Two types of glucose effects on beta-galactosidase synthesis in a membrane fraction of Escherichia coli: correlation with repression observed in intact cells.

A membrane fraction obtained from an osmotic lysate of Escherichia coli spheroplasts retains capability to synthesize beta-galactosidase. The system also retains cellular regulatory functions, one of which is known as catabolite repression. Two types of repression of beta-galactosidase synthesis were observed in this membrane system: one was caused by the addition of 2-deoxyglucose or glucose at a low concentration (3 times 10- minus 4 M), and the other was caused by glucose-6-phosphate or glucose at a high concentration (3 times 10- minus 2 M). In the presence of cyclic adenosine 3',5'-monophosphate (10 mM), repression caused by the former was completely reversed, whereas repression by the latter was only partially reversed. Conditions in intact cells causing transient and permanent repression were also investigated. Upon addition of 2-deoxyglucose or glucose at a low concentration to intact cells, only transient repression of beta-galactosidase synthesis was observed. Glucose at a high concentration caused both transient and subsequent permanent repression, and intensity of permanent repression depended upon glucose concentration, whereas duration and intensity of transient repression were independent of glucose concentration. Mutants deficient in phosphoenolpyruvate-phosphotransferase system (Hpr minus and enzyme I minus) showed transient repression but failed to show permanent repression. In mutants deficient in glucose catabolism beyond glucose-6-phosphate, both transient and permanent repression were observed. Correlation between the observations in the membrane system and in intact cells is discussed. The results obtained here strongly suggest that transient repression is caused by glucose itself, and that permanent repression is caused by glucose-6-phosphate of high intracellular levels of glucose.     

Role of the Cytoplasmic Membrane in the Synthesis of Ribonucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Disrupted spheroplast preparations of Pseudomonas schuylkilliensis strain P contained fragments of cytoplasmic membrane and approximately 82% of the total cellular phospholipid. The protoplast-bursting factor (PB-factor), partially purified from pig pancreas, and a heat-treated pancreatic lipase fraction both inhibited ribonucleic acid (RNA) synthesis by disrupted spheroplasts but did not inhibit or only slightly inhibited RNA synthesis by intact cells or intact spheroplasts. The PB-factor preparation and the heat-treated pancreatic lipase fraction catalyzed partial (15 to 50%) deacylation of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine in disrupted spheroplasts but not in intact spheroplasts. Phospholipase A activity was demonstrated in the PB-factor preparation by use of isolated phospholipids as substrates. Treatment of disrupted spheroplasts with the PB-factor preparation caused a 70% inhibition in oxidative phosphorylation and RNA synthesis, but had little effect on electron transport. Addition of adenosine-5'-triphosphate or adenosine-5'-diphosphate and a mixture of ribonucleosides after treatment with the PB-factor preparation partially restored oxidative phosphorylation but did not relieve the inhibition in RNA synthesis. The most reasonable explanation for the latter observation appears to be that the concentrations of newly synthesized nucleotides retained by the preparations with partially deacylated membrane phospholipids were insufficient to permit the synthesis of RNA.     

Lipoprotein synthesis in Escherichia coli spheroplasts: accumulation of lipoprotein in cytoplasmic membrane.

Synthesis of cell envelope proteins was studied in ethylenediaminetetraacetic acid-lysozyme spheroplasts of Escherichia coli ML30. The rate of incorporation of [3H]arginine into proteins in spheroplasts was about 30% of that of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized in spheroplasts revealed the preferential synthesis of five polypeptides, one of which has been identified as the free form of murein lipoprotein. Lipoprotein synthesized in spheroplasts was found to be of same molecular size as that of mature lipoprotein. No prolipoprotein was observed even with a short pulse-labeling with [3H]arginine. On the other hand, significant accumulation of newly synthesized lipoprotein in the cytoplasmic membrane fraction of spheroplasts was observed. These results suggest that the processing of prolipoprotein occurs in the cytoplasmic membrane fraction of the cell envelope.     

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.     

Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating (32)P-orthophosphate into RNA and DNA, (3)H-adenosine or (3)H-uridine into RNA, and (3)H-leucine or (14)C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A(3), streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.     

Effect of glucose and its analogues on the accumulation and release of cyclic adenosine 3'',5''-monophosphate in a membrane fraction of Escherichia coli: relation to beta-galactosidase synthesis.

Correlation between beta-galactosidase synthesis and cyclic adenosine 3',5'-monophosphate (cAMP) levels in a membrane fraction obtained from disrupted spheroplasts of Escherichia coli was investigated. Repression of beta-galactosidase synthesis in the membrane fraction by glucose-6-phosphate and by 2-deoxyglucose differed in sensitivity to reversal by cAMP. The difference between the two repressions could be due to the fact that glucose-6-phosphate inhibited severely the accumulation of exogenous [3-H]cAMP by the membrane fraction, whereas 2-deoxyglucose had little effect on the accumulation of the nucleotide. On the other hand, a quick decrease in the level of [3-H]cAMP preaccumulated in the membrane fraction resulted from addition of either glucose-6-phosphate or 2-deoxyglucose. Results reported here suggest that repression of beta-galactosidase synthesis is associated with anabrupt decrease in cAMP levels at the intramembranal sites where beta-galactosidase is synthesized, and the major, if not sole, mechanism which leads to instantaneous drop of cAMP level is via the release of cAMP, but not by degradation of the nucleotide since the membrane fraction retained less than 10 percent of cellular cyclic phosphodiesterase and the activity of the enzyme was not affected by repressing sugars.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

Expression in Escherichia coli of chemically synthesized gene for a novel opiate peptide alpha-neo-endorphin

Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13. The resulting recombinant DNA was used to transform E. coli cells. Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E. coli cell. One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification. From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin.     

Mechanical properties of the lateral cortex of mammalian auditory outer hair cells.

Mammalian auditory outer hair cells generate high-frequency mechanical forces that enhance sound-induced displacements of the basilar membrane within the inner ear. It has been proposed that the resulting cell deformation is directed along the longitudinal axis of the cell by the cortical cytoskeleton. We have tested this proposal by making direct mechanical measurements on outer hair cells. The resultant stiffness modulus along the axis of whole dissociated cells was 3 x 10(-3) N/m, consistent with previously published values. The resultant axial and circumferential stiffness moduli for the cortical lattice were 5 x 10(-4) N/m and 3 x 10(-3) N/m, respectively. Thus the cortical lattice is a highly orthotropic structure. Its axial stiffness is small compared with that of the intact cell, but its circumferential stiffness is within the same order of magnitude. These measurements support the theory that the cortical cytoskeleton directs electrically driven length changes along the longitudinal axis of the cell. The Young's modulus of the circumferential filamentous components of the lattice were calculated to be 1 x 10(7) N/m2. The axial cross-links, believed to be a form of spectrin, were calculated to have a Young's modulus of 3 x 10(6) N/m2. Based on the measured values for the lattice and intact cell cortex, an estimate for the resultant stiffness modulus of the plasma membrane was estimated to be on the order of 10(-3) N/m. Thus, the plasma membrane appears to be relatively stiff and may be the dominant contributor to the axial stiffness of the intact cell.     

Transfection of Escherichia coli and Salmonella typhimurium Spheroplasts: Host-Controlled Restriction of Infective Bacteriophage P22 Deoxyribonucleic Acid

Under proper conditions, one infective center was obtained for 3 x 10(8) molecules of P22 phage deoxyribonucleic acid (DNA) when lysozyme-ethylenediaminetetraacetic acid spheroplasts of Escherichia coli were transfected in the presence of 25 mug of protamine sulfate per ml. A 3- to 50-fold B-specific and K-specific E. coli restriction of the incoming P22 DNA was observed. When P22 DNA-infected E. coli spheroplasts were plated with infertile r(LT) (+)m(LT) (+)Salmonella typhimurium indicator, an additional 70-fold restriction was observed. In the presence of protamine sulfate, penicillin spheroplasts of S. typhimurium SB1330 could be transfected b P22 DNA with efficiencies sometimes approaching those obtained with the E. coli spheroplasts; thus, facilitation of transfection by protamine sulfate is not limited to E. coli or to lysozyme-ethylenediaminetetraacetic acid spheroplasts. The application of these results to studies of transfection among other genuses and to studies of in vitro host-controlled restriction and modification for the two loci in S. typhimurium and the one locus in E. coli is discussed.     

Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method.

We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes. Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion. Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone. Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity. After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.     

Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2.

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.     

Permeability of the membrane of the Escherichia coli envelope for ethidium bromide

The interaction of ethidium bromide, a fluorescent dye, with Escherichia coli cells was studied. The envelope of intact cells was shown to be impermeable for ethidium bromide molecules. The dye penetrated however into E. coli spheroplasts. The barrier properties of the cell envelope against ethidium bromide were ruptured if the cells were treated with EDTA. The results suggest that the outer membrane serves as a principal barrier against penetration of ethidium bromide inside the cells while the cytoplasmic membrane of E. coli is permeable for the dye.     

Binding and enrichment of Escherichia coli spheroplasts expressing inner membrane tethered scFv antibodies on surface immobilized antigens

Anchored periplasmic expression (APEx) is a new method for the isolation of high affinity ligand-binding proteins from large combinatorial libraries (Harvey et al., 2004, Proc Natl Acad Sci USA 101(25): 9193-9198). In APEx, proteins are expressed as fusions to a membrane anchor that tethers them onto the periplasmic side of the Escherichia coli inner membrane. Conversion of the cells into spheroplasts and incubation with soluble fluorescently conjugated ligands results in the specific labeling of cells expressing ligand-binding proteins and their subsequent isolation by flow cytometry. Here we show that scFv antibody fragments expressed in the APEx format allow the binding of spheroplasts to immobilized ligands. ScFv antibodies specific for the cardiac glycoside digoxin or for the protective antigen (PA) of Bacillus anthracis as a negative control were expressed in E. coli as fusions to either N-terminal or C-terminal membrane anchoring domains. Only the C-terminally anchored fusions resulted in specific recognition and binding of spheroplasts onto TentaGel beads with immobilized antigen. Following three rounds of flow cytometric screening, spheroplasts expressing anti-digoxin scFvs were enriched 950-fold from a large excess (1,000 x) of spheroplasts expressing anti-PA antibodies. These results indicate that the APEx technology may be employed for the screening of libraries based on binding to insoluble antigens possibly including antigens on cell surfaces.     

Orientation of Membrane Vesicles from Escherichia coli as Detected by Freeze-Cleave Electron Microscopy

The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K(+)-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na(+)-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K(+)-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that "dislocation" of membrane proteins occurs during osmotic lysis of spheroplasts.     

Investigation of osmotically stable spheroplasts from two strains of Escherichia coli ML-35, W7-M5.

Osmotically stable spheroplasts were produced from Escherichia coli ML-35 and W7-M5 using either 1 min exposure to polymyxin B or 10 min exposure to Tris/EDTA, followed by 1 to 3 h incubation with lysozyme. Spheroplast membrane permeability studies were conducted using paired radioactive probes with E. coli ML-35. Experiments with 14C-sucrose-16 kD 3H-dextran indicated that the outer membrane had lost its barrier to 16 kD dextran. Parallel experiments with 81 kD 3H-dextran indicated that the outer membrane was impermeable to the larger dextran. EDTA treated cells also showed outer membrane permeability to 16 kD dextran. Cytoplasmic membrane integrity was confirmed using 14C-sucrose and 3H2O before and after exposure to polymyxin B and EDTA. Scanning electron microscopy showed that a rough surface on polymyxin B produced spheroplasts while Tris/EDTA spheroplasts showed the same smooth surface as control cells.     

