Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli.

The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.

Localization of clavulones, prostanoids with antitumor activity, within the Okinawan soft coral Clavularia viridis (Alcyonacea, Clavulariidae): preparation of a high-purity Symbiodinium fraction using a protease and a detergent.

To investigate the localization of clavulones (CV), prostanoids with antitumor activity, in the Okinawan soft coral Clavularia viridis, we developed a method for the isolation of Symbiodinium cells from the coral, i.e., treatment of a coral homogenate with a protease, pronase E, and a detergent, Nonidet P-40. The conditions for the treatment were optimized by monitoring the morphology microscopically and the amount of chlorophyll in the Symbiodinium fraction (SymF) optically. To evaluate the purity of SymF and a Symbiodinium-free coral fraction (CorF), we analyzed them for proteins and lipids using cultivated Symbiodinium as a reference. TLC of lipids revealed that SymF contained a greater amount of glycolipids, whereas CorF comprised mostly phospholipids. SDS-PAGE of proteins in SymF and CorF revealed their distinct profiles. Thus, we could obtain each fraction with high purity; we reached the conclusion that CV and arachidonic acid, their possible precursor, are localized exclusively in the insoluble fraction of host coral cells.

Isolation of three marine prostanoids, possible biosynthetic intermediates for clavulones, from the Okinawan soft coral Clavularia viridis.

Three marine prostanoids, 1, 2, and 3, were isolated from the extract of the Okinawan soft coral Clavularia viridis. The structures of these compounds were assigned based on the results of spectroscopic analysis. Compound 1 was shown to be preclavulone-A methyl ester, and this is the first isolation of the ester of preclavulone-A as a natural product. Preclavulone-A is proposed to be the key intermediate in the biosynthesis of marine prostanoids exemplified by clavulones in C. viridis. The new prostanoid 3 was suggested to be a biosynthetic intermediate from preclavulone-A to clavulones, and a possible biogenetic pathway via 3 is proposed.

[Studies on discovery and synthesis of bioactive marine organic molecules].

This paper describes the discovery and total synthesis of bioactive marine natural products conducted in our laboratory. Clavulone, chlorovulone, bromovulone, and iodovulone are antitumor marine prostanoids isolated from the Okinawan soft coral Clavularia viridis. The synthesis of clavulone and chlorovulone was achieved from chiral 4-hydroxy-2-cyclopentenone. Marine prostanoid punaglandins 3 and 4 were synthesized via similar methodology. The chemical structures of punaglandins 3 and 4 were revised by these syntheses. Dollaberane-type diterpenoid stolonidiol and claenone were isolated from Okinawan soft coral Clavularia sp. Stolonidiol showed potent choline acetyltransferase-inducible activity in cultured basal forebrain cells. The synthesis of stolondiol and claenone was conducted via sequential Michael reaction and retro-aldol reaction. Aragusterols, isolated from the Okinawan marine sponge Xestospongia sp., are structurally unique steroids possessing a rare 26,27-cyclo structure in the side chain. Aragusterols express potent in vivo antitumor activity against L1210 leukemia in mice. The synthesis of aragusterols was carried out via steroselective construction of the side chain and stereocontrolled coupling reaction with the steroid skeleton. Kalihinane-type diterpenoid kalihinol A, isolated by Scheuer, has remarkable in vitro antimalarial activity. The absolute configuration of kalihinol A was determined by applying the CD exciton chiral method. Synthesis of kalihinene X, a kalihinane-type diterpenoid, was achieved. This synthesis involves the regioselective coupling reaction of carbanion of alkyl sulfone with epoxyalcohol and construction of cis-decalin by an intramolecular Diels-Alder reaction.

Evidence for the formation of a novel cyclopentenone isoprostane, 15-A2t-isoprostane (8-iso-prostaglandin A2) in vivo.

A2/J2-Isoprostanes (IsoPs) are prostaglandin (PG) A2/J2-like compounds that are produced in vivo as dehydration products of D2/E2-IsoPs. One A2-IsoP that should be formed is 15-A2t-IsoP (8-iso-PGA2). Analogous to cyclopentenone PGs, 15-A2t-IsoP readily undergoes nucleophilic addition to various biomolecules suggesting the compound is capable of exerting potent bioactivity. However, proof that it is definitively formed in vivo is lacking. Evidence is now presented that 15-A2t-IsoP, in fact, is generated in vivo by demonstrating that an endogenous A2-IsoP with a retention time on capillary GC identical with that 15-A2t-IsoP co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabeled 15-A2t-IsoP.

Prostaglandins and the tumor-dependent growth inhibitory activity of Yoshida ascites fluid.

