Purification and characterization of flavone synthase I, a 2-oxoglutarate-dependent desaturase

Soluble flavone synthase I from illuminated parsley cells was purified to near homogeneity by a six-step procedure. A molecular mass of 48 +/- 2 kDa was determined by gel permeation chromatography and denaturing polyacrylamide gel electrophoresis. A single protein with an isoelectric point at pH 4.8 +/- 0.1 was detected on isoelectric focusing gels, which catalyzed the overall conversion of 2S-flavanones into the corresponding flavones in the presence of molecular oxygen, 2-oxoglutarate, ferrous ion, and ascorbate. Apparent Michaelis constants for 2S-naringenin, 2S-eriodictyol, and 2-oxoglutarate were determined as 5, 8, and 16 microM, respectively. (+)-Dihydrokaempferol and 2R-naringenin were not accepted as substrates. The enzyme was strongly inhibited by Cu2+ and Zn2+. Potent competitive inhibition with respect to 2-oxoglutarate was observed with 2,4-pyridinedicarboxylate (Ki = 1.8 microM). With crude extracts as well as with the purified enzyme neither the hypothetical intermediate 2-hydroxyflavanone nor a dehydratase activity capable of converting the chemically synthesized compound to flavone could be observed. Moreover, the introduction of the double bond into the substrate naringenin was not altered by addition of chemically synthesized 2-hydroxynaringenin into the reaction mixture. Therefore, 2-hydroxyflavanones are apparently not freely dissociable intermediates in the biosynthesis of flavones in parsley and are not capable of entering the active site of the enzyme to compete with the flavanone. It is postulated that flavone synthase I catalyzes double-bond formation by direct abstraction of vicinal hydrogen atoms at C-2 and C-3 of the substrate. Thus, flavone synthase I is a member of a novel subgroup within the 2-oxoglutarate-dependent dioxygenases that can be referred to as 2-oxoglutarate-dependent desaturases.     

Three 2-oxoglutarate-dependent dioxygenase activities of Equisetum arvense L. forming flavone and flavonol from (2S)-naringenin

Equisetum arvense L. (Equisetaceae-horsetail) accumulates various flavones and flavonols in infertile shoot. Enzyme assays conducted with crude extracts of the green tissue revealed chalcone synthase activity and also three further activities assigned to flavonoid biosynthesis and identified as flavone synthase I, flavanone 3β-hydroxylase and flavonol synthase. The latter three activities were characterized as soluble, 2-oxoglutarate-dependent dioxygenases by their typical cofactor requirements and peculiar inhibition. Notably, this is the first report of flavone synthase I which had been considered to be restricted solely to species of the Apiaceae from a distant plant taxon.     

Cloning of parsley flavone synthase I

A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.     

Molecular evolution of flavonoid dioxygenases in the family Apiaceae

Plant species of the family Apiaceae are known to accumulate flavonoids mainly in the form of flavones and flavonols. Three 2-oxoglutarate-dependent dioxygenases, flavone synthase or flavanone 3 beta-hydroxylase and flavonol synthase are involved in the biosynthesis of these secondary metabolites. The corresponding genes were cloned recently from parsley (Petroselinum crispum) leaves. Flavone synthase I appears to be confined to the Apiaceae, and the unique occurrence as well as its high sequence similarity to flavanone 3beta-hydroxylase laid the basis for evolutionary studies. In order to examine the relationship of these two enzymes throughout the Apiaceae, RT-PCR based cloning and functional identification of flavone synthases I or flavanone 3beta-hydroxylases were accomplished from Ammi majus, Anethum graveolens, Apium graveolens, Pimpinella anisum, Conium maculatum and Daucus carota, yielding three additional synthase and three additional hydroxylase cDNAs. Molecular and phylogenetic analyses of these sequences were compatible with the phylogeny based on morphological characteristics and suggested that flavone synthase I most likely resulted from gene duplication of flavanone 3beta-hydroxylase, and functional diversification at some point during the development of the apiaceae subfamilies. Furthermore, the genomic sequences from Petroselinum crispum and Daucus carota revealed two introns in each of the synthases and a lack of introns in the hydroxylases. These results might be explained by intron losses from the hydroxylases occurring at a later stage of evolution.     

L-type glycogen synthase. Tissue distribution and electrophoretic mobility

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.     

Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms.

Nitric oxide (NO) is generated from L-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Anti-sera A and B reacted with a approximately 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M(r) between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the approximately 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Abstract Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-β-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica . The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-β-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-β-glucan synthase activity.     

Cystathionine β-synthase from human liver: Improved purification scheme and additional characterization of the enzyme in crude and pure form

The previously published procedure (Kraus et al. (1978) J. Biol. Chem.253, 6523–6528) for the purification of cystathionine β-synthase [l-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5′-phosphate-dependent enzyme from human liver has been modified. The new procedure, starting with a liver homogenate “aged” for 7 days at 4 °C, yielded homogeneous enzyme purified over 3000-fold with a much improved yield. “Aging” of the enzyme in crude homogenates yields a form apparently smaller by gel electrophoresis and with significantly increased activity and antigenicity. This species of cystathionine β-synthase does not form stable complexes with other proteins during purification as does the previously employed, freshly used species. An absorption spectrum and an amino acid composition of the pure enzyme were determined; the amino-terminal residue was shown to be methionine. The isoelectric points of holosynthase and aposynthase were estimated to be 5.2 and 5.6, respectively. Rabbit antiserum raised against the pure cystationine β-synthase was characterized using as antigen crude synthase from five different mammalian species as well as the pure human enzyme.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.     

