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A Hermetter

Publications and source records attributed to A Hermetter.

At least 19 recordsLinked to original sources
PubMed2006

Activity-based proteomics: enzymatic activity profiling in complex proteomes.

In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.

Animals↗
PubMed2003

Changes of compressibility of low density lipoproteins following copper mediated oxidation.

The methods of measuring the ultrasound velocity and density were used for study the adiabatic compressibility of low density lipoproteins (LDL) during their oxidation. We showed, that copper-mediated oxidation of LDL resulted in a decrease of apparent specific compressibility, phi(k)/beta0, of lipoproteins. The changes of ultrasound velocity and phi(k)/beta0 value started much earlier than the beginning of propagation phase corresponding to the fast increase in concentration of conjugated dienes, measured by absorption at 230 nm. It was assumed that the changes of compressibility could be in particularly due to increase in ordering of the phospholipids during reductive activation of Cu2+.

Copper↗
PubMed2003

Effects of oxidation on changes of compressibility of bovine serum albumin.

The methods of ultrasound velocity and density measurements were used to study the adiabatic compressibility of bovine serum albumin (BSA) during its oxidation by the prooxidants Cu2+ and 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). We did not find changes of compressibility of BSA in the presence of copper ions at rather high molar ratio Cu2+/BSA = 0.66 mol/mol. This can be explained by binding of the Cu2+ to the binding site of BSA and thus protecting the prooxidant action of the copper. However, AAPH-mediated oxidation of BSA resulted in an increase of its apparent specific compressibility (psik/beta0). These changes could be caused by the fragmentation of the protein.

Amidines↗
PubMed2002

Green tea extract only affects markers of oxidative status postprandially: lasting antioxidant effect of flavonoid-free diet.

Epidemiological studies suggest that foods rich in flavonoids might reduce the risk of cardiovascular disease and cancer. The objective of the present study was to investigate the effect of green tea extract (GTE) used as a food antioxidant on markers of oxidative status after dietary depletion of flavonoids and catechins. The study was designed as a 2 x 3 weeks blinded human cross-over intervention study (eight smokers, eight non-smokers) with GTE corresponding to a daily intake of 18.6 mg catechins/d. The GTE was incorporated into meat patties and consumed with a strictly controlled diet otherwise low in flavonoids. GTE intervention increased plasma antioxidant capacity from 1.35 to 1.56 (P<0.02) in postprandially collected plasma, most prominently in smokers. The intervention did not significantly affect markers in fasting blood samples, including plasma or haemoglobin protein oxidation, plasma oxidation lagtime, or activities of the erythrocyte superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase. Neither were fasting plasma triacylglycerol, cholesterol, alpha-tocopherol, retinol, beta-carotene, or ascorbic acid affected by intervention. Urinary 8-oxo-deoxyguanosine excretion was also unaffected. Catechins from the extract were excreted into urine with a half-life of less than 2 h in accordance with the short-term effects on plasma antioxidant capacity. Since no long-term effects of GTE were observed, the study essentially served as a fruit and vegetables depletion study. The overall effect of the 10-week period without dietary fruits and vegetables was a decrease in oxidative damage to DNA, blood proteins, and plasma lipids, concomitantly with marked changes in antioxidative defence.

Adult↗
PubMed2002

High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates.

Sphingomyelinases are important enzymes of signal transduction. They catalyze the hydrolysis of sphingomyelin, giving rise to the intracellular formation of biologically active ceramide and phosphatidylcholine. Here we report on a fluorescence method for the fast and accurate determination of this enzyme in biological samples. The assay is based on a fluorescent sphingomyelin analog carrying fluorescent 7-nitro-2-1,3-benzooxadiazolyl amino-dodecanoic acid instead of an aliphatic acyl chain at the nitrogen atom. The fluorescent substrate is hydrolysed by sphingomyelinases to form fluorescent ceramide, which can be separated from the remaining substrate using TLC on silica gel. The fluorescence intensity pattern obtained on the TLC plate can accurately be determined using a CCD camera. Typically, a large number of samples can be analyzed simultaneously. Examples for the quantitative analysis of sphingomyelinases from freshly prepared cellular homogenates as well as from commercial sources are given.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

High-throughput fluorescence screening of antioxidative capacity in human serum.

