Pharmacokinetics of propofol in rainbow trout following bath exposure.

Propofol, 2,6-diisopropylphenol, seems to be a good candidate as a fish anaesthetic, however, no study regarding propofol influence on fish has yet been reported. The aim of this study was to examine propofol pharmacokinetics in rainbow trout (Oncorhynchus mykiss) following bath exposure. Fish (n = 100) were exposed to an aqueous propofol bath at 12°C and 17°C; propofol concentration in the bath was 10 mg L(-1). Plasma concentration-time profiles were determined using LC-MS, and pharmacokinetic parameters were calculated. Propofol was absorbed quickly at both temperatures. Its concentration reached 13.8 ± 2.7 µg mL(-1) and 16.1 ± 2.1 µg mL(-1) at 12°C and 17°C, respectively, during the first minute of exposure. Blood plasma propofol decreased rapidly to 6.8 ± 0.7 µg mL(-1) and 6.3 ± 2.2 µg mL(-1) at 12°C and 17°C respectively, during the first 10 minutes of the recovery. The half-life time of propofol was 1.5 h and 1.1 h at 12°C and 17°C, respectively. We found propofol anaesthesia in trout effective and safe. However, it caused a gradual decrease of respiratory rate, and therefore a specific anaesthesia protocol should be developed.

Protease expression in interface tissues around loose arthroplasties.

To determine whether cathepsins and matrix metalloproteinase-1 are involved in accelerating tissue destruction, we examined, immunohistochemically, the expression of matrix metalloproteinase-1 and cathepsins B, D, L, and X in periprosthetic synovial-like interface tissues from 14 patients with failed prosthetic hips and in the synovial membranes of hips from 18 patients with rheumatoid arthritis and 25 patients with primary osteoarthritis. The expression levels of all these proteases in the interface tissue were higher than in the synovial membrane of osteoarthritis. The expression levels of cathepsins B and X in the interface tissue were higher than in the rheumatoid synovium. The results show similarities in the expression patterns of cathepsins D and L and matrix metalloproteinase-1 between aseptic prosthetic loosening and rheumatoid arthritis. In addition, these data suggest that the impact of cathepsins B and X on tissue degradation is more pronounced in aseptic prosthetic loosening than in rheumatoid arthritis.

Substrate specificity of the streptococcal cysteine protease.

The streptococcal pyrogenic exotoxin B (SpeB) is an important factor in mediating Streptococcus pyogenes infections. SpeB is the zymogen of the streptococcal cysteine protease (SCP), of which relatively little is known regarding substrate specificity. To investigate this aspect of SCP function, a series of internally quenched fluorescent substrates was designed based on the cleavage sites identified in the autocatalytic processing of SpeB to mature SCP. The best substrates for SCP contain three amino acids in the nonprimed position (i.e. AIK in P(3)-P(2)-P(1)). Varying the length of the substrate on the primed side of the scissile bond has a relatively lower effect on activity. The highest activity (k(cat)/K(M) = 2.8 +/- 0.6 (10(5) x m(-1)s(-1)) is observed for the pentamer 3-aminobenzoic acid-AIKAG-3-nitrotyrosine, which spans subsites S(3) to S(2)' on the enzyme. High pressure liquid chromatography and mass spectrometry analyses show that the substrates are cleaved at the site predicted from the autoprocessing experiments. These results show that SCP can display an important level of endopeptidase activity. Substitutions at position P(2) of the substrate clearly indicate that the S(2) subsite of SCP can readily accommodate substrates containing a hydrophobic residue at that position and that some topological preference exists for that subsite. Substitutions in positions P(3), P(1), and P(1)' had little or no effect on SCP activity. The substrate specificity outlined in this work further supports the similarity between SCP and the cysteine proteases of the papain family. From the data regarding the identified or proposed natural substrates for SCP, it appears that this substrate specificity profile may also apply to the processing of mammalian and streptococcal protein targets by SCP.

Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities.

Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.

Autocatalytic processing of the streptococcal cysteine protease zymogen: processing mechanism and characterization of the autoproteolytic cleavage sites.

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP.

Purification and characterization of a Penicillium sp. lipase which discriminates against diglycerides.

A lipase was isolated from Penicillium sp. strain UZLM-4 and characterized. This lipase has a molecular weight of 27,344 (determined by mass spectrometry) and hydrolyzes triglycerides in preference to mono- and diglyceride substrates. Among various triglyceride substrates, tributyrin is hydrolyzed about four times faster than any other tested. The lipase has a preference for hydrolysis at the 1,3 positions of the lipids and shows a weak stereoselectivity for the S enantiomer. Unlike most other lipases, this lipase is stable and has a high activity at low surface pressures (5-10 mN/m).

Redesigning the active site of Geotrichum candidum lipase.

