Reconstitution of denatured E. coli alkaline phosphatase with E. coli ribosome.

E. coli alkaline phosphatase was denatured by physical/chemical means. In vitro reconstitution of this denatured enzyme was assisted by 70S E. coli ribosome, as shown by the recovery of its catalytic competence. Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.     

3-13C-methionine-labelled E. coli alkaline phosphatase

3-13C-methionine has been biosynthetically incorporated into E. coli alkaline phosphatase using strain CW3747 which is auxotrophic for Met. 13C NMR of the dimeric native enzyme labelled at the eight methionine residues of the primary structure shows a dispersion of resonance signals permitting resolution of at least five methionine environments, none of which coincide with the chemical shift position of free methionine. At acid pH, 13C signal intensity is shifted to a chemical shift consistent with solvent exposure. However, three discrete resonances are observed, suggesting a retention of defined structure. The labelled protein thus can serve as a probe of conformational alterations of the enzyme.     

Phospholipids of E. coli and activity of alkaline phosphatase]

The effects of liposomes prepared from the E. coli lipids on the activity of soluble alkaline phosphatase and on the complementation reaction between its subunits were studied. It was shown that the liposomes nonspecifically catalyze the dimerization of the enzyme subunits without changing the dimer activity. The effects of phospholipases A2 and C on the activity of membrane-bound alkaline phosphatase were studied. An interrelationship was found between the level of hydrolysis of membrane phosphatidyl glycerol (PG) by these enzymes and the changes in the activity of membrane-bound alkaline phosphatase. It was also shown that PG is less accessible to the effects of phospholipases in the cells with derepressed biosynthesis of alkaline phosphatase. It is assumed that the membrane PG interacts with the membrane-bound alkaline phosphatase during its translocation into the periplasm.     

Hysteresis on heating and cooling of E. coli alkaline phosphatase

Measurements of [theta](222) of E. coli phosphatase on heating from 20 degrees to 90 degrees and subsequent cooling to 20 degrees shows a gradual increase in [theta](222) on heating, while cooling shows a symmetric transition centered at 45 degrees . Reheating and cooling shows the same phenomenon. Enzyme heated and cooled once is fully active. The activity of the enzyme depends on its storage conditions (buffer and pH for example), but such changes are least to some extent reversible, especially by heating in different solvents. We conclude the enzyme exists in several forms which are in slow equilibrium with each other, so that the enzyme responds slowly when heated and hence is not at equilibrium during heating/cooling experiments.     

Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase.

1. An alkaline phosphatase was partially purified from extracts of Halobacterium cutirubrum. 2. The enzyme has a mol.wt. of 15 500 and is therefore less than one-quarter of the size of other known bacterial alkaline phosphatases. 3. It is stimulated up to ten-fold by Mn2+, but not by Ca2+ or Mg2+. 4. The activities with and without Mn2+ cannot be separated by gel filtration and have similar restricted substrate specificities. 5. The only substrates for the enzyme that have so far been found are p-nitrophenyl phosphate, 5'-dATP, 5'-dTMP and 5'-dTTP.     

Factors affecting the zinc content of E. coli alkaline phosphatase

Through experiments with radioactively labeled EDTA, it has been shown that alkaline phosphatasc from E. coli has a high affinity for binding EDTA, requiring extensive dialysis for removal. This paper reviews the results of zinc analyses of E. coli alkaline phosphatase prepared in the presence and absence of EDTA. The presence of EDTA in most preparations of alkaline phosphatase accounts for previous overestimates of the zine content of the enzyme.With radioactively labeled EDTA, direct evidence for the binding of EDTA to the metal-free alkaline phosphatase is presented. It has been shown that the apoprotein binds two EDTA molecules rather strongly. Addition of four metal ions are necessary for reactivation of this EDTA-contaminated apoenzyme. However, when the EDTA-contaminated apoenzyme is subject for extensive dialysis and EDTA is removed, the addition of two zinc ions restores the enzyme activity completely.     

Reactivation of denatured fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase with E. coli ribosome.

Fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase were denatured either by physical or by chemical means. In vitro reconstitution of these denatured enzymes was assisted by 70S E. coli ribosome, as shown by the recovery of their catalytic competence. Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively.     

Isolation and properties of fecal proteins and fecal alkaline phosphatase from germfree and conventional rats.

Fecal proteins from germfree and conventional rats were isolated. The proteins from the two kinds of feces differed in molecular weight, judging from Sephadex gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The conventional feces contained a greater amount of high-molecular-weight and a lesser amount of low-molecular-weight proteins than did the germfree feces. The fecal proteins of both kinds contained carbohydrates. Both feces contained considerable enzyme activity. The germfree feces contained extremely high activity in alkaline phosphatase and leucine aminopeptidase. Both feces showed the same level of trehalase activity. The conventional feces contained higher levels of activity of protease and acid phosphatase than did the germfree feces. Lactase activity was observed only in the conventional feces. The fecal alkaline phosphatase resembled the intestinal enzyme in response to L-phenylalanine inhibition and urea denaturation. From these results it was inferred that the germfree feces contained some of the intestinal proteins and that the conventional feces contained bacterial proteins in addition to intestinal proteins.     

Metal-ion induced conformational changes in alkaline phosphatase from E. coli assessed by limited proteolysis

Alkaline phosphatase (AP) displays significant structural changes during metal-ion binding, supporting cooperative interactions between the subunits of the dimeric enzyme. Here, we present data on the dynamic properties of AP from E. coli, and characterize the structural changes that accompany variations in metal-ion content, combining limited proteolysis and MALDI-TOF mass spectrometry. Limited proteolysis revealed an internal cleavage site at Arg-293, reflecting a position of conformational flexibility supporting subunit communication essential for catalysis. A specific shielding of a region distant from the metal-binding site has been demonstrated, implying transmission of conformational changes, induced by metal-ion binding to the adjacent subunit, across the subunit interface.     

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.     

Zinc and magnesium content of alkaline phosphatase from Escherichia coli.

Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-aton of magnesium per molecular weight 80,000 (Plocke, D.J., Levinthal, D., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared.     

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.     

Plasmid expression vectors with elements of the E. coli alkaline phosphatase gene

A family of plasmid expression vectors, containing fragments of the E. coli alkaline phosphatase gene (phoA), has been constructed for the lambda PR promoter-directed thermoinducible superproduction of fusions of heterologous polypeptides to the N- or C-terminus of E. coli alkaline phosphatase and its leader peptide. Effective expression and export to periplasm of resulting fusion proteins, which may retain enzymatic activity of the phosphatase, has been shown.     

Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants

Summary In continuous cultures, alkaline phosphatase was synthesised and excreted for more than 250 h by immobilized growing cells in contrast to free cells for which the excretion decreased after 150 h of culture. This observed increase in alkaline phosphatase synthesis and excretion by immobilized cells may have resulted from growing conditions within the gel beads.Offprint requests to: C. Manin