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D S Clark

Publications and source records attributed to D S Clark.

At least 55 records · Page 3Linked to original sources
PubMed1998

Long-range acoustic detection and localization of blue whale calls in the northeast Pacific Ocean.

Analysis of acoustic signals recorded from the U.S. Navy's SOund SUrveillance System (SOSUS) was used to detect and locate blue whale (Balaenoptera musculus) calls offshore in the northeast Pacific. The long, low-frequency components of these calls are characteristic of calls recorded in the presence of blue whales elsewhere in the world. Mean values for frequency and time characteristics from field-recorded blue whale calls were used to develop a simple matched filter for detecting such calls in noisy time series. The matched filter was applied to signals from three different SOSUS arrays off the coast of the Pacific Northwest to detect and associate individual calls from the same animal on the different arrays. A U.S. Navy maritime patrol aircraft was directed to an area where blue whale calls had been detected on SOSUS using these methods, and the presence of vocalizing blue whale was confirmed at the site with field recordings from sonobuoys.

Acoustics↗
PubMed1997

Feasibility of an immunoassay for mevalonolactone.

Mevalonic acid is a key intermediate in a broad spectrum of cellular biological processes and their regulation. Availability of a rapid, sensitive and accurate method for its assay would be highly useful. Therefore, the feasibility of developing an immunoassay for mevalonic acid in biological samples was explored. The strategy employed was to synthesize several racemic haptens structurally resembling R-mevalonolactone, the cyclic form of mevalonic acid present at lower pH and presumed to be more antigenic. Two of these haptens were coupled to keyhole limpet hemocyanin, and the resulting conjugates were used successfully to generate antibodies in rabbits. The first antiserum bound to R,S-mevalonolactone much more effectively at pH 4.0 than at pH 6.0, consistent with the structural resemblance of the haptens to the lactone form. This antiserum also bound the free hapten from which it was generated and two others of different structure with comparable effectiveness; and slightly better than it bound R,S-mevalonolactone at pH 4.0. Similar results were obtained with the antiserum to the second hapten. The binding of either antiserum to the natural enantiomer, R-mevalonolactone, was 20 times weaker than to R,S-mevalonolactone, suggesting that the nonbiological enantiomer was more antigenic. Nevertheless, the results demonstrate that an immunochemical approach to accurate quantitation of mevalonic acid in biological samples is feasible.

Animals↗
PubMed1997

Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen.

We describe the properties of a hyperthermophilic, barophilic protease from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This enzyme is the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. The partially purified enzyme has a molecular mass of 29 kDa and a narrow substrate specificity with strong preference for leucine at the P1 site of polypeptide substrates. Enzyme activity increased up to 116(deg)C and was measured up to 130(deg)C, one of the highest temperatures reported for the function of any enzyme. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125(deg)C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barophilic and thermophilic behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously (J. F. Miller et al., Appl. Environ. Microbiol. 54:3039-3042, 1988).

Journal Article↗
PubMed1997

Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture.

A fluorescent pseudomonad (strain CW-96-1) isolated from a deep-sea vent sample grew at 30 degrees C under aerobic conditions in an artificial seawater medium and tolerated cadmium concentrations up to 5 mM. After 140 h, strain CW-96-1 removed > 99% of the cadmium from solution. Energy dispersive microanalysis revealed that the cadmium was removed by precipitation on the cell wall; sulfide production was confirmed by growth on Kligler's agar. Based on 16S ribosomal DNA sequencing and fatty acid analysis, the microorganism is closely related to Pseudomonas aeruginosa.

Aerobiosis↗
PubMed1995

Probing enzymic transition state hydrophobicities.

