NO did not cause inhibition of complex I activity within isolated mitochondria, but 0.34 mM peroxynitrite, 1 mM S-nitroso-N-acetylpenicillamine or 1 mM S-nitrosoglutathione did cause substantial inhibition of complex I activity. Inhibition by these reagents was reversed by light, dithiothreitol or glutathione-ethyl ester, either partially or completely, depending on the reagent used. The rapid inhibition of complex I activity by S-nitroso-N-acetylpenicillamine also occurred in conditions where there was little or no release of free NO, suggesting that the inhibition was due to transnitrosylation of the complex. These findings have implications for the physiological and pathological regulation of respiration by NO and its derivatives.

The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2).

A human lens proteinase was purified by a five-step procedure that included two consecutive size-exclusion agarose A 1.5 m chromatographies, a preparative non-denaturing gel-electrophoretic separation, HPLC on a size-exclusion column (TSK G-3000 PW(XL)) followed by preparative isoelectric focusing. A 2300-fold purified enzyme showed a major band of 22 kDa during SDS-PAGE, a pH optimum of 7.8, pI between 4.5 and 5.0, a loss of activity above 45 degrees C and a serine type nature. The partial N-terminal sequence of the enzyme, i.e. P-M-P-G-S-L-G-P-W, matched with the sequence of human lens betaA3/A1-crystallin starting at residue No. 23. Based on the Western blot results of the enzyme with five different site-specific polyclonal antibodies raised against betaA3/A1-crystallin, it was concluded that the 22 kDa crystallin enzyme had a cleaved N-terminus but an intact C-terminus. The betaA3/A1-crystallin, isolated from human lenses, also exhibited proteinase activity following detergent activation and size-exclusion chromatography. The mouse recombinant betaA3/A1-crystallin proteinase was purified by the above five-step procedure, from a homogenate of Sf-9 cells transfected with baculovirus containing the full length coding sequence of betaA3/A1-crystallin. The mouse 22 kDa species also exhibited proteinase activity and immunoreactivity with anti-betaA3/A1-C-terminal antibody. Together, the data suggest that a truncated species of betaA3/A1-crystallin exhibits proteinase activity.

FliI is a key component of the flagellar export apparatus in Salmonella typhimurium. It catalyzes the hydrolysis of ATP which is necessary for flagellar assembly. Affinity blotting experiments showed that purified flagellin and hook protein, two flagellar axial proteins, interact specifically with FliI. The interaction of either of the two proteins with FliI, increases the intrinsic ATPase activity. The presence of either flagellin or hook protein stimulates ATPase activity in a specific and reversible manner. A Vmax of 0.12 nmol Pi min-1 microgram-1 and a Km for MgATP of 0.35 mM was determined for the unstimulated FliI; the presence of flagellin increased the Vmax to 0.35 nmol Pi min-1 microgram-1 and the Km for MgATP to 1.1 mM. The stimulation induced by the axial proteins was fully reversible suggesting a direct link between the catalytic activity of FliI and the export process.

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.