The correcting role of autonomous 3'-->5' exonucleases in mammalian multienzyme DNA polymerase complexes

A study was made of the correcting role of autonomous 3'-->5' exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes. DNA was synthesized on phage psi X174 amber3 or M13mp2 primer-templates, and used to transfect Escherichia coli spheroplasts. Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis. The mistake rate of the hepatocytic complex was estimated at 3 x 10(-6) with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3'-->5' exonuclease activity of the complex were removed. The fidelity of DNA synthesis was completely restored in the presence of exogenous AE (epsilon subunit of E. coli DNA polymerase III). Nuclear (Pol delta n) and cytosolic (Pol delta c) forms of DNA polymerase delta were isolated from calf thymocytes. The former was shown to contain an AE (TREX2) absent from the latter. As compared with Pol delta c, Pol delta n had a 20-fold higher exo/pol ratio and allowed 4-5 times higher fidelity of DNA synthesis. The mistake rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.     

Release of periplasmic enzymes from Escherichia coli by penicillin-ethylenediaminetetraacetate treatment.

When Escherichia coli cells are converted into spheroplasts by penicillin treatment, none of the periplasmic enzymes is released. However, incubation of the penicillin-induced spheroplasts in a medium containing ethylenediaminetetraacetic acid caused the release of endonuclease I and cyclic phosphodiesterase but not of beta-galactosidase.     

Purification and some characteristics of a recombinant dimeric rhizobium meliloti beta-galactosidase expressed in escherichia coli

A recombinant Rhizobium meliloti beta-galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl beta-D-galactopyranoside (PNPG) and o-nitrophenyl beta-D-galactopyranoside (ONPG) with K(m)(PNPG) and K(m)(ONPG) of 1 mM at 25 degrees C. The k(cat)/K(m) ratios for both substrates were approximately 70 mM(-1) sec(-1), indicating no clear preference for either PNPG or ONPG, unlike E. coli beta-galactosidase. After non-denaturing electrophoresis, active beta-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl beta-D-galactopyranoside (BNG) and diazo blue B.     

Properties of D-arabinose isomerase purified from two strains of Escherichia coli

d-Arabinose isomerase (EC 5.3.1.3) has been isolated from l-fucose-induced cultures of Escherichia coli K-12 and d-arabinose-induced cultures of E. coli B/r. Both enzymes were homogeneous in an ultracentrifuge and migrated as single bands upon disc electrophoresis in acrylamide gels. The s(20,w) was 14.5 x 10(-13) sec for the E. coli K-12 enzyme and 14.3 x 10(-13) sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 +/- 0.06 x 10(5) for the E. coli K-12 enzyme and 3.42 +/- 0.04 x 10(5) for the enzyme isolated from E. coli B/r. Both enzyme preparations were active wth l-fucose or d-arabinose as substrates and showed no activity on any of the other aldopentoses or aldohexoses tested. With the E. coli K-12 enzyme, the K(m) was 2.8 x 10(-1)m for d-arabinose and 4.5 x 10(-2)m for l-fucose; with the E. coli B/r enzyme, the K(m) was 1.7 x 10(-1)m for d-arabinose and 4.2 x 10(-2)m for l-fucose. Both enzymes were inhibited by several of the polyalcohols tested, ribitol, l-arabitol, and dulcitol being the strongest. Both enzymes exhibited a broad plateau of optimal catalytic activity in the alkaline range. Both enzymes were stimulated by the presence of Mn(2+) or Co(2+) ions, but were strongly inhibited by the presence of Cd(2+) ions. Both enzymes were precipitated by antisera prepared against either enzyme preparation. The amino acid composition for both proteins has been determined; a striking similarity has been detected. Both enzymes could be dissociated, by protonation at pH 2 or by dialysis against buffer containing 8 m urea, into subunits that were homogeneous in an ultracentrifuge and migrated as single bands on disc electrophoresis in acrylamide gels containing urea. The molecular weight of the subunit, determined by high-speed sedimentation equilibrium, was 9.09 +/- 0.2 x 10(4) for the enzyme from E. coli K-12 and 8.46 +/- 0.1 x 10(4) for the enzyme from E. coli B/r. On the basis of biophysical studies, both isomerases appear to be oligomeric proteins consisting of four identical subunits.