Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme.

A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment. Therefore, the purification was carried out in 2 M NaCl. Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0. Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function. Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C. Peroxidase activity decreased rapidly above its maximum at 40 degrees C. An activation energy of 2.5 kcal (ca. 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca. 17 kJ)/mol was calculated for peroxidase. Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki [KiApp] of 50 and 67.5 microM, respectively). Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM). The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite. A heme content of 1.43 per tetramer was determined. The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.     

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.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

An endogenous regulator of the guanylate cyclase activity of human platelets

Chromatography of soluble human platelet guanylate cyclase (105,000 g supernatant) on DEAE-cellulose in a linear gradient of NaCl (0-0.5 M) in 50 mM Tris-HCl buffer pH 7.6 gave two protein peaks, I and II, of which only peak II possessed the guanylate cyclase activity (0.18-0.22 M NaCl). The protein fraction I was found to possess an inhibiting activity; its addition to the partially purified enzyme decreased the guanylate cyclase activity by 60-70% in the presence of Mg2+ with no effect on the enzyme activity in the presence of Mn2+. The isolated enzyme lost (by approximately 80%) its ability to be activated by sodium nitroprusside; the latter was reconstituted after addition of the inhibiting fraction. The data obtained testify to the heme origin of the endogenous inhibitor of human platelet guanylate cyclase.     

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.     

Preparation of iduronate sulfatase from the human placenta]

The preparation of the enzyme iduronate sulfatase from human placenta has been undertaken. The substrate O-(alpha-L-idopyranosyluronic acid 2-sulfate) (1 leads to 4)-2,5-anhydro-D-[3H]mannitol 6-sulfate was used to measure the enzymatic activity. The enzyme shows a pH optimum of 4.0 in 0.1 M sodium formiate or acetate buffer. Chromatography on DE-52 gives a 5.4 fold purification. The enzyme is inhibited by NaCl or KCl: in 20 mM salt the reaction rate was only 63% and 34% respectively. Inhibition by salt can be removed by extensive dialysis after the chromatographic step.     

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.     

Purification and characterization of a pyruvated-mannose-specific xanthan lyase from heat-stable, salt-tolerant bacteria

A xanthanase complex secreted by a consortium of heat-stable, salt-tolerant bacteria includes a lyase that specifically removes terminal pyruvated beta-d-mannose residues from the side chains of xanthan gum. The enzyme was purified to homogeneity from the culture broth following ion-exchange chromatography and gel permeation chromatography. It consists of a single subunit of molecular weight 33,000. The enzyme is stable to 55 degrees C for more than 6 h in 20 mM sodium phosphate buffer (pH 5.0) containing 0.25 M NaCl. Optimal enzyme activity was observed at 0.05 M NaCl and a pH of 5. The enzyme has a pI of 3.7. It does not remove unsubstituted terminal beta-d-mannose residues from xanthan side chains nor does it hydrolyze p-nitrophenyl-beta-d-mannose. Treatment of xanthan with purified lyase results in a polysaccharide containing side chains terminating in an unsaturated 4,5-ene-glucuronic acid.     

Purification and characterization of a catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T) exhibiting high catalase activity

Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T), which was isolated from an environment exposed to H(2)O(2) and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U. mg of protein(-1) under standard reaction conditions at 40 degrees C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1(T) is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1(T) catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1(T) should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1(T) is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.     

Production of Aspergillus niger catalase under various stress conditions

The conidia of Aspergillus niger were cultured under various stress conditions. The highest yield of catalase activity was obtained after addition 1.2 M NaCl to 6-h-old culture, which improved the activity of the enzyme by about 2.8-fold in comparison with the control medium without this depressor. A significantly lower increase (1.3- to 1.7-fold) in catalase activity was reached when spores were incubated under oxidative stress (100-200 mM H2O2) or at low pH (2 and 3). Respiratory activities in A. niger culture show important changes with the osmotic stress increase.     

