Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes

The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such ‘isoelectrically immobilized’ reactor, the enzyme β-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5β-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3β-hydroxy-7,12-dioxo-5β-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD⁺) collected, separately, into the two neighbouring chambers at the anodic side. Advantages: in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.     

Heterogeneity of staphylococcal enterotoxin A on isoelectric focusing and disc electrophoresis.

Heterogeneity of purified staphylococcal enterotoxin A, obtained from a culture supernatant of Staphylococcus aureus, strain 13N-2909, was demonstrated by isoelectric focusing. The toxin was composed of three immunologically identical fractions with isoelectric points of 6.5, 7.0 and approximately 8.0. Heterogeneity of the toxin was also shown by disc electrophoresis. At pH 8.0 and 9.4 two major bands and a faint minor band were observed, while at pH 4.3 only one band was observed. The faster-moving band for the anode in disc electrophoresis at pH 9.4 was found to correspond with the pI 6.5 component from isoelectric focusing, while the slower-moving band corresponded with the pI 7.0 component. From the results of the electrophoretic migration tests of the toxin, the components corresponding to the two major bands found in disc electrophoresis at pH 9.4 were considered to be charge isomers.     

Heterogeneity of human erythrocyte band 3 analyzed by two-dimensional gel electrophoresis

Human erythrocyte membrane and purified band 3 were separated initially by isoelectric focusing and then examined in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Band 3 was segregated into three major bands whether the protein was contained within the membranes or was present in the isolated state. The isoelectric points of these major bands were 5.25, 5.35 and 5.70. Of chymotryptic fragments of band 3, the 60-kDa fragment was also separated into three major bands whose pI values were 4.75, 5.10 and 5.30. The multiplicity of band 3 appears to be due to different charges carried by the peptide(s) and is not ascribed to oxidation of band 3 during its preparation. Isoelectric points of the purified 60-kDa fragment were different from the pI values of the fragment coexisting with the complementary 35-kDa fragment, in which case the pI values were exactly the same as those of intact band 3. This suggests that these fragments interact tightly in situ even after being cleaved by chymotrypsin, and the tight interaction must still be present during electrophoresis in the first dimension.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient

A new method is described for preparative protein purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification never enters the Immobiline gel and thus does not have to be extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

Isoelectric focusing in immobilized pH gradients in the pH 10-11 range

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10 degrees C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 15.74 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the net positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).     

Recombinant-derived interleukin-1 alpha stabilized against specific deamidation

Recombinant-derived human interleukin-1 alpha (IL-1 alpha), purified from Escherichia coli, was resolved by isoelectric focusing on polyacrylamide gels into two species of isoelectric points (pI) 5.45 and 5.20, which constituted approximately 75% and approximately 25% of the total IL-1 alpha protein respectively. The pI 5.45 and pI 5.20 species were separated by chromatofocusing and subjected to N-terminal sequence analysis. The pI 5.45 species contained the expected Asn residue at position 36 of the mature protein sequence whereas the pI 5.20 species contained an Asp residue at the same position. A mutant protein in which Asn-36 was substituted for a Ser residue was isolated from E. coli and shown to be homogeneous on isoelectric focusing analysis with a pI = 5.45. 1H-n.m.r. and circular dichroism analyses of wild-type and the mutant IL-1 alpha indicated a similar conformation which was also indicated by the identical receptor binding affinities of IL-1 alpha with Asn, Asp or Ser in position 36. The mutant protein was stabilized against specific base-catalysed and temperature-induced deamidation, and may be more suitable than the wild-type position for physical and structural studies.     

Immunological properties of O2.- generating oxidase from bovine neutrophils

Two antisera have been prepared against the O2.- generating oxidase purified from bovine polymorphonuclear neutrophils (PMNs). The first antiserum was directed against the enzymatically active fraction obtained after isoelectric focusing (pI oxidase), which consisted of a major protein of Mr 65,000 [(1985) Biochemistry 24, 7231-7239]. The second antiserum was directed against the 65 kDa band excised from an SDS-polyacrylamide gel after electrophoresis of the pI oxidase preparation. The pI oxidase antiserum inhibited O2.- generation by PMN cells, PMN membranes and detergent-solubilized membranes. The 65 kDa band antiserum was virtually non-inhibitory against PMN cells; in contrast, it was nearly as potent as the pI oxidase antiserum on PMN membranes and detergent-solubilized membranes. Inhibition of O2.- generation by the pI oxidase antiserum was correlated with the immunoreactivity of four membrane-bound proteins of 65, 54, 18 and 16 kDa; the 65 kDa band antiserum reacted only with the two proteins of 65 and 54 kDa. It is concluded that the 18 and 16 kDa proteins, present in trace amounts in the pI oxidase preparation, are probably potent catalysts of the respiratory burst.     