Hydrophobic interactions are important in numerous biological processes; however, the nature and extent of hydrophobic interactions in nonaqueous enzymology remain poorly defined. We have estimated the free energies of enzyme--substrate hydrophobic interactions for a model reaction catalyzed by subtilisin BPN'(from Bacillus amyloliquefaciens) in various solvents. Transition state stabilization of subtilisin in water has contributions from both ground state destabilization of hydrophobic substrates and intrinsic enzyme--substrate hydrophobic interactions. Both contributions are evident even in hydrophobic organic solvents and can be modified by protein engineering of the enzyme's binding site, as well as by changing the hydrophobicity of the reaction medium. We have also developed a method to estimate the hydrophobicity of the enzymic transition state involving systematic variation of the substrate and solvent hydrophobicities. The observed binding pocket hydrophobicities were directly affected by replacing the Gly166 residue, located at the back of hydrophobic S1 binding pocket of subtilisin BPN', with more hydrophobic amino acids such as alanine and valine. Thus, the observed S1 binding pocket hydrophobicities of the wild-type, G166A, and G166V mutants were measured to be 1.2, 1.8, and 2.6 log P units, respectively. Our method of calculating effective binding pocket hydrophobicity was found to be applicable to other enzymes, including horseradish peroxidase and alpha-chymotrypsin. Measurements of the binding pocket hydrophobicities have significant implications toward tailoring enzyme function in aqueous as well as nonaqueous media.

Bacillus subtilis↗
PubMed1995

Synthesis and characterization of polyamides containing unnatural amino acids.

We describe the synthesis of several polyamides that retain the secondary structure of proteins and contain derivatizable side chains. The derivatizable side chain allows for further reaction of the polymer chain (e.g., chain cross-linking or addition of pendant groups). Polymers of alpha-amino acids containing a terminal unsaturated bond on the side chain have been synthesized. Poly-L-pentenyl glycine, poly-L-propargyl glycine, and poly-L-allyl glycine were synthesized chemically via Leuchs' anhydrides and enzymatically using subtilisin Carlsberg. Poly-L-propargyl glycine and poly-D,L-allyl glycine folded into the beta-sheet configuration whereas poly-L-pentenyl glycine assumed a helical conformation. The secondary structure of poly-L-allyl glycine and poly-D,L-pentenylglycine could not be determined conclusively. Comparison of properties between the polymers obtained chemically and enzymatically is provided.

Amino Acids↗
PubMed1995

Enzymatic synthesis of peptides containing unnatural amino acids.

Several proteases were studied as potential catalysts for the enzymatic synthesis of oligopeptides containing the unnatural amino acid allylglycine, the overall objective being the synthesis of a reactive tetrapeptide that could be chemically polymerized into a potentially biocompatible or biodegradable material. Commercially available enzymes were screened for esterase activity toward the methyl ester of the amino acid allylglycine (DL-AgOMe) to identify potential catalysts for dipeptide synthesis. Proteases from Aspergillus oryzae and Aspergillus sojae, pronase E and protease Nagarse synthesized the protected dipeptide Cbz-allylglycine-phenylalaninamide (Cbz-L-Ag-L-PheNH2) from Cbz-DL-AgOMe and L-PheNH2. However, the same enzymes were not able to catalyze the synthesis of Cbz-phenylalanine-allylglycine ethyl ester (Cbz-L-Phe-L-AgOEt). Thus, although these enzymes could use allylglycine as the acyl donor they could not employ it as the acyl acceptor in peptide synthesis. In contrast, chymotrypsin was able to use allylglycine ethyl ester (DL-AgOEt) as the acyl acceptor in the synthesis of Cbz-L-Phe-L-AgOEt, but was not able to synthesize Cbz-L-Ag-L-PheNH2. The two dipeptides, Cbz-allylglycine-phenylalanine and phenylalanine-allylglycine ethyl ester, served as substrates for the thermolysin-catalyzed synthesis of the tetrapeptide Cbz-L-Ag-L-Phe-L-Phe-L-AgOEt.

Allylglycine↗
PubMed1995

Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.

Adenylate Kinase↗
PubMed1995

Pressure effects on the composition and thermal behavior of lipids from the deep-sea thermophile Methanococcus jannaschii.

The deep-sea archaeon Methanococcus jannaschii was grown at 86 degrees C and under 8, 250, and 500 atm (1 atm = 101.29 kPa) of hyperbaric pressure in a high-pressure, high-temperature bioreactor. The core lipid composition of cultures grown at 250 or 500 atm, as analyzed by supercritical fluid chromatography, exhibited an increased proportion of macrocyclic archaeol and corresponding reductions in aracheol and caldarchaeol compared with the 8-atm cultures. Thermal analysis of a model core-lipid system (23% archaeol, 37% macrocyclic archaeol, and 40% caldarchaeol) using differential scanning calorimetry revealed no well-defined phase transition in the temperature range of 20 to 120 degrees C. Complementary studies of spin-labeled samples under 10 and 500 atm in a special high-pressure, high-temperature electron paramagnetic resonance spectroscopy cell supported the differential scanning calorimetry phase transition data and established that pressure has a lipid-ordering effect over the full range of M. jannaschii's growth temperatures. Specifically, pressure shifted the temperature dependence of lipid fluidity by ca. 10 degrees C/500 atm.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Can immobilization be exploited to modify enzyme activity?