Diphenylhexatriene-labeled phosphatidylcholine and propionic acid have been established as selective fluorescence markers for the continuous determination of oxidation processes in the lipid and aqueous phases of unfractionated human serum. Oxidation of the respective fluorophores leads to a decrease in fluorescence intensity from which the time-dependent degradation of the marker molecule can be determined. The lag times preceding the propagation of oxidation are representative for the antioxidative capacity of the system, which may be influenced by exogenous factors, e.g., the antioxidants from the diet. Supplementation of human serum by quercetin, rutin, vitamin E, vitamin C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the oxidation marker and the hydrophobicity of the antioxidant. Quercetin and vitamin E showed a higher in vitro capacity of protecting lipoproteins against oxidation. In contrast, rutin and vitamin C were more efficient as inhibitors in the aqueous phase. The same effect on serum was found after dietary consumption of apples. This result is in line with the known observation that intake of plant polyphenols leads to an increase in serum levels of hydrophilic antioxidants.

Antioxidants↗
PubMed2001

Powerful probes for glycosidases: novel, fluorescently tagged glycosidase inhibitors.

Amino-1,2,5-trideoxy-2,5-imino-D-mannitol was fluorescently tagged by reaction with dansyl chloride at N-1 or by attachment of a dansyl amide bearing spacer to this centre. Compounds obtained are highly potent inhibitors of beta-glucosidase exhibiting Ki values in the single figure nanomolar range. The 1-N-dansyl substituted inhibitor was successfully exploited for binding studies with beta-glucosidase from Agrobacterium sp. employing fluorescence spectrometric methods.

Binding, Competitive↗
PubMed2001

Modified phosphatidylethanolamine as the active component of oxidized low density lipoprotein promoting platelet prothrombinase activity.

We analyzed the influence of the atherogenic oxidized low density lipoproteins (LDL) on the activity of the platelet prothrombinase complex, a major contributor to overall thrombin formation in vivo. Platelet dependent thrombin generation was found to be strongly stimulated by in vitro oxidized LDL. The enhancement was additive to that observed with the platelet agonist thrombin. Oxidized LDL increased the platelet binding of annexin-V, suggesting that the augmented surface exposure of aminophospholipids promoted the prothrombinase activity. All of the stimulatory activity of the oxidized LDL could be recovered in the microemulsions prepared from the lipid portion of the modified particles. Phospholipid vesicles were prepared containing the total lipids of the oxidized LDL but lacking specifically in one lipid component. Following the selective removal of the ethanolamine phospholipids (PE) from the LDL lipids, the platelet-dependent thrombin formation was markedly reduced. Vesicles enriched with the isolated PE fraction alone enhanced the thrombin generation. Analyses with autoxidized phospholipids indicated that oxidation products of unsaturated diacyl-PE were mainly responsible for the increased prothrombinase activity. Oxidized LDL and its PE fraction lost their stimulatory activity after treatment with NaCNBH(3), a chemical reductant of Schiff base adducts. Phospholipid vesicles supplemented with synthetic aldehyde-PE adducts largely reproduced the stimulation of the thrombin generation. We conclude that the oxidized LDL particles elicit a pronounced prothrombotic response by increasing the activity of the platelet prothrombinase complex. Specific oxidative modifications of the LDL-associated ethanolamine phospholipids are mainly responsible for this stimulation.

Blood Platelets↗
PubMed2001

The alpha-amino group of L-arginine mediates its antioxidant effect.