Preparative isoelectric focusing and some properties of solubilized adenylate cyclase from rat brain

A new procedure for the purification of rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) is presented. The enzyme solubilized in Lubrol PX was purified either by molecular sieving or by hydrophobic chromatography, followed by a preparative isoelectric focusing step. For this purpose, a new isoelectric focusing technique was developed which allows a good resolution of adenylate cyclase in a short period of time. When resolved by this procedure, the enzyme migrated as a single molecular species with a pI of 6.3. When isoelectric focusing was performed in the presence of EGTA, two distinct peaks of activity could be detected at pI 6.1 and 7.3. This suggests that adenylate cyclase consists of two subunits held together by divalent ions. It is shown that the purified adenylate cyclase has a smaller sedimentation coefficient and is less hydrophobic than the native one. We conclude that the adenylate cyclase containing complex was at least partially disaggregated by this procedure.     

Isoelectric focusing of oat root membranes

The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad-range (pH 3-10) or narrow-range (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient. Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures.     

Functional and biochemical characterization of B-cell differentiation factor (BCDF) produced by an HTLV-I-transformed human T-cell clone and demonstration of specific binding of the factor to a BCDF responsive cell line

We have previously described YA2, a human T-cell clone that secretes B-cell differentiation factor (BCDF) but not B-cell growth factor (BCGF). The BCDFs secreted by YA2 and HTLV-I-transformed YA2 (TYA2) were functionally similar in their ability to stimulate Ig secretion by Staphylococcus aureus Cowan strain I-activated B cells and IgM secretion by SKW6.4 cells. In addition, they were biochemically similar with a MW of 30 kDa by high-performance liquid chromatography (HPLC) sieving, and a pI of 6.0-6.8 by isoelectric focusing. The BCDF activity was not blocked by antibodies to interleukin 2 and BCGF. BCDF was purified from TYA2 supernatant by sequential media protein immunoadsorption, flat bed isoelectric focusing, HPLC TSK 2000 sieving, and repeated immunoadsorption and was then iodinated. The iodinated material had functional BCDF activity and migrated to a single band at MW 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at pI of 6.8 by polyacrylamide gel isoelectric focusing. 125I-BCDF purified in this manner bound specifically to a BCDF-responsive cell line and not to phytohemagglutinin-activated T cells. 125I binding to the BCDF-responsive cell line was competitively inhibitable by the addition of cold BCDF. Thus we have purified and characterized a factor with BCDF activity and demonstrated that this factor binds specifically to a BCDF-responsive cell line.     

The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point

The murine lymphocyte pore-forming protein (PFP) was purified to apparent homogeneity by successive steps of liquid chromatography. Monospecific antibodies were raised against purified PFP that detect only one protein band in murine CTL lines. 25% of the primary sequence of PFP (134 amino acids) was determined by amino terminal analysis of the purified protein and of some of its enzymatic cleavage products. These primary sequences were identical to sequences deduced by cDNA cloning. By isoelectric focusing, PFP was found to have a pI of 6.4. On the chromatofocusing column Mono P, however, PFP was found to elute at pH 4.7. This suggests a tertiary structure for monomeric PFP that is enriched in surface acidic amino acids.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Biochemical discrimination of the predominant pertussis toxin substrate of rabbit neutrophils from brain Gi and Go: isoelectric focusing improves resolution of pertussis toxin substrates

In the present study, the predominant pertussis toxin substrate in rabbit neutrophils, Gn, was biochemically compared to Gi and Go purified from brain, after being [32P]ADP-ribosylated by activated pertussis toxin and [32P]NAD. On SDS-polyacrylamide gels, a poorly resolved doublet from neutrophil membranes was observed; the upper band, corresponding to approximately equal to 25% labeling, comigrated with Gi-alpha and the predominant lower band, Gn, migrated intermediately between Gi-alpha and Go-alpha. Peptide maps generated by limited-digestion of the labeled Gn, Gi and Go with S. aureus V8 protease were slightly, but definitively and reproducibly different. Isoelectric focusing clearly distinguished Gn from the other two pertussis toxin substrates. The pI value of Gn, 5.60, was distinctly different from those of Gi, 5.75 and 5.80. Although the pI values for Go and Gn were similar (5.60), the patterns of the two proteins were qualitatively different, with Go being resolved into an equal doublet (pI = 5.55 and 5.60) while Gn appeared predominantly as a single band. Thus, Gn is biochemically distinguishable from Gi and Go of brain and these structural differences are most clearly evident following isoelectric focusing.     

A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein.

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.     

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.     

Acid triacyglycerol lipase inhibitor in chicken plasma: purification and properties

1. Acid triacylglycerol lipase inhibitor was highly purified from chicken plasma by ammonium sulfate fractionation (0.6-1 saturation) followed by successive chromatographies on Hydroxyapatite, Blue-Cellulofine, Phenyl-Sepharose and Cellulofine GCL-2000 columns, and isoelectric focusing. 2. The lipase inhibitor showed its inhibitory action on triacylglycerol lipases in chicken erythrocytes ghosts and in chicken liver lysosome, but did not on pancreatic lipase, Rhizopus arrhizus lipase, or wheat germ lipase. 3. The inhibitor showed its molecular weight of 32,000 by Cellulofine GCL-2000 gel filtration. The inhibitor showed some heterogeneity on isoelectric focusing, and the main band had a pI of 5.10. 4. The lipase inhibitor did not show any inhibitory action on trypsin or chymotrypsin.