Immobilization has long been recognized as a useful tool for retaining enzymes in bioreactors and enabling the continuous operation of enzymic processes. However, the potential of immobilization for modifying enzyme activity in an advantageous, if not predictable, manner is less-well appreciated. This review summarizes selected studies that have used immobilization to tailor the catalytic properties of enzymes, and highlights the application of immobilization to the rational design of biocatalysts.

Alcohol Dehydrogenase↗
PubMed1994

Pressure stabilization of proteins from extreme thermophiles.

We describe the stabilization by pressure of enzymes, including a hydrogenase from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This is the first published report of proteins from thermophiles being stabilized by pressure. Inactivation studies of partially purified hydrogenases from an extreme thermophile (Methanococcus igneus), a moderate thermophile (Methanococcus thermolithotrophicus), and a mesophile (Methanococcus maripaludis), all from shallow marine sites, show that pressure stabilization is not unique to enzymes isolated from high-pressure environments. These studies suggest that pressure stabilization of an enzyme may be related to its thermophilicity. Further experiments comparing the effects of increased pressure on the stability of alpha-glucosidases from the hyperthermophile Pyrococcus furiosus and Saccharomyces cerevisiae support this possibility. We have also examined pressure effects on several highly homologous glyceraldehyde-3-phosphate dehydrogenases from mesophilic and thermophilic sources and a rubredoxin from P. furiosus. The results suggest that hydrophobic interactions, which have been implicated in the stabilization of many thermophilic proteins, contribute to the pressure stabilization of enzymes from thermophiles.

Journal Article↗
PubMed1993

Hepatic uptake and metabolism of ingested 24-hydroxycholesterol and 24(S),25-epoxycholesterol.

Although two hepatic sterol metabolites, 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol, are thought to be important regulators of cholesterol biosynthesis, nothing is known of their degradation and disposal in liver, nor of the mechanisms that regulate their levels. As an initial approach to these questions the two sterols were administered intragastrically, as a bolus, to mice and their hepatic accumulation and conversion to more polar compounds were examined as a function of time. These results were compared to those obtained for cholesterol and for the unnatural epimer of one of the oxysterols, 24(R)-hydroxycholesterol. Maximum concentrations of the three oxysterols in liver were reached by approx. 4 h and then declined to control levels by 8 h. More polar neutral and acidic metabolites were found in the liver extracts. Radiolabeled oxysterols and their metabolites were found in bile glands. In comparison, the amounts of hepatic free and esterified cholesterol and of acidic products formed from it increased gradually over the measured period of time. Rates of conversion of the two 24-hydroxycholesterol epimers into acidic compounds by a liver mitochondrial fraction in vitro exceeded those of 24(S),25-epoxycholesterol and cholesterol. 24(S)-Hydroxycholesterol did not lower the level of hepatic HMG-CoA reductase activity, consistent with the absence of any significant accumulation of the free sterol. Accumulation of appreciable amounts of free 24(S),25-epoxycholesterol was associated with lowered levels of reductase. The existence of hepatic systems for the rapid inactivation and degradation of the oxysterols is consistent with their postulated role in the regulation of cholesterol synthesis.

Animals↗
PubMed1993

Random and site-specific immobilization of catalytic antibodies.

The effects of immobilization on the immunologic and catalytic activity of a catalytic antibody were compared for randomly immobilized (via glutaraldehyde) whole antibody and site-specifically immobilized (via the reactive sulfhydryl group at the base of the fragment) Fab' fragments. Upon immobilization, the specific binding capacity (n) and the catalytic activity decreased significantly for both systems. Increases in the Michaelis constant (KM) were accompanied by corresponding decreases in the equilibrium binding constant determined through immunoassays. For the immobilized Fab', n decreased dramatically with increased protein loading, suggesting that, despite the site-specific attachment and favorable orientation, molecular crowding denatured the Fab' fragments. These results also show that there is an optimal surface coverage, not necessarily at the maximum loading, for both immunologic and catalytic activity. Finally, the combining/active site conformation was probed using electron paramagnetic resonance (EPR) spectroscopy. In all antibody samples, there was no spectral evidence of conformational changes in the antibody active site.

