Cloning and characterization of chironomidae ferrochelatase: Copper activation of the purified ferrochelatase

All organisms utilize ferrochelatase (EC 4.99.1.1) to catalyze the insertion of ferrous iron into protoposphyrin IX in the terminal step of the heme biosynthetic pathway. Different metal-binding affinity for the enzyme leads to changes in enzyme activity. In this work, we have cloned and over-expressed the enzyme from chironomidae in E. coli. The enzyme was purified and characterized. The recombinant enzyme showed higher enzymatic activity (four-fold increase) in the presence of copper ions and unaffected by calcium ions. Other divalent metal ions including magnesium, manganese, lead, reduced the enzyme activity by >60%. Over 90% of the enzyme activity was inhibited by Zn2+. The sequence alignment of amino acid residues reveals 83% homology with other ferrochelatases. The results of electron proton resonance (EPR) analysis showed that Fe2+ ion was present in the cluster of the recombinant enzyme complex. The recombinant enzyme also contained the [2Fe-2S] center with two-fold higher enzymatic activity than human ferrochelatase.     

Pre and post cloning characterization of a β-1,4-endoglucanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over a pH range of 6.0–8.0. The K _m and V _max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min⁻¹, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.     

Phosphoprotein phosphatase activity of the progesterone-induced purple glycoprotein of the porcine uterus.

The iron-containing, progesterone-induced, purple glycoprotein from the pig uterus catalyzes the hydrolysis of phosphate groups from phosphvitin. This enzyme which can be purified in large amounts, has a pH optimum at 4.6 and an apparent K_m of 0.1 mM for bound phosphate. Its V_max with phosphvitin as substrate is about 200, μmoles Pi released/min/μmole enzyme and is at least twenty-fold lower than its activity towards p-nitrophenylphosphate. The enzyme is activated by 2-mercaptoethanol and inhibited by molybdate, fluoride and arsenate. The resemblance of this protein to a number of other acid phosphatases of similar substrate specificity in other organisms, suggests that a broad class of such enzymes might exist in nature.     

The role of arginyl residues in porphyrin binding to ferrochelatase

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.     

Purification and characterization of an isoflavones conjugate hydrolyzing β-glucosidase (ICHG) from Cyamopsis tetragonoloba (guar)

A β-glucosidase with high specific activity towards isoflavone glycosidic conjugates was purified from seeds of Guar (Cyamopsis tetragonoloba) by ammonium sulphate precipitation followed by size exclusion and ion exchange chromatography. The pH and temperature optima of the purified Isoflavones conjugate hydrolyzing β-glucosidase (ICHG) were found to be pH 4.5 and 37 °C, respectively. The enzyme was relatively stable at higher temperatures. Effect of different divalent metal ions was studied and it was found that Cobalt and Mercury ions completely inhibited the enzyme activity. K_m and V_max of the purified isoflavones conjugates hydrolyzing β-glucosidases (ICHG) was 0.86 mM and 6.6 IU/mg respectively. The enzyme was most likely a trimer (approximate Mr 150 kDa) with potential subunits of 50 kDa. The purified enzyme showed activity against isoflavone conjugate glycosides viz daidzin and genistin but was inactive towards other flavonoid conjugates. The product conversion was confirmed by HPTLC and HRMS analysis. The MALDI-TOF analysis of the ICHG showed a score greater than 78 with 20 matches in MASCOT software. The five resultant peptides obtained had highest similarity in sequence with β-glucosidase from Cicer arietinum. The β-glucosidase from the C. arietinum has also been reported to exhibit the isoflavone conjugate hydrolyzing properties thus confirming the nature of the enzyme purified from the Guar seeds.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Inhibition of chick embryo DNA ligase by dATP: its use in enzyme purification.

