Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.     

Purification and characterization of an aminopeptidase from the chloroplast stroma of barley leaves by chromatographic and electrophoretic methods

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a M_r of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The M_r of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed.     

Rapid purification and properties of human glycodelin (endometrial α2-globulin)

The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at M_r 28 000 after sodium dodecyl sulphate–polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with pI values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of β-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of α-helix conformation.     

Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48

Quantitation of plasma apo B-48 is currently performed by densitometric analysis of SDS–PAGE zones stained with Coomassie Brilliant Blue, using standard solutions of purified apo B-48. Here, preparative gel electrophoresis with a continuous elution system was used for purifying apo B-48. A chylomicron fraction was isolated by 107 000 g ultracentrifugation of a chylous ascite. The proteins were delipidated and precipitated in ethanol–diethyl ether (3:1, v/v), subjected to preparative electrophoresis in a 5% polyacrylamide gel and eluted in 0.1% SDS. The peak containing apo B-48 was eluted at a retention time of 445–480 min. The purity of apo B-48 in this fraction was assessed by the detection of a single band (M_r 260 000) after silver staining and Coomassie staining of 4–15% gradient SDS–PAGE. It was confirmed by the absence of apo B-100 contaminant in Western blot of the purified protein preparation. A linear relationship was observed between the densitometric analysis of SDS–PAGE bands and the apo B-48 in a protein range of 0–3 μg. In conclusion, preparative gel electrophoresis was used in a single step purification of apo B-48 that was adapted to the preparation of a standard solution.     

Purification and characterization of laccase from Monocillium indicum Saxena

Summary An ascomycete Monocillium indicum Saxena producing extracellular laccase was isolated. The culture filtrate on native polyacrylamide gel electrophoresis (PAGE) revealed four bands of activity, one of which was a major one. The major laccase band, a glycoprotein, was purified and characterized. Gel filtration chromatography showed that the relative molecular weight (M_r) of laccase was 100 000. On sodium dodecyl sulphate (SDS)-PAGE the major laccase band further resolved into three proteins of M_r 72 000, 56 000 and 24 000. The enzyme had a pH optimum of 3.0 and was active on a number of o-phenols and aromatic acids. The 72 000 M_r protein was found to share common immunological properties with laccases of Coriolus versicolor, Agaricus bisporus and lignin peroxidase of Phanerochaete chrysosporium. Correspondence to: K. Koteswara Rao     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Purification and partial characterisation of α2-antiplasmin and plasmin(ogen) from ostrich plasma

This study reports the isolation and partial characterisation of the ostrich serpin, α₂AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich α₂AP was purified using lysine–Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI–Sepharose chromatographies. It revealed a M_r of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine α₂AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human α₂AP, DFP and EACA. Ostrich plasminogen was highly purified after lysine–Sepharose chromatography and showed a M_r of 92 K, a total of 775 amino acids and its N-terminal sequence showed 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M_r of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40°C, respectively.     

Purification and properties of two truncated endoglucanases produced in Escherichia coli harbouring Clostridium cellulolyticum endoglucanase gene celCCD

The endoglucanase gene, celCCD, of Clostridium cellulolyticum has been expressed in Escherichia coli. Multiple active polypeptides were detected in the E. coli cells. The relative molecular mass (M_r) of two major active polypeptides were 56 000 (D56) and 38 000 (D38), which were smaller than the deduced M_r of the mature protein (63 401). D56 and D38 were purified from the periplasmic fraction. The N-terminal sequences of the two purified polypeptides were identical to that of the mature endoglucanase (Ala-Ile-Asn-Ser-Gln-Asp-Met-Val---) deduced from the nucleotide sequence. These data indicated that these polypeptides were produced by processing the original mature protein in the C-terminal region. The enzymatic properties of these two polypeptides were very similar, except that the specific activity of D38 was 2–3.5-fold higher than that of D56, and D38 was more heat stable than D56. Correspondence to: T. Kodama     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA₆₃)₇, in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA₆₃)₇ incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF_N (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF_N adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF_N in the normal direction but also can, at least partially, translocate LF_N in the opposite direction.     

Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran inSphingomonas sp. strain RW1

Sphingomonas sp. strain RW1, when grown in salicylate-salts medium, synthesized the enzymes for the degradation of dibenzofuran. The reaction subsequent tometa cleavage of the first benzene ring was found to be catalyzed by two isofunctional hydrolases, H1 and H2, which were purified by chromatography on anion exchange, hydrophobic interaction and gel filtration media. Each enzyme was able to hydrolze 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate to produce salicylate and benzoate, respectively. SDS/PAGE of each purified enzyme showed a single band ofM _r 31 000 (H1) or 29 000 (H2). The N-terminal amino acid sequences of the two proteins showed 50% homology.Abbreviations DHB 2,3-dihydroxybiphenyl - DSM German Culture Collection (Braunschweig) - FPLC protein liquid chromatograph(y) - HOHPDA 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - THB 2,2,3-trihydroxybiphenyl     

Purification and biochemical characterization of lysosomal acid phosphatases (EC 3.1.3.2) from blood stream forms, Trypanosoma brucei brucei

Three Acid phosphatases (ACP) were isolated and characterized from the lysosomes of blood stream forms of Trypanosoma brucei by a combination of isopynic and differential centrifugation through Ficoll, organic solvent precipitation, ion exchange on DEAE cellulose 52 and size exclusion chromatography on Sephadex G-75 columns. The purified ACP emerged as three distinct peaks (ACP I, ACP II and ACP III) with high specific activities and they moved homogenously on 12% SDS-PAGE each as a single band with relative molecular weight of 36 kDa, 25 kDa and 45 kDa respectively. The purified enzymes were active at an optimum pH and temperature of 5.5 and 40 °C respectively. The enzyme activities appeared to be ACP because their activities were enhanced at low pH values and inhibited by the acid phosphatase inhibitor, sodium fluoride. ACP I and ACP II were sensitive to l-tartrate while ACP III was insensitive to l tartrate. The kinetic analysis of the purified enzyme (ACP I, ACP II and ACP III) determined using para-nitrophenylphosphate as substrate gave K_M values of 0.2 mM, 0.15 mM and 0.5 mM. Monofunctional group sulfhydryl group inhibitors; HgCl₂, and AgCl₂ strongly inhibited the activity of ACP III and millimolar concentrations of dithiothreitol and iodoacetamide activated and inhibited the activity of the ACP III respectively, suggesting the involvement of thiol groups at the active site of the enzyme. Thus, differentiating it from ACP I and ACP II. The implication of these findings in relation to the pathology of trypanosomosis is discussed.     

Purification of an elicitor-induced glucan synthase (callose synthase) from suspension cultures of French bean (Phaseolus vulgaris L.): purification and immunolocation of a probable Mr-65 000 subunit of the enzyme

Membrane preparations from suspension-cultured cells of French bean (Phaseolus vulgaris L.) contained callose synthase (EC 2.4.1.34) activity which was preserved upon solubilisation. Following elicitor treatment of cell cultures, increased activity could be extracted and this increase was maintained during purification. The enzyme was purified by high-pressure liquid chromatography and active fractions showed a variable association of two polypeptides of relative molecular masses (M_r) 55 000 and 65 000, the latter being in excess. The M_r-65 000 polypeptide was purified to homogeneity and an antibody raised to it. This antibody showed complex effects on callose synthase activity when incubated with membrane and soluble extracts. In comparison with other systems, the M_r-55 000 subunit is likely to represent the catalytic subunit while the M_r-65 000 polypeptide is a possible regulatory subunit. The M_r-65 000 polypeptide was immunolocated in membranes at sites of callose synthesis in the plant, in cell plates, in sieve plates, at the plasma membrane-wall interface of wounded cells and in papillae in infected cells. Received: 18 January 1997 / Accepted: 8 May 1997     

Isolation and partial characterization of nigrin b,a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

The bark ofSambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b.In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (M _r 58 000) contains two subunits, A (M _r 26 000) and B (M _r 32 000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.     

Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Purification of theN-acetylglucosaminide α(1–3/4) fucosyltransferase of human milk

TheN-acetylglucosaminide (1–3/4)fucosyltransferase has been purified 1.8×10⁶-fold from human milk by ion-exchange chromatography, affinity chromatography of GDP-agarose and HPLC. The (1–3/4)fucosyltransferase behaves in gel filtration-HPLC as a molecule of M_r 98 000, and differs from the (1–3)fucosyltransferase which behaves like a molecule of about M_r 47 000. The enzyme is a glycoprotein, and the purified preparation appears in SDS polyacrylamide gel electrophoresis as a band of M_r 44 000. The results present the first purification of human milk (1–3/4)fucosyltransferase to apparent homogeneity, and suggest that the (1–3/4)- and (1–3)fucosyltransferases of human milk differ in their native molecular sizes, the former being a dimer of two subunits.     

Improved purification and biochemical characterization of extracellular amylopullulanase from Thermoanaerobacter ethanolicus 39E

A maltose-limited chemostat culture was used to investigate the expression and excretion of amylopullulanase by Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). In maltose-limited continuous culture, amylopullulanase was produced and secreted at tenfold higher levels than in batch culture. The extracellular amylopullulanase was purified to homonogeneity by using an inhibitor-linked affinity column matrix. The purified amylopullulanase had a specific activity of 480 units (U)/mg protein for pullulanase and 175 U/mg protein for -amylase. -Cyclodextrin inhibited both -amylase and pullulanase activities, with a substrate inhibition constant (K _i) of 0.065 mg/ml.Amylopullulanase had a relative molecular mass (M_r) of 140 000 using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and an M_r of 133 000 using gel-filtration chromatography. The N-terminal sequence of the enzyme was Glu-Thr-Asp-Thr-Ala-Pro-Ala. The purified enzyme displayed Michaelis constant (K _m) values of 0.35 mg/ml for pullulan and 1.00 mg/ml for amylose. The enzyme had an isoelectric point (pI) of 4.0, and displayed an optimum pH for stability and activity of 6.2 and 5.5, respectively. The enzyme was stable up to 85° C in the presence of Ca²⁺, and had a half-life of 40 min at 90° C (pH 6.2). Ca²⁺ was required for thermal stability, but not for activity. Amylose, glycogen, and amylopectin were degrade to maltose, maltotriose, and maltotetraose, whereas only maltotriose was formed from pullulan. Correspondence to: J. G. Zeikus     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

Simultaneous preparation of the three biotin-containing mitochondrial car☐ylases from rat liver

Affinity chromatography on avidin-Sepharose column was used to bind the biotin-containing car?ylases from rat liver. With a biotin gradient (0–0.3 mM), peaks of activity of pyruvate, propionyl CoA and β-methylcrotonyl CoA car?ylases co-eluted. Subsequent separation of the three car?ylases was attained using DEAE-Sepharose chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed each of the enzymes to be pure, with pyruvate car?ylase giving a single subunit band (M_r 130 000), propionyl-CoA car?ylase giving two bands (M_r 73 000and56500) and β-methylcrotonyl-CoA car?ylase giving two bands (M_r 75 000and60 000. The specific activity of propionyl-CoA car?ylase (15.8 munits/mg) and β-methylcrotonyl-CoA car?ylase (24.2 munits/mg) were comparable with reported activities for these purified enzymes, while that of pyruvate car?ylase (1.25 munits/mg) was low. This is a suitable method for the simultaneous preparation of purified car?ylases for the specific purpose of raising antisera to these enzymes.