Antioxidant effects may constitute part of the possible antiatherogenic effects of the amino acid L-arginine. These antioxidant properties were further characterized in a model of lipoprotein oxidation. Oxidation of lipoproteins in unfractionated human serum was continuously monitored by a fluorescent probe. The antioxidant effects of L-arginine, N-alpha-acetyl-arginine and vitamin E in combination with L-arginine were measured after initiation of free radical generation with either copper or 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). The half-time of the fast propagation rate for copper-induced lipoprotein oxidation increased after incubation with L-arginine in a dose-dependent manner (P < 0.01). N-alpha-acetyl-arginine did not show such effects. Vitamin E and L-arginine show different effects on copper-induced oxidation, the former increasing only lag-time, the latter increasing only propagation rate, and do not have reciprocal effects. In contrast to copper-induced oxidation, L-arginine increased the lag-time of AAPH-induced lipoprotein oxidation (P < 0.01), with no effect on the propagation rate at physiological concentrations. Again, N-alpha-acetyl-arginine did not show any antioxidant effects. Our experiments provide further evidence that mechanisms other than serving as a substrate for the NO-synthase could be involved in the antiatherosclerotic effect of L-arginine. In addition, our experiments clearly show, that the antioxidant effect of L-arginine is due to a chemical moiety different from that serving as the substrate for NO biosynthesis.

Amidines↗
PubMed2000

Lateral microheterogeneity of diphenylhexatriene-labeled choline phospholipids in the erythrocyte ghost membrane as determined by time-resolved fluorescence spectroscopy.

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.

Choline↗
PubMed1999

Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes.

We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.

Binding Sites↗
PubMed1999

Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.

2-Propanol↗
PubMed1999

Synthesis and intermembrane transfer of pyrene-labelled liponucleotides: ceramide phosphothymidines.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against human immunodeficiency virus (HIV) in vitro. Here we report on the synthesis and characterization of two pyrene containing conjugates: 2-N-(4-(pyren-1-yl)butanoyl)ceramide 5'-phosphothymidine (Pbs-Cer-P-T) (XII) and 2-N-(10-(pyren-1-yl)decanoyl)ceramide 5'-phosphothymidine (Pds-Cer-P-T) (XIII). These fluorescent labelled conjugates served as model compounds to study incorporation of sphingoliponucleotides into membranes. The complex compounds were prepared by condensation of 3'-acetylthymidine and labelled ceramides using the phosphite triester coupling procedure. UV absorption, fluorimetry as well as 1H-, 31P-, 13C-NMR analyses were used for structure confirmation of the synthesized substances. When incorporated into small unilamellar 1-palmitoyl-2-oleoyl-glycerophosphatidyl-choline (POPC) vesicles and incubated with unlabelled acceptor POPC vesicles, the compounds (XII) and (XIII) exhibited spontaneous transfer. Kinetic data suggest that transfer from donor to acceptor vesicles occurred via the intervening aqueous phase. The non-specific lipid transfer protein from bovine liver stimulated the transfer of Pds-Cer-P-T between phospholipid vesicles in a concentration dependent manner.

Animals↗
PubMed1999

Novel reversible, irreversible and fluorescent inhibitors of platelet-activating factor acetylhydrolase as mechanistic probes.

Phosphatidylcholines (1-O-alcoxy-2-amino-2-desoxy-phosphocholines and 1-pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from plasma, and with macrophage-like U937 cells. Novel phosphatidylcholines containing a sn-2-carbamoylester group such as 1-O-hexadecyl-2-desoxy-2-amino-methylcarbamoyl-2-methyl-rac-glycer o-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with Ki-values up to 83 (K(irev)) and 177 (Ki(inact)) microm. The compounds exhibit only marginal inhibition of Ca2+-dependent phospholipases. Kinetic data show that phosphocholines carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an effectivity similar to analogs with an aliphatic chain. 1-O-Decyloxy-[10-(4-pyrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-me thyl-rac(-glycero-3-phosphocholine 13 could be used for enzyme labeling and to demonstrate an inhibitor-enzyme stoichiometry of 0.7:1. At 8 degrees C, the compound accumulated in the membranes of U937 cells, at 37 degrees C it was internalized into intracellular compartments. Structure activity studies in a mixed micelle assay indicated that the inhibition power of reversible and irreversible inhibitors increases along with the (sn)-1-chain length similar to the structure-dependent binding of ether phospholipids to the PAF-receptor. Unlike the situation at the (sn)-1-position, increasing chain length at the sn-2-position, or an alkyl branching of the glycerol backbone significantly reduced the inhibitory potency.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1999

Interactions of fluorescent triacylglycerol analogs covalently bound to the active site of a lipase from Rhizopus oryzae.

