Kinetic properties of catecholoxidase activity of tarantula hemocyanin

Phenoloxidases occur in almost all organisms, being essentially involved in various processes such as the immune response, wound healing, pigmentation and sclerotization in arthropods. Many hemocyanins are also capable of phenoloxidase activity after activation. Notably, in chelicerates, a phenoloxidase has not been identified in the hemolymph, and thus hemocyanin is assumed to be the physiological phenoloxidase in these animals. Although phenoloxidase activity has been shown for hemocyanin from several chelicerate species, a characterization of the enzymatic properties is still lacking. In this article, the enzymatic properties of activated hemocyanin from the tarantula Eurypelma californicum are reported, which was activated by SDS at concentrations above the critical micellar concentration. The activated state of Eurypelma hemocyanin is stable for several hours. Dopamine is a preferred substrate of activated hemocyanin. For dopamine, a K(M) value of 1.45 +/- 0.16 mm and strong substrate inhibition at high substrate concentrations were observed. Typical inhibitors of catecholoxidase, such as l-mimosine, kojic acid, tyramine, phenylthiourea and azide, also inhibit the phenoloxidase activity of activated hemocyanin. This indicates that the activated hemocyanin behaves as a normal phenoloxidase.     

Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms

Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.     

SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.     

Enzymatic properties of pyruvate kinase from the rumen ciliates genus Entodinium

1. Pyruvate kinase from the rumen ciliates genus Entodinium was partially purified and the enzymatic properties were investigated. 2. Three types of pyruvate kinase (type I, II and III) on DEAE-cellulose column were eluted with a linear gradient of KCl. The enzymatic properties differed among the types of enzyme, especially type I and type III displayed different kinetic properties to each other. The enzymatic property of type II enzyme was an intermediate between type I and III. 3. The principal enzyme, type I, required a divalent cation, Mg2+ and was activated with AMP and FDP. ATP was a potent inhibitor. The saturation curves for the substrates, PEP and ADP, were hyperbolic and Km values were 0.15 and 0.27 mM, respectively.     

Structural and immunochemical properties of rat liver tyrosine aminotransferase

Our results show for the first time sequence data of the N-terminal part of tyrosine aminotransferase. This unblocked form of TATase, which has never been detected before, starts with a serine. This serine was found at position 29 of the primary structure of the enzyme deduced from the cDNA. We suggest that this free N-terminal amino acid is the extremity of a TATase form generated by proteolysis during the process of purification. Thus, proteolysis does not occur at the C-terminal as has been suggested before, but rather at the N-terminal region of the enzyme. To confirm this possibility, a peptide corresponding to the sequence of the seven carboxy-terminal amino acids of TATase was synthesized. It was coupled to ovalbumin or keyhole limpet hemocyanin, and the resulting conjugates were used to raise anti-peptide antibodies. The crude sera obtained were purified and their abilities to recognize TATase in ELISA and dot-blot experiments were proven. Our results demonstrate that the C-terminal part of the enzyme is present and well-recognized by the anti-peptide serum prepared. Furthermore, the anti-peptide serum reacts with TATase without inhibiting its enzymatic activity.     

Quinoprotein D-glucose dehydrogenase of the Acinetobacter calcoaceticus respiratory chain: membrane-bound and soluble forms are different molecular species

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases, while other oxidative bacteria contain the membrane-bound enzyme exclusively. The two forms of glucose dehydrogenase were believed to be the same enzyme or interconvertible forms. Previously, Matsushita et al. [(1988) FEMS Microbiol. Lett 55, 53-58] showed that the two enzymes are different with respect to enzymatic and immunological properties, as well as molecular weight. In the present study, we purified both enzymes and compared their kinetics, reactivity with ubiquinone homologues, and immunological properties in detail. The purified membrane-bound enzyme had a molecular weight of 83,000, while the soluble form was 55,000. The purified enzymes exhibited totally different enzymatic properties, particularly with respect to reactivity toward ubiquinone homologues. The soluble enzyme reacted with short-chain homologues only, whereas the membrane-bound enzyme reacted with long-chain homologues including ubiquinone 9, the native ubiquinone of the A. calcoaceticus. Furthermore, the two enzymes were distinguished immunochemically; the membrane-bound enzyme did not cross-react with antibody raised against the soluble enzyme, nor did the soluble enzyme cross-react with antibody against the membrane-bound enzyme. Thus, each glucose dehydrogenase is a molecularly distinct entity, and the membrane-bound enzyme only is coupled to the respiratory chain via ubiquinone.     