The content of prostaglandins in Yoshida tumor ascites fluid was evaluated by extraction of the arachidonic acid metabolites by means of octadecylsilyl-silica chromatography (ODS-extracts). The pattern of association of the icosanoids to ascites fluid colloids was determined by sequential precipitation with polyethylene glycol and extraction of the precipitates with methanol (M-extracts). Thin layer chromatographic analysis of these extracts demonstrated in the ODS-extract the presence of chromatographic fractions corresponding to prostaglandin E2 (4.6 micrograms/mL) and prostaglandin A2 (3.0 micrograms/mL), while in the M-extract only the fraction corresponding to prostaglandin A2 (9.2 micrograms/mL) was detected. This fraction was as the growth inhibitory activity reduced by approximately 60% in M-extracts from animals treated with Indomethacin. These results support previous indications that arachidonic acid metabolites are involved in the cytostatic activity of ascites fluid extracts. The preferential association of prostaglandin E2 to the ascites macroglobulin fraction could be a consequence of the presence of an unidentified tumor-dependent amphiphilic polymer in the Yoshida ascites fluid.

Tumor cell biotransformation products of prostaglandin A1 with growth inhibitory activity.

The growth inhibitory effect and the fate of prostaglandin A1 (10(-6) M) were followed in cultures of rat B104 neuroblastoma and C6 glioma cells. More than 40% and 85% of the drug were neither recognized by a prostaglandin A1 antiserum nor extracted from the acidified medium with ethyl acetate, after 6 h and 24 h-incubation, respectively. When the supernatant of cells cultured in the presence of prostaglandin A1 during 24 hours was transferred to other cells and used as culture medium, the same growth inhibitory effect as with prostaglandin A1 was observed even when no prostaglandin A1 was added. After extensive purification and reverse phase HPLC of supernatant, four peaks more polar than prostaglandin A1 were shown; two of them were still active as growth inhibitors. This biotransformation was not observed with normal cells like L 929 or chick embryo fibroblasts, for which prostaglandin A1 had no inhibitory effect. The identification of these metabolites will allow the study of the structure-activity relationship.

Use of mini-columns packed with acid-washed Florisil for the rapid separation of A, E and F series prostaglandins.

A new procedure was developed for the rapid isolation of the A, E and F series prostaglandins from plasma extracts. This new procedure involves the use of mini-columns packed with acid-washed Florisil to rapidly separate the A, E and F series prostaglandins. These new mini-columns give flow rates of around 1 ml/min. and they generate a well-resolved chromatographic pattern while at the same time producing good recoveries for [3H]-labeled prostaglandins of the A, E and F series. Using these new mini-columns, large numbers of plasma extracts can be processed in a short period of time.

Utilization of cyclodextrin complexation for separation of E, A, and B prostaglandins by ion-exchange liquid chromatography.

Application of cyclodextrin complexation to the separation of E-, A-, and B-type prostaglandins by ion-exchange liquid chromatography is demonstrated. The addition of alpha-or beta-cyclodextrin into the mobile phase on an anion-exchange support decreased the retention times of the prostaglandins significantly because of soluble complex formation. Chromatographic separation behavior is discussed on the basis of the stability of the inclusion complex. A rapid and sensitive method for the separation and quantification of the prostaglandins, using beta-cyclodextrin in the mobile phase, is described.

Separation of A and E prostaglandins by celite partition chromatography for radioimmunoassay.

This report describes the use of celite partition chromatography to separate the A and E prostaglandins. The stationary phase consists of a glycine-HCL buffer, pH 3.6-3.7. The mobile phase consists of mixtures of ethyl acetate in toluene. At this critical hydrogen ion concentration of the stationary phase, the two prostaglandin groups are clearly separated on the basis of their difference in polarity, with greater than 95% recoveries and only 5% overlap of the elution patterns. It is anticipated that specific radioimmunoassays can be achieved by combining this system with partially specific antisera.

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins.

Antiserum against PGE2 was raised in rabbits following immunization with prostaglandin-hen-gamma-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE1 and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE2 from PGE1 and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE2. The precision of the method in the rage 10-500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE2 in serum. In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE2 obtained were 239 +/- 25 pg/ml and 250 +/- 58 pg/ml, respectively.

High performance liquid chromotography of prostaglandins: biological applications.

Two procedures are described for separation and purification of prostaglandins by high performance liquid chromatography. Both systems show excellent resolution of PGA2, PGE2 and PGF2a. Peak definition on the micro-particle silicic acid system is particularly good with the PGs appearing in 2-3 ml of organs effluent. Studies on reproducibility showed that PGE2 and PGE2a could be recovered with a retention volume of 54.2+/-0.76 ml and 64+/-0.6 ml, respectively (n=7, mean +/-50) with good recovery. The column can be run in about one hour and can be regenerated indefinitely (greater than 200 times). The degree of purification is compatible with analysis by gas chromatography-mass spectrometry. Examples showing the application of this chromatographic method to human seminal fluid, human renal tissue, platelet rich plasma and human urine samples indicate that it makes possible analysis of these samples even at low levels.