Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium.

When subjected to the stress of growth in a relatively low-salt environment (1.25 M NaCl), the halophilic bacterium Halobacterium halobium induces a catalase. The protein has been purified to electrophoretic homogeneity and has an M(r) of 240,000 and a subunit size of approximately 62,000. The enzyme is active over a broad pH range of 6.5 to 10.0, with a peak in activity at pH 7.0. It has an isoelectric point of 4.0. This catalse, which is not readily reduced by dithionite, shows a Soret peak at 406 nm. Cyanide and azide inhibit the enzyme at micromolar concentrations, whereas maleimide is without effect. The addition of 20 mM 3-amino-1,2,4-triazole results in a 33% inhibition in enzymatic activity. The tetrameric protein binds NADP in a 1:1 ratio but does not peroxidize NADPH, NADH, or ascorbate. Although the enzymatic activity is maximal when assayed in a 50 mM potassium phosphate buffer with no NaCl, prolonged incubation in a buffer lacking NaCl results in inactive enzyme. Moreover, purification must be performed in the presence of 2 M NaCl. Equally as effective in retaining enzymatic function are NaCl, LiCl, KCl, CsCl, and NH4Cl, whereas divalent salts such as MgCl2 and CaCl2 result in the immediate loss of activity. The catalase is stained by pararosaniline, which is indicative of a glycosidic linkage. The Km for H2O2 is 60 mM, with inhibition observed at concentrations in excess of 90 mM. Thus, the mesohalic catalase purified from H. halobium seems to be similar to other catalases, except for the salt requirements, but differs markedly from the constitutive halobacterial hydroperoxidase.     

The physical properties of NAD-dependent l-fucose dehydrogenase from sheep liver

Sheep liver l-fucose (d-arabinose) dehydrogenase has been purified to homogeneity as indicated by polyacrylamide disc-gel electrophoresis and sedimentation velocity ultracentrifugation experiments. The enzyme possesses an apparent molecular weight of 123,000 and is composed of four subunits of molecular weight approximately 30,000. The pI of the enzyme is 5.8. The enzyme is stable at high temperatures, retaining 65% of its original activity after 15 min at 60 °C. High ionic strength (μ = 1.0–1.3) in the assay medium stimulates the enzymatic activity and lowers the pH values at which maximal enzymatic activity is observed.     

Purification and renaturation of Dictyostelium recombinant alkaline phosphatase by continuous elution electrophoresis

A 1583 bp fragment of Dictyostelium alp cDNA (94% of the gene) was cloned in pET32a+. The enzyme was expressed in an inactive form in the inclusion body of the expression host BL21-CodonPlus (DE3)-RIL. The recombinant ALP constituted more than 50% of the total protein in the inclusion body and 25-30% of the total protein in the expression host after 3 h induction with IPTG at 37 degrees C. A continuous elution polyacrylamide gel electrophoresis procedure was used to purify the recombinant enzyme. This technique yielded a homogeneous protein that retained enzymatic activity after dialysis without further treatment. A yield of 5mg per liter of culture broth was obtained with a specific activity of approximately 0.7 nmol/min/mg protein (0.7 mU/mg). Immunoinhibition studies using a polyclonal antibody produced against the recombinant protein showed complete inhibition of enzymatic activity when the enzyme was preincubated with the antibody at a 1:1000 dilution. The enzyme exhibited a pH optimum of approximately 9.0. The substrate specificity indicated that the Dictyostelium enzyme is a typical broad range alkaline phosphatase.     

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight? and compared with the activity of Fructozyme?. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme?, which is important for its potential application in the tequila industry.     

Purification and characterization of a phosphatidylinositol 4-kinase from bovine uteri

Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.     

Isolation and partial characterization of trehalase from Ascaris muscle.

The tissues of female Ascaris suum were assayed for alpha,apha'-glucoside 1-D-glucohydrolase (trehalase) activity. A soluble from of the enzyme was isolated from muscle tissue and purified approximately 37-fold. The enzyme was specific for trehalose as substrate. The pH optimum for enzymatic activity was found to be 6.0, and the apparent Km for trehalose was estimated to be 2.1 x 10-4 M. The product of the reaction was identified as D-glucose by chemical, chromatographic and enzymatic methods.     