Latent phenoloxidase activity and N-terminal amino acid sequence of hemocyanin from Bathynomus giganteus,a primitive crustacean

N-terminal amino acid sequences for the two hemocyanin subunits from the deep-sea crustacean Bathynomus giganteus have been determined by Edman degradation, providing the first sequence information for a hemocyanin from an isopod. In addition, purified hemocyanin from B. giganteus exhibited phenoloxidase activity in the presence of sodium dodecyl sulfate. Although a natural activator has not yet been identified, a preliminary study of the enzyme indicated a K(m) of 5mM for dopamine and an initial rate of 0.1 micromol per min per mg protein, values consistent with a significant role for this enzyme in the innate immune system of B. giganteus. Moreover, after separation of hemolymph by alkaline polyacrylamide gel electrophoresis, the only detectable phenoloxidase activity coincided with the two hemocyanin subunits. The hemocyanin of this primitive crustacean may fulfill dual functions, both as oxygen carrier and as the phenoloxidase crucial for host defense.     

Purification and some properties of thermostable invertase from wine

Invertase from a white table wine made from Semillon grapes was purified to homogeneity on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the enzyme were compared with those of invertase purified from Semillon grape juice. The invertases from the two sources showed similar properties, suggesting that the wine invertase originated from the juice and was stable during the vinification and aging processes.     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Tyrosinase activity and hemocyanin in the hemolymph of the slipper lobster <Emphasis Type="Italic">Scyllarides latus</Emphasis>

The respiratory protein hemocyanin is present in molluscans and in some species of arthropods, and its dioxygen binding site strongly resembles that of the monophenol-hydroxylating and catechol-quinonising enzyme tyrosinase. Moreover, some hemocyanins show a certain extent of tyrosinase activity, so a common ancestry between the two proteins has been suggested. However, in the case purified hemocyanin of Scyllarides latus any attempts to evoke tyrosinase activity failed. A distinct tyrosinase has been purified to homogeneity from the hemolymph, and kinetically characterised. The purified tyrosinase showed both monophenolase and diphenolase enzyme activity and therefore it could be well defined as a true tyrosinase. This finding suggests that in the case of the studied crustacean the evolutionary functional divergence between dioxygen transport and oxidation of phenolics has already reached its completeness.     

Purification and Properties of Gentisate 1,2-Dioxygenase from Rhodococcus erythropolis S-1

Gentisate 1,2-dioxygenase, which participates in salicylate and m-hydroxybenzoate metabolism, was purified from cell-free extracts of Rhodococcus erythropolis S-1, a Gram-positive bacterium. The purified enzyme gave a single band on native PAGE and SDS–PAGE. The molecular mass of the enzyme was estimated to be 328 kDa. The structure of the enzyme appears to be an octamer of identical subunits. The enzyme from this bacterium was similar in general enzymatic properties to a gentisate 1,2-dioxygenase from a Gram-negative bacterium except for molecular mass and structure.     

Isolation and characterization of the hatching enzyme from the amphibian,Xenopus laevis

The proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its ¹²⁵I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn²⁺ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn²⁺ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.     

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.     

Cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp. Strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl diphosphate biosynthesis

In addition to the ubiquitous mevalonate pathway, Streptomyces sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene, dxs, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among dxs homologs from six genera. The dxs gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in Escherichia coli was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a V(max) of 370 U per mg of protein and K(m)s of 65 microM for pyruvate and 120 microM for D-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a K(m) value of 35 mM for D-glyceraldehyde. To compare the enzymatic properties of CL190 and E. coli DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties.     