Fluorescent triacylglycerol analogs were synthesized as covalent inhibitors of lipase activity. The respective 1(3), 2-O-dialkylglycero-3(1)-alkyl-phosphonic acid p-nitrophenyl esters contain a fluorescent pyrenealkyl chain and a long-chain alkyl residue bound to the sn-2 and sn-1(3) positions of glycerol, respectively. The phosphonic acid p-nitrophenyl ester bond is susceptible to nucleophilic substitution by the active serine residue in the catalytic triad of a lipase, leading to inactivation of the enzyme. The fluorescent dialkylglycerophosphonates contain two chiral centers, the sn-2 carbon of glycerol and the phosphorus atom. The (1-O-hexadecyl-2-O-pyrenedecyl-sn-glycero)-O-(p-nitrophenyl)-n-hex yl- phosphonate, first peak during HPLC separation and the (3-O-hexadecyl-2-O-pyrenedecyl-sn-glycero)-O-(p-nitrophenyl)-n-hex yl- phosphonate, second peak during HPLC separation were found to be potent lipase inhibitors. After incubation of an equimolar amount of these isomers with lipase from Rhizopus oryzae complete inactivation was observed. Stable conjugates containing a 1 : 1 molar ratio of lipid to protein were formed. The spatial proximity of the fluorescently labeled sn-2 alkyl chain of the inhibitor and tryptophan residues of the lipase was assessed by fluorescence resonance energy transfer. The extent of tryptophan fluorescence quenching and the concomitant increase in pyrene fluorescence upon excitation of lipase tryptophans was found to be similar for the above-mentioned isomers. Thus, the (labeled) sn-2 alkyl chains of a triacylglycerol analog are likely to interact with the same binding site of the R. oryzae lipase, irrespective of their steric configuration. However, it was shown that the extent of resonance energy transfer is strongly influenced by the reaction medium, indicating conformational changes of the lipase in different environments.

Binding Sites↗
PubMed1998

Platelet agonists enhance the import of phosphatidylethanolamine into human platelets.

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.

Adenosine Diphosphate↗
PubMed1997

The Effect of Ternary Complex Formation on the Partitioning of Pyrene and Anthracene in Aqueous Solutions Containing Sodium Dodecyl Sulfate and M-gamma-cyclodextrin

The partitioning behavior of pyrene and anthracene between n-decane and aqueous solutions containing a partially methylated gamma-cyclodextrin (M-gamma-CD) and sodium dodecyl sulfate (SDS) was studied by extraction experiments. At concentrations below the aqueous solubility of pyrene and anthracene both were found to form 1:1 complexes with M-gamma-CD. Formation constants were determined as 1843 M-1 for pyrene and 624 M-1 for anthracene. In micellar solutions of SDS pyrene was found to be better solubilized than anthracene. At constant concentrations of M-gamma-CD the dependence of the partition coefficients on increasing concentrations of SDS could be divided into three linear regions. At concentrations below a critical surfactant concentration (CSCSDS) the partition coefficients of both polyaromatic hydrocarbons as well as the fluorescence intensity of pyrene increased linearly with the addition of SDS. In the fluorescence spectra of anthracene the addition of SDS led to a shift of the maxima to higher wavelengths. These data suggest the formation of ternary complexes of M-gamma-CD with each of the polyaromatic hydrocarbons and SDS. The ternary complex with anthracene was found to be more stable than that of pyrene, which resulted in higher partition coefficients than seen with pyrene. Increasing the surfactant concentrations above the CSCSDS the partition coefficients remained constant until micelles were formed and another linear increase of the partition coefficients could be observed. CSCSDS and the critical micellization concentration (CMC) were found to increase linearly with cyclodextrin concentration. For CSCSDS a molar ratio of 3/1 was found for M-gamma-CD/SDS. Mixed micelles between SDS and gamma-CD are supposed to be formed above CSCSDS. Copyright 1997Academic Press

Journal Article↗