Purification of extracellular alkaline phosphatase released by Escherichia coli excretory mutants

Summary Alkaline phosphatase (APase) is the major protein released into the extracellular medium by strain 706, a periplasmic-excretory (lky) mutant of Escherichia coli K12. We developed a rapid three step procedure for APase purification from culture supernatants of lky mutants. Two ultrafiltration stages and an heat treatment were sufficient to obtain a 99% pure enzyme preparation. Batch culture conditions of strain 706 in a 15 l fermentor leading to an extracellular APase yield of 1250 U/ml were determined.Abbreviation APase E. coli alkaline phosphatase     

Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Purification of synthetic oligodeoxyribonucleotides by ion-exchange high-performance liquid chromatography

Synthetic oligodeoxyribonucleotides rangin from 11 to 37 nucleotides in length and with varying base compositions, prepared by both the phosphotriester and phosphite procedures, have been purified by ion-exchange high-performance liquid chromatography on Whatman Partisil 10/SAX columns using phosphate buffer gradients. The effects of different buffer systems on elution times and resolution have been evaluated. Oligomer composition and length had a marked effect on the resolution achieved. In general the use of formamide buffers gave the best results, particularly in the case of 2′-deoxyguanosine-rich sequences. These methods have also been successfully applied to the purification of mixtures of synthetic oligodeoxynucleotides.     

Proteolytic modification of Escherichia coli alkaline phosphatase

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.     

Studies on alkaline phosphatase. Transientstate and steady-state kinetics of Escherichia coli alkaline phosphatase

1. Reduction of a 19s immunoglobulin M with 3mm-mercaptoethanol or 0.05-0.5mm-dithiothreitol followed by alkylation gave sedimentation patterns indicating products compatible with structures consisting of one, two, three, four and five 7s sub-units. This supports the concept of a five-sub-unit structure for immunoglobulin M. 2. Reduction with 0.125mm-dithiothreitol or 20mm-cysteine produced 7s sub-units that could not be dissociated into chains in m-propionic acid. 3. By labelling (with iodo[2-(14)C]acetic acid) the thiol groups liberated during reduction with 0.125mm-dithiothreitol, it was possible to identify the tryptic peptides involved in the disulphide bridges that link the 7s sub-units together (inter-sub-unit bridges). 4. By further reducing and labelling (with iodo[2-(14)C]acetic acid) the 7s sub-units produced by 0.125mm-dithiothreitol, it was possible to identify tryptic peptides derived from intra-sub-unit bridges. 5. Sub-units produced by reduction with 20mm-cysteine proved to be unsuitable for distinguishing between inter-sub-unit bridges and intra-sub-unit bridges. 6. The possible arrangement of the interchain disulphide bridges was deduced.     

Preparative high-performance liquid chromatographic separation of proteins with HyperD ion-exchange supports

HyperD ion-exchange media combine the mechanical strength of a rigid polystyrene-mineral composite skeleton with the high protein-binding capacity of a three-dimensional soft gel located inside the skeleton. The skeleton solid matrix is completely filled with functionalized, highly hydrophilic, chemically stable ion-exchange hydrogels. These materials gave very efficient columns for protein separation with superior dynamic capacity, high resolving power and excellent protein recovery. Various protein mixtures were used to study the chromatographic performance of these new stationary phases. Comparisons between different particle size packing materials demonstrated the potential of this ion-exchange material for use on a large scale.     

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.     

A hybrid Escherichia coli alkaline phosphatase formed on proteolysis

Cleavage of bacterial alkaline phosphatase by trypsin at the R-11, A-12 bond of both subunits results in changes in the structure and function of the enzyme as previously reported (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1985) J. Biol. Chem. 260, 7557-7561). A hybrid dimer has been formed by cleaving the R-11, A-12 bond of only one of the two subunits. This enzyme species has been purified and characterized to investigate subunit interactions of this hybrid dimeric enzyme species. Subunit interactions were observed using various methods to study functional and structural properties of the enzyme. In a kinetic study the T-2/A-12 hybrid enzyme was found to have a Vmax similar to the A-12 fully trypsin-modified enzyme. On exposure to EDTA the hybrid was found to lose activity at essentially the same rate as the A-12 enzyme presumably as a consequence of loss of metal ions required for function. On adding metal ions back to the apoenzyme form, activity of the hybrid was reconstituted to a degree similar to that of the native enzyme whereas the activity of the A-12 enzyme was reconstituted to a much lesser extent. The Tm of the hybrid measured by differential scanning calorimetry was closer to the value obtained for the A-12 enzyme than the T-2 enzyme but circular dichroic spectra indicated secondary structural features of the hybrid different from both symmetrical forms of the enzyme. These results provide evidence for strong subunit interactions in the alkaline phosphatase dimer.     

Molecular asymmetry in alkaline phosphatase of Escherichia coli

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.     

Determination of thiol proteins using monobromobimane labeling and high-performance liquid chromatographic analysis: application to Escherichia coli thioredoxin

A highly sensitive and specific assay for Escherichia coli thioredoxin was developed using the thiol-specific reagent monobromobimane. Treatment of dithiothreitol-reduced thioredoxin with an excess of monobromobimane in Tris buffer (pH 8.0, 23 degrees C) for 30 min resulted in the formation of a stable derivative which was quantitated by reverse-phase high-performance liquid chromatography with fluorescence detection providing sensitivity in the low picomole range. This method was applied to the determination of intracellular levels of thioredoxin in E. coli. Cell extracts were heated, treated with dithiothreitol, reacted with monobromobimane, and desalted to give a solution which was analyzable for thioredoxin using the chromatographic procedure.     

Microbore high-performance liquid chromatographic determination of cisapride in rat serum samples using column switching

For the determination of cisapride from serum samples, an automated microbore high-performance liquid chromatographic method with column switching has been developed. After serum samples (100 μl) were directly injected onto a Capcell Pak MF Ph-1 pre-column (10×4 mm I.D.), the deproteinization and concentration were carried out by acetonitrile–phosphate buffer (20 mM, pH 7.0) (2:8, v/v) at valve position A. At 2.6 min, the valve was switched to position B and the concentrated analytes were transferred from MF Ph-1 pre-column to a C₁₈ intermediate column (35×2 mm I.D.) using washing solvent. By valve switching to position A at 4.3 min, the analytes were separated on a Capcell Pak C₁₈ UG 120 column (250×1.5 mm I.D.) with acetonitrile–phosphate buffer (20 mM, pH 7.0) (5:5, v/v) at a flow-rate of 0.1 ml/min. Total analysis time per sample was 18 min. The linearity of response was good (r=0.999) over the concentration range of 5–200 ng/ml. The within-day and day-to-day precision (CV) and inaccuracy were less than 3.7% and 3.8%, respectively. The mean recovery was 96.5±2.4% with the detection limit of 2 ng/ml.     

Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.     

Three-minute high-performance liquid chromatographic assay for NMN adenylyltransferase using a 20-mm-long reversed-phase column

NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly catalyzes the synthesis of NAD from ATP and NMN. In this paper, we describe a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C₁₈ reversed-phase (RP) column. The activity was measured by separating in less than 3 min the substrates (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22°C. NAD was directly quantitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfully applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) cells.