Type I phosphodiesterase in the isolated, brush-border membrane of Hymenolepis diminuta

The isolated, brush-border membrane of Hymenolepis diminuta contained an enzyme which hydrolyzed phosphodiester bonds. This enzyme appeared to be a Type I phosphodiesterase (E. C. 3.1.4.1) (produces nucleoside 5'-phosphates) and had no activity against synthetic, Type II phosphodiesterase substrates (mononucleotides substituted at the 3' position). The effects of various potential inhibitors of enzymatic activity, and cation requirements of this enzyme, demonstrated a distinct difference between the phosphodiesterase and alkaline phosphatase activities of the isolated, brush-border membrane. SDS-polyacrylamide gel electrophoresis of the isolated membrane preparation, followed by localization of phosphodiesterase activity in the gels, indicated the enzyme had a molecular weight of approximately 87,000. Thus, the phosphodiesterase activity represents a previously undescribed, membrane-bound enzyme of the brush-border of Hymenolepis diminuta.     

NADH oxidase activity of soybean plasma membranes oscillates with a temperature compensated period of 24 min

The enzymatic activity of a hormone-stimulated and growth-related protein disulfide-thiol oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity of etiolated hypocotyls of soybean oscillates with a period of about 24 min or 60 times per 24 h day. The oscillations were temperature compensated such that the period remained constant at about 24 min between 17 and 37°C, a temperature range over which enzymatic activity varied approximately fourfold (Q₁₀ of 2). The oscillations were observed with intact tissue sections, with isolated plasma membrane vesicles, and with the detergent-solubilized and partially purified enzyme. The oscillations were observed both with the oxidation of NADH and in the restoration of activity to scrambled ribonuclease used as a measure of the protein disulfide-thiol interchange activity. The enzymatic activity which is located at the cell surface may represent a biochemical reaction with a potential function as an ultradian oscillator of circadian time keeping.     

Chemical modification of Corynebacterium sarcosine oxidase: role of sulfhydryl and histidyl groups

Sarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 0.2% SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (I), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%. The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds. The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.     

Further enzymatic characteristics of a thylakoid protein kinase

The enzymatic characteristics of a protein kinase purified from thylakoids are further described. ATP (KM approximately 30 microM) and Mg2+ ion (greater than 1.0 mM) were required for activity, while ADP was a competitive inhibitor (Ki = 100 microM). Activity was 55% inhibited by the sulfhydryl inhibitor p-chloromercuribenzoate (1 mM) and was less sensitive to substituted maleimides. Lysine-rich histones (H1) were utilized as an exogenous phosphorylation substrate both by thylakoid-bound kinase and by isolated enzyme; threonine was predominantly phosphorylated by the in situ enzyme, whereas the isolated enzyme phosphorylated closely related serine residues as determined by peptide mapping. Detergents that proved useful in extracting the kinase from thylakoids markedly inhibited activity of the isolated enzyme, whereas Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid had little effect. The enzyme could be freed from detergent and behaved as an active monomer on size-exclusion chromatography. The phosphate contents of the light-harvesting chlorophyll a/b protein complex of photosystem II isolated from maximally phosphorylated thylakoid membranes of spinach and pea were equivalent to approximately 6% and approximately 19% phosphorylation, respectively. Corresponding values for nonphosphorylated membranes were approximately 3% and approximately 14.5%.     

Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.     

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.     

Evidence for a tyrosine residue at the triphosphopyridine nucleotide-binding site of 6-phosphogluconate dehydrogenase

Treatment of the 6-phosphogluconate dehydrogenase from Candida utilis with tetranitromethane results in the partial inactivation of the enzyme. The nitration of approximately one tyrosine residue per enzyme subunit accounts for the loss of 70% of the enzymatic activity. The reduction of the nitrotyrosyl to aminotyrosyl residue does not induce a recovery of activity.     

A truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase with improved enzymatic activity and thermotolerance

As an approach to improving Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase (Fsbeta-glucanase) for use in industry and to studying the structure-function relationship of the C-terminus in the enzyme, a C-terminally truncated ( approximately 10 kDa) Fsbeta-glucanase was generated using a PCR-based gene truncation method and then overexpressed in either Escherichia coli BL21(DE3) or Pichia pastoris strain X-33 host cells. The initial rate kinetics, protein folding, and thermostability of the wild-type and truncated glucanases were characterized. The truncated enzyme expressed in Pichia cells was found to be glycosylated and composed of two dominant polypeptide bands as judged by SDS-PAGE. An approximate 3-4-fold increase in the turnover rate (k(cat)), relative to that of the full-length enzyme, was detected for the purified truncated glucanases produced in E. coli (designated TF-glucanase) or Pichia host cells (designated glycosylated TF-glucanase). The glycosylated TF-glucanase is the most active known 1,3-1,4-beta-d-glucanase, with a specific activity of 10 800 +/- 200 units/mg. Similar binding affinities for lichenan (K(m) = 2.5-2.89 mg/mL) were detected for the full-length enzyme, TF-glucanase, and glycosylated TF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymatic activity after a 10 min incubation at 90 degrees C, whereas the full-length enzyme possessed only 30% of its original enzymatic activity after the same treatment. This report demonstrates that deletion of the C-terminal region ( approximately 10 kDa) in Fsbeta-glucanase, consisting of serine-rich repeats and a basic terminal domain rich in positively charged amino acids, significantly increases the catalytic efficiency and thermotolerance of the enzyme.     

An enzyme from the fungus Aspergillus niger that hydrolyzes choline ethers]

The acetylthiocholine-hydrolyzing enzymatic activity inhibited by the neostigmine and partly physostigmine has been found in extracts from mycelium of fungus Aspergillus niger. The enzyme has been isolated and 15-20 fold purified. The cholinesterase activity of the protein (Kmu 7.10-7 M) is comparable with known for analogous enzymes from higher plants, for its inhibition high concentrations of substrate (greater than 10-3M) are required. The enzyme hydrolyzes acetylthiocholine with rate approximately 1.5 times higher than butyrylthiocholine. Molecular mass of native protein is approximately 600 kDa, subunits -63 and 44 kDa.     

Polyenzymatic activation of cellular hydrolases induced by interferon; its role in cell lysis and other interferon--related phenomena

Treatment of human cultured T cells by interferon (IFN), which enhances cell-mediated cytotoxicity directed against human K562 cell targets (NK-like activity) induces an activation of cell hydrolases. Treatment of mouse cultured L929 cells by IFN and double strand RNA enhancing cell autolysis induces first an activation of tested hydrolases (hexosaminidase, beta-glucuronidase, acid and alkaline phosphatases). Polyenzymatic activation of hydrolases induced by IFN is similar to coordinate enzymatic induction described by Hosli which occurs by lack of specific enzyme when non-digestible substrates are absorbed by cells. It is hypothesized that the polyenzymatic activation which accounts for cell lysis is a general defense mechanism which might also explain other actions related to IFN such as antiviral effect, inhibition of cell division, diminution of protein synthesis and cell surface alteration.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.     

Reversible dissociation of gamma-glutamylcysteine synthetase into two subunits

gamma-Glutamylcysteine synthetase (rat kidney; Mr approximately 104,000) is composed of 2 nonidentical subunits. In the present work, a procedure was developed for the reversible dissociation of the enzyme into its subunits (Mr = 73,000 and 27,700) under nondenaturing conditions. Students in which gel electrophoresis was used, in conjunction with an enzyme activity stain and elution and re-electrophoresis of protein bands, showed that the heavy subunit contains all of the structural requirements for enzymatic activity and also for feedback inhibition of the enzyme activity by glutathione. The light subunit, which may be formed from a precursor protein, has a significantly lower content of Trp, Phe, Tyr, Val, and Ala residues than the heavy subunit, while its content of Lys, His, Met, and Asx residues is higher.