Highly purified chick embryo DNA ligase (EC.6.5.1.1) obtained in our laboratory using classical methods, mainly column chromatographies shows a bimodal pH activity and an high affinity inhibition by dATP. A single step passage of crude extract containing DNA ligase through an anion exchange resin (Dowex AG₁X₂) saturated with dATP allows an important purification of the enzyme retained on the column at pH 7.5 and eluted at pH 8.6. Specific activity of the purified enzyme preparation is more than 600 fold higher than that of the crude extract. Analysis of the eluant by polyacrylamide gel electrophoresis shows a main protein containing the enzyme activity.     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

Specificity of a Nuclease from Penicillium citrinum

The enzyme with high milk clotting activity produced by Irpex lacteus was partially purified by a CM-cellulose chromatography. Throughout the over-all process, the enzyme was purified approximately 9-fold from a crude powder with about 22.8% recovery of the original activity. The MCA/PU ratio of this fraction was 2.51 and the specific milk clotting activity was 188.7.

The purified enzyme is a sort of acid protease with optimum pH of 2.5 for casein digestion and 4.0 for hemoglobin digestion. The Lineweaver-Burk plot, when casein was used as a substrate, showed that the Km value of the enzyme was about 0.07% and the V_max value was 0.4. The molecular weight of the enzyme is about 34,000, the isoelectric point is pH 5.2 and a ultraviolet absorption maximum is at 277 mμ. The enzyme has not yet been crystalized but seems to be a sort of glycoprotein, because the Molish reaction was positive at the present purification stage.

Some enzymological properties of the enzyme was studied and compared with those of a calf rennet and Mucor rennet. In some respects such as pH optima, pH stability, thermostability and temperature optima, the enzyme is Mucor rennet alike. On the other hand, as to the increase in activity along with decrease in pH of milk and the increase in activity along with the addition of Ca ion, the enzyme is not very different from the calf rennet. However, proteolysis of milk casein by the enzyme was fairly higher than by the calf rennet.

As to the production of enzymes, I. lacteus can produce at least three types of proteases into liquid media. When, for example, R medium was used, only one type of protease, that is the fraction A, could mainly be produced and it was this enzyme that assumed to be a rennet like enzyme.     

Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.     

Purification and properties of polyvinyl alcohol oxidase with broad substrate range obtained from Pseudomonas vesicularis var. povalolyticus PH

Extracellular PVA oxidase produced by Pseudomonas vesicularis var. povalolyticus PH was purified to homogeneity by ammonium sulphate fractionation followed by successive column chromatography, and a study made of its characteristics. The molecular weight of the purified enzyme was estimated to be 75,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consists of monomeric protein. Its isoelectric point was 5.7. The purified enzyme was colourless, and contained one atom of iron per molecule. It exhibited a broad pH activity profile with maximum activity at pH 10.0, and was stable between pH 6.0 and 10.0. The optimum temperature for enzyme activity was 40°C, with stability up to 45°C. The enzyme activity was inhibited strongly by Fe2+, Hg2+ and Sn2+, and weakly by Cu2+, EDTA, thiourea and IAA. The enzyme exhibited activity toward several secondary alcohols, suggesting that it was a secondary alcohol oxidase. In particular, the enzyme exhibited strong activity towards the larger secondary alcohols such as 2-octanol and 4-decanol, and relatively strong activity towards cyclohexanol and benzyl alcohol.     

Enhanced activity and stability of cellobiase (β-glucosidase: EC 3.2.1.21) produced in the presence of 2-deoxy-d-glucose from the fungus Termitomyces clypeatus

Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-d-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett.2006, 28, 1773-1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. K_m and V_max of the purified enzyme were measured as 0.187 mM and 0.018 U mg⁻¹, respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 °C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 °C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The β-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.     

Activation of Chloroplast Fructose-1, 6-Bisphosphatase of Pisum sativum by Mole Mass Change

Separation of extracts, obtained from isolated intact P. sativum chloroplasts, by fast protein liquid chromatography (FPLC) on superose 6, reveals a 1,400 kDa-FBPase II form at pH 6.0 and a 380 kDa form at pH 7.5. Addition of F1,6P₂, Mg⁺⁺ and ATP cause dissociation of the large form into the smaller one, which leads to an approximate 4-fold increase in activity. Reversibility of the mole mass change could be shown for the influence of pH and of fructose-1, 6-bisphosphate on purified enzyme samples, separated from crude leaf extracts. Compared to thelarge enzyme form, the small form has higher activity and is specific for the substrate fructose-1, 6-bisphosphate, while the large form is not. Activation of FBPase II in the light and inactivation in the dark is discussed on the basis of different oligomeric forms of the enzyme caused by changes in the concentration of intermediates and effectors in the chloroplast stroma. The conclusion is drawn that oligomerization of key enzymes might provide an effective mechanism for enzyme activation/inactivation in vivo.     