Purification and characterization of catalase from chard (Beta vulgaris var. cicla)

Catalase is a major primary antioxidant defence component that primarily catalyses the decomposition of H(2) O(2) to H(2) O. Here we report the purification and characterization of catalase from chard (Beta vulgaris var. cicla). Following a procedure that involved chloroform treatment, ammonium sulfate precipitation and three chromatographic steps (CM-cellulose, Sephadex G-25, and Sephadex G-200), catalase was purified about 250-fold to a final specific activity of 56947 U/mg of protein. The molecular weight of the purified catalase and its subunit were determined to be 235 000 and 58 500 daltons, indicating that the chard catalase is a tetramer. The absorption spectra showed a soret peak at 406 nm, and there was slightly reduction by dithionite. The ratio of absorption at 406 and 275 nanometers was 1.5, the value being similar to that obtained for catalase from other plant sources. In the catalytic reaction, the apparent Km value for chard catalase was 50 mM. The purified protein has a broad pH optimum for catalase activity between 6.0 and 8.0. The enzyme had an optimum reaction temperature at 30 degrees C. Heme catalase inhibitors, such as azide and cyanide, inhibited the enzyme activity markedly and the enzyme was also inactivated by ?-mercaptoethanol, dithiothreitol and iodoacetamide.     

Purification and characterization of catalase from a facultative alkalophilic Bacillus

Catalase was purified to an electrophoretically homogeneous state from the facultative alkalophilic bacterium, Bacillus YN-2000, and some of its properties were studied. Its molecular weight was 282,000 and its molecule was composed of four identical subunits. The enzyme contained two protoheme molecules per tetramer. The enzyme showed an absorption spectrum of typical high-spin ferric heme with a peak at 406 nm in the oxidized form and peaks at 440, 559, and 592 nm in the reduced form. In contrast to the typical catalases, the enzyme was reduced with sodium dithionite, like peroxidases. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The amino acid composition of Bacillus YN-2000 catalase was very similar to those of catalase from Neurospora crassa and peroxidase from Halobacterium halobium. The catalase content in the soluble fraction from the bacterium was higher with the cells grown at pH 10 than with the cells grown at lower pHs (pH 7-9).     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

Hydroxyapatite chromatography of phage-display virions

Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.     

Halophilic Nuclease from a Moderately Halophilic Micrococcus varians

The moderately halophilic bacterium Micrococcus varians, isolated from soy sauce mash, produced extracellular nuclease when cultivated aerobically in media containing 1 to 4 M NaCl or KCl. The enzyme, purified to an electrophoretically homogeneous state, had both ribonuclease and deoxyribonuclease activities. The nuclease had maximal activity in the presence of 2.9 M NaCl or 2.1 M KCl at 40 C. The enzymatic activity was lost by dialysis against low-salt buffer, whereas when the inactivated enzyme was dialyzed against 3.4 M NaCl buffer as much as 77% of the initial activity could be restored.     

Study of betacyanin-discolorating enzyme]

An enzyme catalyzing the discoloration and breakdown of betacyanins was isolated from beet roots Beta vulgaris by centrifugation in sucrose density gradient (2.5 M, 2.0 M, 1.5 M, 1.0 M, tris-HCl buffer, 0.05 M, pH 7.2), and purified 100-fold. The enzyme activity induced the discoloration of betanin, betanidin. It was found that the beet root enzyme exists in an insoluble state and is firmly bound with subcellular structures, which were isolated by centrifugation in sucrose gradient. The optimal activity of the enzyme was observed at pH 3.4, +40 degrees C. The dependence of the enzymatic reaction on the enzyme concentration showed a linearity. Studies of the enzyme inhibition by sodium azide, sodium diethyldithiocarbamate, thiourea, demonstrated that the active site of the enzyme contains a metal. The enzymatic discoloration of betanin is followed by the oxygen uptake.     

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.     

Purification and characterization of 3-methyladenine-DNA glycosylase from calf thymus

The 3-methyladenine-DNA glycosylase from calf thymus has been purified and characterized. Two species of Mr = 42,000 and 27,000 +/- 5% and Stokes radius of 27.5 and 22.4 A, respectively, were found. Only the lower molecular weight species were present in the nucleus; it was bound to chromatin and could be dissociated in the presence of 0.25 M KCl. The enzymatic properties of the two species appeared to be identical. Both enzyme species released 3-methyladenine, 7-methylguanine, and 3-methylguanine, listed in the order of decreasing activity. The chromatin-associated enzyme was purified to apparent homogeneity and found to be a basic protein having a pI greater than 9. It was completely inhibited by p-hydroxymercuribenzoate, but this inhibition could be fully reversed by addition of excess 2-mercaptoethanol. Kinetic studies, heat inactivation, and inhibition experiments demonstrated that the 3-methyladenine and 7-methylguanine releasing activities were located on the same protein molecule. The enzymes showed no activity on methylated single-stranded DNA. No product inhibition was observed for any of the enzyme species, and the enzyme activity was optimal when the incubation was performed in the presence of 50 mM NaCl or KCl at pH values between 8 and 9.