Purification and mode of action of a microsomal endoribonuclease from rat liver

An endoribonuclease has been purified nearly to homogeneity from rat liver microsomes, and its mode of action and general properties were studied. The enzyme had an apparent molecular weight of 58 000, as estimated by both gel filtration and SDS-polyacrylamide gel electrophoresis and produced oligonucleotides from poly(A), poly(U) and poly(C). No mononucleotide was obtained by the enzymatic hydrolysis of the above substrates. The enzyme made endonucleolytic cleavages which generated 5'-phosphate-terminated oligonucleotides. It was suggested that the existence of at least (Ado5'P)2 residues at both sides of the cleavage bond was necessary for the action of the endoribonuclease. Divalent cations (Mg2+ or Mn2+) were required for the enzymatic activity, while K+ inhibited the enzyme. Spermine stimulated the enzymatic activity in the presence of 1 mM Mg2+.     

Immobilization of phosphorylase B and study of its enzymatic and immunological properties]

The properties of phosphorylase B (PhB) immobilized on an agar derivative were studied. It was shown that the enzyme activity makes up to 15-20% as compared to that of the soluble enzyme, the Km value for glucose-1-phosphate is increased 1.5-fold and the pH optimum remains unchanged, whereas the thermostability of enzyme shows a considerable increase. PhB immobilized on a highly activated sorbent completely losses its enzymatic activity but retains its antigenic properties and binds 1.6-2 mol antibodies (per monomer). Using immunosorbents, purified antibodies homogeneous during electrophoresis in polyacrylamide gel were isolated. The immunosorbent capacity is 500-800 mg of antibodies per 1 g of dry weight. The purified antibodies are characterized by a lower inhibitory power upon interaction with soluble PhB. The type of inhibition of both immobilized and soluble enzyme is similar. It is assumed that immobilization produces conformational changes only at the active site of enzyme, which is spatially separated from the antibody binding site.     

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4x6-meric protein of Upogebia pusilla (Crustacea)

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4x6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4x6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4x6 hemocyanins. Electron micrographs are in agreement with a model in which four 2x6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4x6-meric hemocyanin into hexamers occurs without any intermediate 2x6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found.     

Leukotriene A4 hydrolase in the human B-lymphocytic cell line Raji: indications of catalytically divergent forms of the enzyme

Leukotriene A4 hydrolase was purified 1400-fold, with an approximate yield of 25%, to apparent homogeneity from the human B-lymphocytic cell line Raji. The purification included ammonium sulfate precipitations followed by anion exchange, hydrophobic interaction, and molecular exclusion fast protein liquid chromatography. Kinetic properties at 2 degrees C varied between different enzyme preparations. Two patterns were observed, one with a Km of about 12 microM and Vmax of about 1.1 mumol LTB4/mg protein/min which correlated well with the properties of the human leukocytic LTA4 hydrolase. In other enzyme preparations a higher catalytic activity was observed. These enzyme batches did not obey Michaelis-Menten kinetics but were compatible with a mixture of enzymatic species. Heat treatment (60 degrees C) led to a time-dependent decline in catalytic activity. However, certain enzyme preparations contained a subfraction of enzymatic activity which was more resistant to heat treatment, yielding a biphasic inactivation pattern. It is thus suggested, on the basis of the kinetic properties and the heat-inactivation pattern, that these enzyme preparations contained an addition form of LTA4 hydrolase.     

An iron dioxygenase from Alcaligenes faecalis catalyzing the oxidation of pyruvic oxime to nitrite

Abstract An enzyme which participated in the oxidation of hydroxylamine to nitrite from was partially purified Alcaligenes faecalis , and some of its properties were studied. The enzyme oxidized aerobically pyruvic oxime to nitrite in the presence of hydroxylamine or ascorbate. As molecular oxygen equimolar to nitrite formed was consumed in the enzymatic oxidation of pyruvic oxime to nitrite, the enzyme was thought to be a dioxygenase. It was an iron protein, and a reducing reagent was required to keep the iron in the ferrous state for the action of the enzyme.