Evaluation of alpha-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of alpha-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against alpha-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.     

Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemmal vesicles

The intrinsic cAMP-dependent protein kinase activity of highly purified cardiac sarcolemmal vesicles was characterized. The sarcolemmal protein kinase was specifically activated by cAMP. Binding of cAMP to the kinase was saturable and occurred exclusively to a protein of Mr = 55,000 intrinsic to the vesicles. This binding of cAMP to the sarcolemmal vesicles caused a selective release of catalytic activity from the membranes, which was capable of phosphorylating several endogenous sarcolemmal substrates as well as one additional substrate, which was also identified in purified vesicles of cardiac sarcoplasmic reticulum. Unmasking experiments conducted with the ionophore alamethicin demonstrated that the protein kinase activity and its endogenous sarcolemmal substrates were localized on the inner, cytoplasmic surfaces of the vesicles, and, furthermore, suggested that at least 75% of the vesicles were right side out. The major protein substrates phosphorylated in the sarcolemmal fraction exhibited apparent molecular weights of 21,000 and 8,000, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Heating the membranes in the presence of sodium dodecyl sulfate prior to electrophoresis completely converted the 21,000-dalton substrate into the form of higher mobility, suggesting that the two substrates were, in fact, identical proteins. This was supported by the observation that both substrates exhibited identical pI values of approximately 6.7. Although present in the sarcolemmal fraction, these two substrates were not localized exclusively to sarcolemmal membranes. The same two substrates were present in 3-fold higher content in purified cardiac sarcoplasmic reticulum vesicles. Moreover, although phosphorylation of all other sarcolemmal proteins in right side out vesicles by exogenously added protein kinase was increased 4-fold or greater by alamethicin, phosphorylation of the substrates of Mr = 21,000 and 8,000 was not altered appreciably by the ionophore. The results suggest that these two major substrates identified in the sarcolemmal preparations are not intrinsic sarcolemmal proteins.     

Influence of surface energy of modified surfaces on bacterial adhesion

There are a number of contrary reports on the effect of surface energy of substrates on bacterial adhesion. Some reports showed that bacterial adhesion decreased with decreasing surface energy of substrates; while other reports showed that bacterial adhesion decreased with increasing surface energy of substrates. In this study Escherichia coli adhesion on the Ni--P--PTFE coatings with various surface energies was investigated and the extended DLVO theory was used to calculate the interaction energy between bacteria and the substrates in water. The theory explained the effect of surface energy of substrates on bacterial adhesion.     

Differential recognition of natural and nonnatural substrate by molecular chaperone alpha-crystallin-A subunit exchange study

alpha-Crystallin is a molecular chaperone that recognizes proteins substrates in stress. It binds to the unstable conformer of a large variety of related or unrelated substrates and thus prevents them aggregating and holds them in a folding competent state. In this article, we have tried to critically analyze, from experimental point of view, whether alpha-crystallin has any preference for its natural substrates compared to the nonnatural one. Our results clearly show that alpha-crystallin is exceptionally active and sensitive in preventing aggregation of its natural substrates and can fully prevent such an aggregation in a substoichiometric ratio, but nonnatural substrates require a considerably higher amount of alpha-crystallin. Using suitable fluorescent-labeled alpha-crystallins and performing fluorescence resonance energy transfer experiments, we were able to determine the subunit exchange kinetics between the alpha-crystallin oligomers. It was found that while alpha-crystallin was bound to its natural substrate, the rate of subunit exchange was slightly decreased. But, when a nonnatural substrate carbonic anhydrase remained bound to the chaperone, further loss in subunit exchange rate was observed. Nonnatural substrate was found to create higher activation energy barrier for the subunit exchange reaction compared to the native substrates. Similarities in major beta-sheet structure of both alpha-crystallin and its natural substrates may be the reason for the preference in molecular recognition in comparison with the nonnatural substrate.     

Energy requirement of bovine spermatozoa for in vitro capacitation

The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.     

Genome-wide detection and analysis of cell wall-bound proteins with LPxTG-like sorting motifs

Surface proteins of gram-positive bacteria often play a role in adherence of the bacteria to host tissue and are frequently required for virulence. A specific subgroup of extracellular proteins contains the cell wall-sorting motif LPxTG, which is the target for cleavage and covalent coupling to the peptidoglycan by enzymes called sortases. A comprehensive set of putative sortase substrates was identified by in silico analysis of 199 completely sequenced prokaryote genomes. A combination of detection methods was used, including secondary structure prediction, pattern recognition, sequence homology, and genome context information. With the hframe algorithm, putative substrates were identified that could not be detected by other methods due to errors in open reading frame calling, frameshifts, or sequencing errors. In total, 732 putative sortase substrates encoded in 49 prokaryote genomes were identified. We found striking species-specific variation for the LPxTG motif. A hidden Markov model (HMM) based on putative sortase substrates was created, which was subsequently used for the automatic detection of sortase substrates in recently completed genomes. A database was constructed, LPxTG-DB (http://bamics3.cmbi.kun.nl/sortase_substrates), containing for each genome a list of putative sortase substrates, sequence information of these substrates, the organism-specific HMMs based on the consensus sequence of the sortase recognition motif, and a graphic representation of this consensus.     