Animals↗
PubMed1993

Apical sealing ability of metal versus plastic carrier Thermafil obturators.

Sixty straight maxillary canines, 40 straight mandibular incisors, and 40 curved mesial canals of 27 mandibular molars were instrumented and randomly assigned to one of seven groups of 20 canals. Experimental groups were obturated with metal or plastic carrier Thermafil obturators. Control groups were obturated using the lateral condensation technique. An additional group of 20 unobturated curved molar canals served as positive controls. Kerr Pulp Canal Sealer was used in all obturation groups. The presence or absence of apical extrusion of gutta-percha was recorded. Teeth were suspended in black India ink for 2 wk, cleared, and then examined under a dissecting microscope at x20 magnification. No leakage was found in any of the obturated canals, whereas unobturated controls showed total dye penetration. Plastic carrier Thermafil and metal carrier Thermafil groups produced a significantly greater incidence of apical extrusion of gutta-percha compared with lateral condensation groups (p < 0.001). Extrusion for Thermafil groups occurred significantly more frequently in straight compared with curved canals (p = 0.002). The Thermafil groups showed a higher frequency of filled lateral and accessory canals than in the lateral condensation group; however, this was statistically insignificant (p = 0.48).

Chi-Square Distribution↗
PubMed1993

Surface-density and orientation effects on immobilized antibodies and antibody fragments.

The binding parameters of randomly immobilized protein 315 and Fv fragments, as well as site-specifically immobilized Fab' fragments, have been measured for a small hapten (MW = 341 Daltons) and a large synthetic antigen (MW = 50 kD). Immobilized Fv fragments had the highest binding capacities; hence, removing unnecessary protein domains can be beneficial for improving the total capacity of an immunosorbent. For all immunosorbents, high protein loadings led to relatively low specific activities (n values). This effect was reversible, however, as the loss of immobilized antibody upon prolonged storage partially restored the specific activity. At high loadings the specific activity of immobilized whole antibody was lower for the large antigen than for the small hapten, whereas no effect of hapten size on n was evident for either immobilized Fab' or Fv fragments. Although a fraction of immobilized antibody was inactive at the higher loadings, EPR spectroscopy revealed no significant changes in the conformation of active immobilized antibody.

Antigens↗
PubMed1992

Solvent dielectric effects on protein dynamics.

Electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations were used to investigate the dynamics of alpha-chymotrypsin in solvents ranging in dielectric constant from 72 to 1.9. EPR measurements showed that motions in the vicinity of two spin-labeled amino acids (Met-192 and Ser-195) decreased dramatically with decreasing solvent dielectric constant, a trend consistent with changes in the electrostatic force between charged residues of the protein. EPR results and MD simulations revealed a very similar functional dependence between rates of motion in the protein and the dielectric constant of the bulk solvent; however, predicted motions of protein atoms were markedly faster than measured motions of the spin labels. MD calculations for dielectric constants of 5 and 72 showed the greatest differences near the outer surface of the protein. In general, at the lower dielectric constant many atoms of the protein move more slowly, and many of the slowest residues are near the exterior. These results suggest that altered dynamics may contribute to the unusual properties--e.g., modified stereoselectivities--of enzymes in nearly dry organic solvents.

Chymotrypsin↗
PubMed1992

Enzymatic catalysis and dynamics in low-water environments.

Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in enzyme structure and function. Addition of less than 1% (vol/vol) water to tetrahydrofuran containing 1 M 1-propanol leads to a substantial increase in the transesterification activity of subtilisin Carlsberg (from Bacillus licheniformis) that correlates with a sharp increase in the active-site polarity and a 90% decrease in the rotational correlation time (i.e., increase in mobility) of a nitroxide spin label within the active site. Water in excess of 1% has little additional effect on active-site polarity and coincides with a further increase in spin-label mobility, yet the transesterification activity decreases dramatically. Thus, transesterification activity increases and then decreases with increasing enzyme hydration and flexibility (which are presumably coupled through dielectric screening), suggesting that the conformation of partially hydrated subtilisin is different from that of the nearly dry enzyme--i.e., enzyme containing less than 9% (wt/wt) water.

Bacillus↗