Improved procedure for the purification of hepatic lipase from rat liver homogenate

A procedure is described for the purification of hepatic lipase (HL)4 from rat liver homogenate which results in a high yield (41%) of electrophoretically homogeneous enzyme. The method is based on that of Twu et al. (Biochim. Biophys. Acta 1984: 792, 330), but it is more efficient with respect to yield (about 4-fold) and purity (1.6-fold). It includes the preparation of a high-speed supernatant, chromatography in series on octyl-, heparin- and concanavalin A-Sepharose, and finally gel filtration. On SDS-PAGE analysis, the purified enzyme exhibited an apparent molecular mass of 63.6 +/- 3.2 kDa. Heterogeneity was observed, when purified HL was subjected to isoelectric focussing. The enzyme displayed a specific catalytic activity of 23,000 U* (mumol fatty acid released per h at 37 degrees C) per mg protein, when assayed with trioleoyl glycerol suspensions in arabic gum. A highly specific antiserum against rat liver HL, capable of inhibiting 817 mU* HL per microliter antiserum, was raised in rabbits.     

Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution.

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.     

Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme.

An alpha 1,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16,000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-l-deoxymannojirimycin (CP-dMM). On SDS/polyacrylamide-gel electrophoresis under reducing conditions, the isolated enzyme migrated as a single protein band with a molecular mass of 49 kDa. The enzyme does not bind Con A and is not susceptible to glycopeptidase F, indicating that it lacks N-linked oligosaccharides of the high-mannose or complex type. Purified Man9-mannosidase has a pH optimum close to 6.0 and requires bivalent cations for activity, with Ca2+ being most effective. The enzyme is inhibited strongly by basic sugar analogues of mannose such as 1-deoxymannojirimycin (dMM, Ki approximately 5 microM), N-methyl-dMM (Ki approximately 55 microM) and CP-dMM (Ki approximately 150 microM), whereas NN-dimethyl-dMM and the mannosidase II inhibitor swainsonine were hardly or not at all inhibitory. A homogeneous preparation of the 49 kDa enzyme cleaves specifically three of the four alpha 1,2-mannosidic linkages in the natural Man9-GlcNAc2 (M9) substrate. The relative rates by which the parent and intermediate structures are hydrolysed were found to be about 3:2:5 for M9, M8 and M7 respectively. The enzyme displays only marginal activity toward the remaining alpha 1,2-mannosidic linkages in the Man9-GlcNAc2 oligosaccharide (relative rate of M6 hydrolysis approximately 0.02) and is not active against nitrophenyl and methylumbelliferyl alpha-mannosides. This unique substrate specificity suggests that Man9-mannosidase processing differs from that catalysed by other trimming alpha 1,2-mannosidases hitherto reported. A polyclonal antibody raised against the denatured 49 kDa polypeptide not only recognizes a protein band of similar size in Western blots of crude microsomes, but also reacts strongly with a 65 kDa protein species. On trypsin treatment of detergent-solubilized microsomes, the 65 kDa protein is converted specifically into a stable 49 kDa fragment, indicating a precursor-product relationship between the two proteins. We conclude from this observation that the 65 kDa protein represents the intact form of Man9-mannosidase from which the 49 kDa enzyme which we have isolated has been generated, with retention of catalytic activity, by proteolysis during purification. Proteolytic studies with sealed microsomes suggest that the intact 65 kDa enzyme is a protein with a membrane-spanning domain, as well as a cytosolic polypeptide domain of size at least 3 kDa.     

Rapid purification of protein kinase C from rat brain. A novel method employing protamine-agarose affinity column chromatography

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine-coupled to agarose. Sequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and protamine-agarose columns resulted in a 1,500-fold purification of protein kinase C. SDS-PAGE analysis of the purified enzyme resolved a doublet protein of 77-80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments. The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column; phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.     

The 165-kDa DNA topoisomerase I from Xenopus laevis oocytes is a tissue-specific variant.

Two forms of topoisomerase I can be purified from Xenopus laevis. A protein with a molecular mass of 165 kDa has been identified as topoisomerase I in ovaries (Richard and Bogenhagen, 1989. J. Biol. Chem. 264, 4704-4709). When a similar purification is performed using liver tissue, topoisomerase I is purified as a 110-kDa protein. Separate rabbit antisera were raised against oocyte and liver topoisomerase I polypeptides. Each antiserum reacts in immunoblotting or immunoprecipitation procedures only with the tissue-specific topoisomerase I polypeptide against which it was generated. The failure of the antiserum raised against liver topoisomerase I to cross-react with the oocyte enzyme suggests that the smaller topoisomerase I is not derived from the 165-kDa oocyte enzyme by proteolysis. X. laevis tissue culture cells lysed and processed in the presence of SDS contain the 110-kDa form of topoisomerase I. The 165-kDa form of topoisomerase I disappears during oocyte maturation in vitro.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

On the nature of the activating enzyme of the inactive form of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides.

The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase.     

Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver

A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.