A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Investigation of the heterogeneity of heterogalactan from the fruit bodies of Fomitopsis pinicola, by employing concanavalin A-Sepharose affinity chromatography

A heterogalactan was isolated from the hot water extract of fruit bodies of Fomitopsis pinicola by a combination of fractionation procedures including precipitation with ethanol and with Cetavlon, and chromatography on columns of DEAE-cellulose and Sephadex G-100. Despite its apparent homogeneity on gel filtration, zone electrophoresis, sedimentation equilibration, and immunodiffusion analyses, the neutral component of heterogalactan was further fractionated into unbound, weakly bound, and strongly bound forms by affinity chromatography on a column of concanavalin A-Sepharose CL 4B. The former two polysaccharides fractions eluted with 0.1 M phosphate buffer (pH 7.0) were found to be a fucogalactan and a mannofucogalactan, respectively. A more tightly bound fraction (mannofucogalactan) was subsequently eluted with 0.1 M glucose in 1 M NaCl. The results of methylation, complete Smith degradation, and proton and 13C NMR spectroscopic analyses indicated that the three kinds of heterogalactans are all highly branched polysaccharides containing a framework of (1 leads to 6)-linked alpha-D-galactopyranosyl residues, the C-2 positions of which are substituted in different proportions with either single L-fucopyranosyl residues or disaccharide units of 3-O-alpha-D-mannopyranosyl-L-fucopyranose residues.     

Molecular size and shape of beta-connectin, an elastic protein of striated muscle

Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, alpha and beta (also called titins 1 and 2). In the present study, beta-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1 X 10(6) (SDS gel electrophoresis) or 2.7 X 10(6) (sedimentation equilibrium). The sedimentation coefficient (SO20, w) was 17S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s-1, a solution of connectin (0.3 mg/ml) showed a viscosity value of 17,000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that beta-connectin is derived from alpha-connectin, which has an apparent molecular weight of 2.8 X 10(6).     

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.     

A calorimetric study of the interaction of ATP with rabbit muscle phosphofructokinase.

The heat of interaction of ATP with phosphofructokinase from rabbit muscle was determined at 25 degrees C in 0.1 M potassium phosphate, pH 7.0 and 8.0. The limiting value of the enthalpy change at high ATP concentrations was found to be -11.5 kcal mol of enzyme polypeptide chains. Since phosphate and imidazole have very different heats of ionization (+0.8 and +7.5 kcal/mol, respectively), this suggests that the binding of at least two protons to the enzyme occurs concomitantly with the binding of ATP at the regulatory site.     

Developing procedures for the large-scale purification of human serum butyrylcholinesterase

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2× LD₅₀ of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05–0.2 M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.     

An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate

Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we had isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp beta-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we have developed a novel affinity column procedure using an mAb prepared against alpha-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl and 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then prewashed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. As a result, the isolated Hp was devoid of apoA-I and was able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1, or 2-2 was greater than 95% with an yield greater than 50%. The procedure described here is significantly improved in time consumption, recovery, and purity. The rationale, design, and optimization for each step are described in detail.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

Preparation and properties of soluble, immunoreactive apoLDL.

Immunoreactive apo-low density lipoprotein (LDL), soluble in mildly alkaline buffers of low ionic strength, was prepared by attaching LDL to a DEAE-Sepharose column and eluting the lipids with a 0--2% (w/v) gradient of nonionic detergents. Brij-36T, Nonidet P-40, and Triton X-100 gave similar results. After washing the detergent from the column, the bound apoLDL was eluted with 1 M NaCl, pH 7.4, with recoveries up to 85%. This apoLDL could be dialyzed extensively against low ionic strength solutions, and remained soluble as long as the pH was above 7. Spectrophotometric analysis showed that less than 0.1% %w/v) of cholesterol or phospholipids and less than 1% (w/v) of detergent remained associated with the protein. The apoLDL cross-reacted with LDL against antisera prepared vs. intact LDL. Pore-gradient polyacrylamide gel electrophoresis, with SDS and urea, showed that this preparation was less aggregated than organic solvent extracted apolLDL and appeared to be made of oligomers of two monomeric subunits, one with molecular weight around 22,700 and a smaller one of approximately 8000. Isoelectric focusing showed that there also was charge heterogeneity in the soluble apoLDL.     