Attempts to engineer enzymes with unique catalytic properties have largely focused on altering the existing specificities by reshaping the substrate binding pockets. Few experiments have aimed at modifying the configuration of the residues essential for catalysis. The difference in the topological location of the triad acids of Geotrichum candidum lipase (GCL) and the catalytic domain of human pancreatic lipase (HPL), despite great similarities in their topologies and 3-D structures, suggest that these are related enzymes whose catalytic triads have been rearranged in the course of evolution (Schrag et al., 1992). In this study we prepared a double mutant GCL in which the catalytic triad acid is shifted to the position equivalent to the location of the triad acid of HPL. The double mutant maintains approximately 10% of the wild type activity against triglycerides and the fluorogenic ester 4-methylumbelliferyl-oleate. The only significant differences between the 3-D structures of the double mutant and wild type GCL are at the mutated sites. Even the water structure in the region of the triad is unchanged. The hydrogen bonding pattern of the catalytic triad of the double mutant is very similar to that of pancreatic lipase. The acid of the double mutant is stabilized by only two hydrogen bonds, whereas three hydrogen bonds are observed in the wild type enzyme. These results strongly support the hypothesis that the pancreatic lipases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the alpha/beta hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.

Reduction of strong lipase-polyclonal antibodies binding by limited proteolysis.

Initial attempts to purify Geotrichum candidum lipase (EC 3.1.1.3) using immunoaffinity chromatography have been hampered by the tight binding of the lipase to the immobilized rabbit anti-GCL IgG. Stringent elution conditions were unable to release more than a few percent of the antigen. To decrease the tight binding, the immunosorbent was treated with minute amounts of different proteases, of which elastase proved to be most effective. Using the elastase-treated immunosorbent both natural and recombinant GCL II were purified to homogeneity with 30% of lipase recovered from the immunoaffinity chromatography step.

Expression and characterization of Geotrichum candidum lipase I gene. Comparison of specificity profile with lipase II.

Despite tremendous progress in the elucidation of three-dimensional structures of lipases, the molecular basis for their observed substrate preference is not well understood. In an effort to correlate the lipase structure with its substrate preference and to clarify the contradicting reports in the literature, we have compared the enzymic characteristics of two closely related recombinant lipases from the fungus Geotrichum candidum. These enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions with an N-terminal poly(His) tag and were purified in a single step by metal-affinity chromatography. Their specific activities against a series of triacylglycerol substrates were compared using a titrimetric assay. The substrates varied in fatty acyl chain length, number of double bonds and their position along the chain. G. candidum lipases I and II (GCL I and GLC II) are markedly different with respect to their substrate preferences. For unsaturated substrates having long fatty acyl chains (C18:2 cis-9, cis-12 and C18:3 cis-9, cis-12, cis-15), GCL I showed higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains (C8, C10, C12 and C14). We have constructed a hybrid molecule containing the N-terminal portion of GCL I (including the flap covering the active site) linked to the C-terminal portion of GCL II. The hybrid molecule showed a substrate preference pattern identical to that of GCL II. These results indicate that sequence variation within the N-terminal 194 amino acids of G. candidum lipases do not contribute to the observed variation in efficiency by which the lipases hydrolyze their substrates. Moreover, it also shows that the flap region in GCL is not directly involved in substrate differentiation, even though this region is thought to be involved in recognition of the interface and in the activation of the enzyme.

Determination of lipase activity by a rhodamine-triglyceride-agarose assay.

A quantitative fluorescence lipase assay based on the interaction of rhodamine B with fatty acids released during the enzymatic hydrolysis of triglycerides is described. The assay is linear over the range of 0.5-2 mM oleic acid and 0.05-1 microgram pure lipase. The method allows flexibility in the choice of substrate. A large number of samples can be assayed simultaneously, making it practical for assaying lipase activity in column fractions during purification. The substrate profiles of Geotrichum candidum lipase obtained by both a titrimetric assay and the RTA assay indicated the highest activity against triolein. The method is rapid and can be further automated.

Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase.

The structures of Candida rugosa lipase-inhibitor complexes demonstrate that the scissile fatty acyl chain is bound in a narrow, hydrophobic tunnel which is unique among lipases studied to date. Modeling of triglyceride binding suggests that the bound lipid must adopt a "tuning fork" conformation. The complexes, analogs of tetrahedral intermediates of the acylation and deacylation steps of the reaction pathway, localize the components of the oxyanion hole and define the stereochemistry of ester hydrolysis. Comparison with other lipases suggests that the positioning of the scissile fatty acyl chain and ester bond and the stereochemistry of hydrolysis are the same in all lipases which share the alpha/beta-hydrolase fold.

Cloning and expression of Geotrichum candidum lipase II gene in yeast. Probing of the enzyme active site by site-directed mutagenesis.