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.     

Regulation of haem biosynthesis in normoblastic erythropoiesis: role of 5-aminolaevulinic acid synthase and ferrochelatase

The development of haem biosynthetic enzyme activity during normoblastic human erythropoiesis was examined in seven patients. The first and last enzymes of the haem biosynthetic pathway, ALA synthase and ferrochelatase, were assayed by radiochemical/high performance liquid chromatographic (HPLC) methods. An assay for ferrochelatase activity in human bone marrow was developed. Enzyme substrates were protoporphyrin IX and ⁵⁹Fe²⁺ ions. ⁵⁹Fe-labelled haem was isolated by organic solvent extraction/sorbent extraction followed by reversed-phase HPLC. Optimal activity occurred at pH 7.3 in the presence of ascorbic acid, in darkness and under anaerobic conditions. Haem production was proportional to cell number and was linear with time to 30 min. The assay was sensitive to the picomolar range of haem production. ALA synthase and ferrochelatase activity was assayed in four highly purified age-matched erythroid cell populations. ALA synthase activity was maximal in the most immature erythoid cells and diminished as the cells matured with an overall five fold loss of activity from proerythroblast to late erythroblast development. Ferrochelatase activity was, however, more stable with less than a two fold change in activity observed during the same period of erythroid differentiation. Maximal activity occurred in erythroid fractions enriched with intermediate erythroblasts. These results support sequential rather than simultaneous appearance of these enzymes during normoblastic erythropoiesis. Quantitative analysis of relative enzyme activity however indicates that at all times during erythroid differentiation ferrochelatase activity is present in excess to that theoretically required relative to ALA synthase activity since ALA and haem are not produced in stoichiometric amounts. The lability of ALA synthase versus the stability and gross relative excess of ferrochelatase activity indicates a far greater role for ALA synthase in the regulation of erythroid haem biosynthesis than for ferrochelatase.     

Human renal renin. Complete purification and characterization

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.     

Partial purification and characterization of nitrous oxide reductase fromParacoccus denitrificans

We report the first partial purification of nitrous oxide reductase, a unique and labile enzyme of denitrifying bacteria. The procedure, which required anaerobic conditions throughout, resulted in a 60-fold purification relative to crude lysate in the case ofParococcus denitrificans. The molecular weight was estimated by gel exclusion chromatography to be about 85,000. The partially purified enzyme is inactivated rapidly by O₂, dithionite, and mercaptoethanol and is reversibly inhibited by moderate concentrations of common salts. Up to 80% of the original activity can be reconstituted following O₂ inactivation by incubating the enzyme with reduced benzyl viologen for 2 to 3 h. TheV _max pH profile shows a broad maximum at pH 8. The enzyme is irreversibly retained by common anion exchangers in the range pH 7 to 8 but can be eluted in acceptable yield as one of the last components from an imidazole-based anion exchange material by means of a pH gradient. This behavior implies that nitrous oxide reductase is very acidic. Among the several peptides observed by sodium dodecyl sulfate slab electrophoresis, only two, with apparent molecular weights of 58,000 and 25,000, correlated closely with the activity of fractions eluted from the imidazole column. These two peptides together comprised about 30% of the total protein in the fractions with highest specific activity.     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

The reaction of horse-liver alcohol dehydrogenase with glyoxal.

Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10% of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 argi-ine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed.     

In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH₂)-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes—methylviologen-nitrate reductase and NADH-ferricyanide reductase—also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH₂-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH₂-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t_½ [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH₂ do not donate electrons to the same site of the nitrate reductase protein.