Acid unfolding and self-association of recombinant Escherichia coli derived human interferon gamma

The secondary and tertiary structure of recombinant human interferon gamma, determined by far- and near-UV circular dichroism, showed a transition from the native state to an unfolded state below pH 4.5. The acid unfolding was extensively studied at pH 3.5 as a function of NaCl concentration. Addition of 0.05-0.2 M NaCl to a pH 3.5 sample increased the amount of beta-sheet structure with no change in the amount of alpha-helix and also induced reversible self-association of interferon gamma to form large aggregates from the monomer. When samples at pH 3.5 were dialyzed against 0.1 M ammonium acetate (pH 6.9) to refold interferon gamma, the samples that contained NaCl in acid formed aggregates upon dialysis while those without NaCl formed a dimer apparently identical with the starting protein (i.e., before acid treatment). Thus, the self-association of interferon gamma in acid is closely correlated with its aggregation behavior in 0.1 M ammonium acetate after removal of acid.     

Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction

Reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Forward extraction of lipase was found to be maximum using Tris buffer at pH 9.0 containing 0.10 M NaCl in aqueous phase and 0.20 M CTAB in organic phase consisting of isooctane, butanol and hexanol. In case of backward extraction, lipase was extracted from the organic phase to a fresh aqueous phase in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.0 M KCl. The activity recovery, extraction efficiency and purification factor of lipase were found to be 82.72%, 40.27% and 4.09-fold, respectively. The studies also indicated that the organic phase recovered after back extraction could be reused for the extraction of lipase from crude extract.     

Synthesis of [(14)C]pyrroloquinoline quinone (PQQ) in E. coli using genes for PQQ synthesis from K. pneumoniae

Radiochemical forms of pyrroloquinoline quinone (PQQ) are of utility in studies to determine the metabolic role and fate of PQQ in biological systems. Accordingly, we have synthesized [(14)C]PQQ using a tyrosine auxotrophic strain of Escherichia coli (AT2471). A construct containing the six genes required for PQQ synthesis from Klebsiella pneumoniae was used to transform the auxotrophic strain of E. coli. E. coli were then grown in minimal M9 medium containing 3.7x10(9) Bq/mmol [(14)C]tyrosine. At confluence, the medium was collected and applied to a DEAE A-25 anionic exchange column; [(14)C]PQQ was eluted using a KCl gradient (0-2 M in 0.1 M potassium phosphate buffer, pH 7.0). Radioactivity co-eluting as PQQ was next pooled, acidified and passed through a C-18 column; [(14)C]PQQ was eluted with a phosphate buffer (0.1 M, pH 7.0). Reverse phase HPLC (C-18) using either the ion-pairing agent trifluoroacetic acid (0. 1%) and an acetonitrile gradient or phosphoric acid and a methanol gradient were used to isolate [(14)C]PQQ. Fractions were collected and analyzed by liquid scintillation counting. (14)C-labelled compounds isolated from the medium eluted corresponding to the elution of various tyrosine-derived products or PQQ. The radioactive compound corresponding to PQQ was also reacted with acetone to form 5-acetonyl-PQQ, which co-eluted with a 5-acetonyl-PQQ standard, as a validation of [(14)C]PQQ synthesis. The specific activity of synthesized [(14)C]PQQ was 3.7x10(9) Bq/mmol [(14)C]PQQ, equal to that of [U-(14)C]tyrosine initially added to the medium.     

Chicken leg muscle alpha-connectin as studied by a monoclonal antibody to the 1200 kDa fragment.

Chicken leg gracilis muscle contained only alpha-connectin (ca 3000 kDa) without beta-connectin. When myofibrils were kept standing for 20 hr at 4 degrees C, alpha-connectin was degraded to beta-connectin (ca 2000 kDa) and 1200 kDa peptide. The latter was prepared from myofibrils and purified by gel filtration in the presence of SDS. A monoclonal antibody, alpha 7, to this 1200 kDa fragment was prepared. The antibody reacted with the 1200 kDa fragment and its mother molecule alpha-connectin, but not with beta-connectin. Immunoelectron microscopy using alpha 7, as well as other antibodies to chicken breast muscle beta-connectin, revealed that the 1200 kDa peptide covered the portion of alpha-connectin from the Z line to the N2 line region in the I band of chicken leg gracilis muscle sarcomeres. The results were in good agreement with those observed in rabbit skeletal muscle.     

A rapid and gentle method for the salt extraction of chromatin core histones H2A,H2B,H3 and H4 from rat liver nuclei

A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak.     

