Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.     

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Crystal Structures of β-1,4-Galactosyltransferase 7 Enzyme Reveal Conformational Changes and Substrate Binding

The β-1,4-galactosyltransferase 7 (β4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human β4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type β4GalT7 and D211N β4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N β4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an S_N2 type catalytic mechanism for the β4GalT7 enzyme.     

Aryl Acylamidase Activity on Acetylcholinesterase Is High During Early Chicken Brain Development

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to exhibit aryl acylamidase activities (here called AAA(AChe) and AAA(BChe), respectively), which have been suggested to be involved in developmental and pathological processes. We here have investigated the developmental profiles of both AAA(AChe) and AAA(BChe) activities along with their AChE and BChE activities from embryonic days E3 to hatching (E21) in Triton-extracted homogenates from chicken embryonic brains. AAA(AChe) follows continuously an increase that is typical for AChE expression itself, whereas AAA(BChe) was relatively high before E10 to then become negligible toward hatching. Sucrose gradient centrifugation of both homogenized and immunopurified samples from E6-E18 brains showed that all globular forms (G1, G2, G4) of AChE present AAA(AChe) activity. Interestingly, the ratio of AAA(AChe) to AChE is highest at E6, and here again higher on G1/G2- over the G4-form. Noticeably, the sensitivity of AAA(AChe) toward the specific AChE inhibitor BW284c51 at all stages is higher than that of AChE itself. These data of high ratios of AAA associated at young stages with cholinesterases strongly indicate a role of AAA in early brain development.     

Identification of serotonin-sensitive aryl acylamidase activity with cobra venom acetylcholinesterase

Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom antibody and showed the same heat denaturation profile at 40 degrees C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.     

Anaerobic biotransformation of furfural to furfuryl alcohol by a methanogenic archaebacterium

The metabolic conversion of furfural by a methanogenic Archaea, Methanococcus sp., strain B was studied. The organism was grown on H₂–CO₂ in the presence of various concentrations of furfural. Furfural at higher concentrations, namely, 25 and 30 mM inhibited growth of this organism. At concentrations 5, 10, and 15 mM, no inhibition was observed. Furfural was completely (100%) metabolized at the concentration of 15 or <15 mM in the cultures within five days of incubation. The end product observed during furfural metabolism was furfuryl alcohol. An almost stoichiometric quantity of furfuryl alcohol was produced. This biotransformation is likely to be of value in the detoxification of furfural and its ultimate conversion to methane and CO₂ by the anaerobic process.     

Assessment Strategies and the Effects of Fighting Experience on Future Contest Performance in the Green Anole (Anolis carolinensis)

Social experiences can be useful sources of information for animals charged with making fitness‐related decisions. Fighting experience can alter an animal's perception of its fighting ability possibly leading to changes in future contest decisions, which may increase/decrease their probability of winning future contests. Winner and loser effects have been revealed in a wide array of animals, but studies using reptilian models are rare. This study investigated the impact of fighting experience on future contest performance and outcome in the green anole lizard and investigated the assessment strategies used by anoles during contests of different intensities. To determine whether the green anole expresses winner or loser effects, focal animals engaged in a primary contest with a smaller (larger) opponent to gain a winning (losing) experience; opponent size asymmetries were a significant predictor of contest outcome. Focal individuals were isolated for 2 d before being given a secondary contest with a size‐matched, naïve opponent. We found no evidence of winner or loser effects 2 d following a previous contest. Although previous contest outcome did not dictate future contest success, dynamics of the previous contest did. Highly aggressive primary contest losers won a significant proportion of the secondary contests, while less aggressive losers were more apt to lose the secondary contest. Secondary contest success of prior winners was not influenced by earlier contest performance. Further analyses of contest dynamics reveal that individuals may use different assessment strategies depending on the intensity of the contest. Our results demonstrate that future contest success may be driven more by individual performance in a prior contest and less by prior contest outcome.     

Surface functionalized mesoporous activated carbon for the immobilization of acidic lipase and their application to hydrolysis of waste cooked oil: Isotherm and kinetic studies

This study deals with the surface functionalization of mesoporous activated carbon, using ethylenediamine and glutaraldehyde to facilitate the strong immobilization of acidic lipase (AL) onto MAC. The AL was produced from Pseudomonas gessardii by using slaughterhouse lipid waste as the substrate. The AL immobilized on functionalized mesoporous activated carbon (ALFMAC) was applied for the hydrolysis of waste cooked oil (WCO). The optimum conditions for the immobilization of AL onto functionalized mesoporous activated carbon (FMAC) were 90 min; pH 3.5; and 35 °C; which resulted at the maximum immobilization of 5440 U/g of FMAC (3.693 mg of AL/g of FMAC or the yield 2.7% or the expressed activity 103.7% or the activity per unit area of FMAC 1.08 mg of AL/m²). The ALFMAC showed better thermal and storage stabilities than the free AL. The ALFMAC retained a 98% and a 92% initial activity at 40 °C and 50 °C, respectively, while the AL showed the thermal stability (residual activities) 65% and 38%, respectively. The storage stability of ALFMAC at 4 °C showed 100% initial activity up to 15 days from the initial day of the storage, whereas AL showed only 88% initial activity up to 15 days. The FMAC and ALFMAC were characterized by using scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD) analysis. The K_m values of the ALFMAC and AL were 0.112 mM and 0.411 mM, respectively. The v_max values of the ALFMAC and AL were 1.26 mM/min and 0.53 mM/min, respectively. Immobilization of AL onto FMAC obeyed the Freundlich and Redlich–Peterson isotherm models. The non-linear models of pseudo first, and second order, intra-particle diffusion, Bangham, and Boyd plot were also performed to understand the dynamic mechanism of immobilization. ALFMAC showed a 100% hydrolysis of WCO up to 21 cycles of reuse, and 60% up to 45 cycles. The hydrolysis of WCO was confirmed by using FT-IR spectra.     

Percutaneous Transvenous Angioplasty of Left Innominate Vein Stenosis Following Right Side Permanent Pacemaker Implantation- A Left Femoral Vein to Left Axillary Vein Approach

Central venous stenosis after the insertion of a permanent pacemaker is a well recognized complication. This late complication is encountered when there is a need to change the pacemaker lead or extract it. We describe a young male who had such a complication after many years after right side pacemaker implantation. The lesion was managed percutaneously leading to placement of a new lead from the left side.