The three-dimensional structure of lipase II of Geotrichum candidum strain ATCC34614 (GCL II) has provided insights with respect to the nature of the catalytic machinery of lipases. To support these structural observations, we have carried out an analysis of GCL II by mutagenesis. The gene encoding lipase II of Geotrichum candidum strain ATCC34614 (GCL II) was amplified using the polymerase chain reaction, cloned, and sequenced. The intronless lipase gene was expressed and secreted from Saccharomyces cerevisiae at approximately 5 mg/liter of culture. Recombinant GCL II was purified by immunoaffinity chromatography and characterized using a combination of substrates and independent analytical methods. The recombinant enzyme and the enzyme isolated from its natural source have comparable specific activities against triolein of about 1000 mumol of oleic acid released/min/mg of protein. The putative catalytic triad Ser217-His463-Glu354 was probed by site-directed mutagenesis. The substitution of Ser217 by either Cys or Thr and of His463 by Ala led to a complete elimination of the activity against both triolein and tributyrin. Substitution of Glu354 by either Ser, Ala or Gln renders the enzyme inactive and also perturbs the enzyme stability. However, the enzyme with the conservative replacement Glu354 Asp is stable and displays only a small decrease of triolein activity but a 10-fold decrease in activity against tributyrin. There was no appreciable difference in esterase activity between the native, recombinant wild type, and Glu354 Asp mutant. These results confirm that the triad formed by Ser217-Glu354-His463 is essential for catalytic activity. They also show that the active site of GCL II is more tolerant to a conservative change of the carboxylic side chain within the triad than are other hydrolases with similar catalytic triads.

Use of delta-(alpha-aminoadipoyl) chromogenic amides in screening for aminoadipoyl amidohydrolases.

The synthesis of delta-(alpha-aminoadipoyl) aromatic amides and their use in screening for enzymes able to cleave delta-(alpha-aminoadipoyl) residues off the synthetic amides and cephalosporin C are described. A number of commercially available proteases and peptidases were not active with delta-(alpha-aminoadipoyl) chromogenic amides. Also, most tested microbial strains known to produce acylases did not hydrolyze these compounds. Only one microbial strain, Xanthomonas maltophila, had an appreciable activity toward the racemic form of chromogenic substrates. Activity measured in crude extracts from Xanthomonas cells indicated that this bacterium produces predominantly L-specific aminoadipoyl amidohydrolase and gamma-glutamyl hydrolase. A low level of cephalosporin C and glutaryl-cephalosporin acylase activities was also found.

Modification of Lignins by Growing Cells of the Sulfate-Reducing Anaerobe Desulfovibrio desulfuricans.

The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans was grown on medium supplemented with either Kraft lignin or lignosulfonate. Only lignosulfonate contributed to the growth of D. desulfuricans cells, by replacing sulfate, a natural electron acceptor for this microorganism. Kraft lignin added to the culture medium could not substitute for lactate or sulfate, both necessary culture medium components. However, it was found to enhance the viability of D. desulfuricans cells. When changes occurring in lignin during growth of Desulfovibrio cultures were monitored, it was found that both lignin preparations could be partially depolymerized. Spectrophotometric and elemental analysis of biologically treated lignins suggested that both the polyphenolic backbone and lignin functional groups were affected by D. desulfuricans. After treatment, a twofold increase in the sulfur content of Kraft lignin and a minor decrease (14%) in the sulfur content of lignosulfonate were observed. After biological treatment, Kraft lignin and lignosulfonate both bound larger quantities of heavy metals.

Relationship between Fungal Biomass Production and the Brightening of Hardwood Kraft Pulp by Coriolus versicolor.

The white-rot fungus Coriolus versicolor increased the brightness of hardwood kraft pulp by two mechanisms depending on the concentration of available nitrogen. In low-nitrogen conditions, the brightening process was a chemical effect mediated by the fungus, associated with the removal of residual lignin in the pulp; kappa number was used as an indicator of lignin concentration. A five-day treatment in low-nitrogen conditions increased the brightness of hardwood kraft pulp from 36.2 to 54.5%, with a corresponding decrease in kappa number from 12.0 to 8.5, equivalent to a reduction in the lignin concentration from ca. 2.0% (wt/wt) to ca. 1.4% (wt/wt). Under these conditions, we concluded that the brightening of the pulp was a secondary metabolic event initiated after the depletion of available nitrogen. This method of brightening has been described as bleaching or biobleaching. By contrast, in high-nitrogen conditions, the brightening was a physical effect associated with the dilution of the dark pulp fibers by the relatively high levels of brighter fungal mycelium produced. Since this method of brightening was not evidently associated with lignin removal, it cannot be described as bleaching. In pulp samples brightened in high-nitrogen conditions, as brightness increased, there was a corresponding increase in kappa number. This observation was explained by the consumption of potassium permanganate by the fungal mycelium, which interfered with kappa number determinations at high fungal biomass levels.

Immobilization of isolated and cellular hydrogenase of D. desulfuricans in radiation-polymerized polyacrylamides.

Purified hydrogenase from Desulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6-7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65 degrees C) and long term storage (4 degrees C) of the hydrogenase activity of both the purified enzyme and whole cells.

Application of hydrophobic chromatography to the large scale purification of Desulfovibrio desulfuricans cytochrome C3.

Hydrophobic chromatography on Phenyl-Sepharose was employed for large scale purification of cytochrome c3 from Desulfovibrio desulfuricans. This chromatographic procedure minimizes operational volumes and eliminates the lengthy dialysis ordinarily used to remove large amounts of salts. Pure cytochrome c3 was obtained after one additional purification by ion-exchange chromatography.