High-speed gel filtration of polypeptides in sodium dodecyl sulfate

The separation of polypeptides treated with SDS was studied using G3000SW packing prepared from silica for high-speed gel filtration. The peaks of ovalbumin, chymotrypsinogen A, cytochrome c, aprotinin, and insulin B chain were completely separated in the presence of 0.1% SDS and 0.05 M sodium phosphate buffer (pH 7.0). A plot of the logarithm of molecular weight of polypeptides versus Kd was linear over a molecular weight range of 3,000 to 50,000 at the above concentrations of SDS and sodium phosphate. The slopes of the plots of log molecular weight versus Kd depend to a significant extent on the concentration of the sodium phosphate buffer (pH 7.0).     

Comparison of 10 nm filaments from three bovine tissues

Enriched fractions of 10 nm filaments were isolated from three bovine tissues and were compared using morphological biochemical, and immunological techniques. We studied keratin filaments from hoof epidermis, 10 nm filaments from corneal epithelium, and 10 nm filaments from brain white matter. The parameters of comparison and results were as follows.

1. 1. Corneal epithelial filaments and keratin filaments repolymerized after a buffered 8 M urea extract of the tissue was dialyzed against a low ionic strength (0.005 M) buffer. However, a greater yield of repolymerized corneal epithelial filaments was obtained if the urea-soluble fraction was dialyzed against the same buffer containing 0.17 M NaCl. Brain filaments harvested by cell fractionation did not repolymerize when similarly treated.

2. 2. Electrophoretic patterns of proteins of filament-enriched fractions from the three sources were different in sodium dodecyl sulphate (SDS) polyacrylamide gels, except for one co-migrating band.

3. 3. Peptide mapping by limited proteolysis of the eluted co-migrating proteins showed few similarities.

4. 4. Amino acid analysis of the co-migrating proteins revealed numerous differences.

5. 5. Antibodies to the co-migrating corneal epithelial filament and brain filament proteins reacted only with their own antigen and whole filament type, and antibody to total keratin filament protein cross-reacted only with keratin filaments.

     

Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.     

Biosynthesis of Streptomycin

A method for the accumulation of the streptomycin precursor (L) in the culture broth of Streptomyces griseus was developed and the precursor was successfully isolated from the broth.

When the microorganism was cultured under shaking in the glucose-meat extract-peptone medium (0.5% glucose, 0.2% yeast extract, 0.2% meat extract, 0.4% peptone, 0.5% sodium chloride, 0.025% magnesium sulfate, pH 7.0), the accumulation of the precursor in the broth was induced by the addition of supplementary glucose (e.g., 2 g glucose per 100 ml broth) 24 hr after inoculation followed by further cultivation for 48 hr. Increased accumulation of L component was obtained merely by increasing glucose content in the culture medium (e.g., 5% glucose-containing medium in the above-indicated one) instead of glucose supplement on the way of fermentation. For the accumulation of a large amount of L component in a culture broth, it looked to be necessary for pH value of the broth to be maintained between 6 and 7 during fermentation.

L component was isolated from the culture broth by adsorption on Amberlite IRC-50 and elution with 2% NaCl solution. The L component was separated on this column from contaminated streptomycin which requires 5% NaCl solution to be eluted. The L component in the 2% NaCl eluate was adsorbed on active carbon at neutral or slightly alkaline pH and eluted with 95% methanol at acidic pH, Partially purified L component precipitated as hydrochloride by addition of acetone to the methanol extract which had been concentrated in vacuo.     

Partial characterization of thymocyte-activating factor derived from MDP-stimulated guinea pig fibroblasts

The activity of fibroblast-derived thymocyte activating factor (FTAF) of the guinea pig was measured, and the factor was partially characterized. The FTAF activity was heat labile, and destroyed by treatment with trypsin, chymotrypsin, and Streptomyces griseus protease, suggesting the protein nature of FTAF. FTAF bound to DEAE-Sepharose CL-6B in Tris-HCl buffer at pH 8.0, and was eluted with 0.1-0.2 M NaCl. FTAF was absorbed with Blue Sepharose CL-6B. The factor bound to a hydroxylapatite column in 10 mM phosphate buffer and was eluted in two major fractions, one fraction with 40 mM phosphate buffer, the other with 70-110 mM phosphate buffer. Finally, FTAF did not have as much effect on the proliferation of lymph node T cells as T-cell-activating monokines which exhibited marked stimulating effects on both T lymphocytes and thymocytes.