Substrate size selectivity of 20S proteasomes: analysis with variable-sized synthetic substrates

Proteasomes are tubular complexes with proteolytic activities on their lumenal surfaces so that large substrates should be sterically hindered from reaching the catalytic sites. Here we examine effects of substrate size on rates of cleavage by 20S proteasomes of Methanosarcina thermophila. Synthetic chromogenic substrates of variable size were prepared by linking a constant substrate group (Ala-Ala-Phe-p-nitroanilide) to a linear polymer (methoxypolyethylene glycol) with variable chain length. The smallest macromolecular substrates were cleaved more efficiently than free tripeptide substrate, and cleavage of macromolecular substrates was saturable, whereas cleavage of free tripeptide substrate was not, indicating mechanistic differences between the cleavage of large and small substrates. Rates of macromolecular substrate cleavage decreased progressively up to 10-fold as the size of the polymeric component of substrates increased. Macromolecular synthetic substrates appear to be better models of proteasome action on natural protein substrates and demonstrate substrate size selectivity of proteasomes.     

Protein substrates of proteinases with fluorescent labels

Protein substrates for proteinases with double fluorescent fluorophors are synthesized. Pyridoxal-5'-phosphate, dansyl chloride and fluorescein isothiocyanate were used as fluorescent sounds for modification of globin. Phosphoyridoxyl fluorophor was present in the both substrates. The second label was either fluoresceinthiocarbamyl or dansyl fluorophor. Spectral characteristics and ability to hydrolysis of obtained substrates have been studied. The influence of some salts on fluorescent characteristics of those substrates have been analyzed. Differentiation of the hydrolyzed substrate from the initial one by ammonium sulphate is shown to be possible.     

Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein

A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold 'better' than those in the glycine series. The S3 subsite was found to 'prefer' lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.     

Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae

Protein kinases mediate much of the signal transduction in eukaryotic cells and defects in kinase function are associated with a variety of human diseases. To understand and correct these defects, we will need to identify the physiologically relevant substrates of these enzymes. The work presented here describes a novel approach to this identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. This approach takes advantage of two catalytically inactive PKA variants, Tpk1K336A/H338A and Tpk1R324A, that exhibit a stable binding to their substrates. Most protein kinases, including the wild-type PKA, associate with substrates with a relatively low affinity. The binding observed here was specific to substrates and was dependent upon PKA residues known to be important for interactions with peptide substrates. The general utility of this approach was demonstrated by the ability to identify both previously described and novel PKA substrates in S. cerevisiae. Interestingly, the positions of the residues altered in these variants implicated a particular region within the PKA kinase domain, corresponding to subdomain XI, in the binding and/or release of protein substrates. Moreover, the high conservation of the residues altered and, in particular, the invariant nature of the R324 position suggest that this approach might be generally applicable to other protein kinases.     

Biochemical methane potential and biodegradability of complex organic substrates

The biomethane potential and biodegradability of an array of substrates with highly heterogeneous characteristics, including mono- and co-digestion samples with dairy manure, was determined using the biochemical methane potential (BMP) assay. In addition, the ability of two theoretical methods to estimate the biomethane potential of substrates and the influence of biodegradability was evaluated. The results of about 175 individual BMP assays indicate that substrates rich in lipids and easily-degradable carbohydrates yield the highest methane potential, while more recalcitrant substrates with a high lignocellulosic fraction have the lowest. Co-digestion of dairy manure with easily-degradable substrates increases the specific methane yields when compared to manure-only digestion. Additionally, biomethane potential of some co-digestion mixtures suggested synergistic activity. Evaluated theoretical methods consistently over-estimated experimentally-obtained methane yields when substrate biodegradability was not accounted. Upon correcting the results of theoretical methods with observed biodegradability data, an agreement greater than 90% was achieved.     

Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates

Kinetic properties of rat liver acid phosphatase were evaluated using the conventional synthetic substrates sodium beta glycerophosphate (betaGP) and p-nitrophenyl phosphate (PNPP) and physiologically occurring phosphate esters of carbohydrates, vitamins and nucleotides. The extent of hydrolysis varied depending on the substrates; phosphate esters of vitamins and carbohydrates were in general poor substrates. Kinetic analysis revealed the presence of two components of the enzyme for all the substrates. Component I had low Km and low Vmas. Opposite was true for component II. The Km values were generally high for betaGP, PNPP and adenosine diphosphate (ADP). Amongst the nucleotides substrates AMP showed high affinity i.e. low Km. The increase in enzyme activity in general at high substrate concentration seems to be due to substrate binding and positive cooperativity. AMP which showed highest affinity was inhibitory at high concentration beyond 1 mM. The results suggest that in situ the nucleotides may be the preferred substrates for acid phosphatase.     

Vitamin K-dependent carboxylase from calf liver: studies on the steady-state kinetic mechanism

The steady-state kinetic mechanism of vitamin K-dependent carboxylase from calf liver has been investigated by initial-velocity measurements with varying concentrations of two carboxylase substrates and constant, nonsaturating concentrations of the other two substrates. With all combinations of the varied substrates tested linear kinetics were obtained with lines intersecting on the left side of the 1/v axis in double-reciprocal plots. Thus the carboxylase has a sequential reaction mechanism which includes the quinternary complex of the enzyme with its four substrates. A mechanism with the ordered steady-state addition of all substrates to the enzyme accords well with the results. A totally random mechanism was excluded but the alternative possibility remained that part of the substrates are added in a rapid-equilibrium random reaction. Experiments with saturating constant concentrations of sodium bicarbonate and varying concentrations of the other substrates suggest that bicarbonate (CO2) is either the first or, more probably, the last substrate bound to the enzyme.     

Probing the efficiency of proteolytic events by positional proteomics

Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with (potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography (1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

Site preparation alters soil distribution of roots and ectomycorrhizae on outplanted western white pine and Douglas-fir

This report documents root and ectomycorrhizal development on container-produced (1-0), outplanted, western white pine and Douglas-fir seedlings growing in site-prepared forest soils typical of the Inland Northwestern US. The following site preparations were used: 1) mounding organic and surface mineral horizons; 2) mounding with subsequent physical removal or chemical control of competing vegetation; 3) scalping to reduce competing vegetation; and, 4) a control or no post-harvest disturbance. Treatments were applied on relatively harsh and moderate sites in northern Idaho. Most ectomycorrhizae on the seedling population were found in the mineral substrates that dominated planting sites. However, compared to mineral substrates, highest seedling ectomycorrhizal tip counts were recorded in organic matter, particularly decayed wood or mixtures containing decayed wood. Strong ectomycorrhizal development was characteristic of western white pine. It supported highest ectomycorrhizal activity in organic substrates on the harshest treatments (scalps). Douglas-fir showed even stronger relative increases of ectomycorrhizae in organic substrates on harsh treatments. Three of the four common ectomycorrhizal morphological types were concentrated in mineral substrates with all treatments. A treatment-induced change of behavior was shown by the principal pine type. It occurred at highest numbers in organic substrates of the mound with competing vegetation treatment and in mineral substrates with the control. If relative availability to seedling roots was considered, organics (especially decomposed wood) were generally equal or superior to mineral substrates for supporting ectomycorrhizal activity on planted seedlings.     

Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer.

The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.     

Reproductive success in female Neolamprologus mondabu (Cichlidae): influence of substrate types

Individual variation in body size, home range size, and reproductive success among Neolamprologus mondabu (Cichlidae) females were investigated with reference to substrate types in the littoral zone of northern Lake Tanganyika. Larger females occupied sandy substrates and smaller ones stony substrates. Female reproductive success, estimated as the number of offspring successfully released per month, was higher on sandy substrates than on stony substrates. This may be attributable to higher spawning intensity and better protection of broods against predators in open, sandy habitats.     

Studies on habitat selection by amphipods using artificial substrates within an estuarine environment

Field studies on habitat selection and community development of four gammarid amphipod species were conducted in a brackish lagoon at Nanakita River Estuary, Japan. Development of amphipod community was studied by focussing mainly on provision of shelter for these invertebrates. Seven artificial substrates (including wire net cage, circular concrete block, dried oyster shell, brush, sponge, blue and green tapes) were used in the field as substitutes of natural substrates (macroalgae). This paper demonstrates that amphipods will mostly select and inhabit substrates with available space that provide them with adequate protection. Brush substrate was ranked the best among the selected substrates, because its filamentous nature like natural substrates (macroalgae) provided suitable habitat for both tube-dwelling and free-living amphipods and accommodated maximum densities of amphipods in all seasons at all stations. Tube-dwelling amphipods (domicolous) constructed their tubes in and on algal substrates and their densities tended to be correlated with algal biomass, whilst free-living species (nestlers) prowled on surfaces, clang to filamentous structures and hid themselves in crevices, holes and boulders. Brush substrates at different stations suggested the importance of site difference.     

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.