Requirement For Divalent Cations Research Articles

Properties of phenylalanine ammonia-lyase from marrow cotyledons

Salinity induced phenylalanine ammonia-lyase (PAL, EC. 4.3.1.1.) from cotyledons of Cucurbita pepo (marrow) up to 200 mM. The effect of stabilizing solutes, glycerol, proline, and betaine against salt in the assay medium on PAL activity was analyzed. Glycerol, betaine and proline enabled a great increase in substrate-dependent salt activation of the enzyme. PAL activity declined following polyethyleneglycol (PEG)-induced water stress. The enzyme was inhibited by β-chloroethyltrimethylammonium (CETA) and induced by GA 3. PAL was purified from marrow ( C. pepo) using (NH 4) 2SO 4 fractionation, ion exchange chromatography using DEAE–cellulose, Sephadex G-100 and Q-Sepharose. The enzyme was purified with 83-fold with specific activity of 26 U mg −1 protein. The enzyme was extremely unstable unless 30% glycerol was added. There was an abrupt discontinuity in Arrhenius plot and the values of activation energy were 2.6 and 12.2 kJ mol −1 for the upper and lower parts of the plot, respectively. PAL activity was strongly inhibited by ethylenediaminetetraacetate (EDTA) and o-phenanthroline. AMP, ADP and ATP inactivated PAL with ATP being the most potent inactivator. Treatment of the pure enzyme with phenylmethyl-sulphonyl fluoride (PMSF), N, N-dicyclohexylcarbodiimide (DCCD), and N-acetyl-imidazole (NAI) resulted in the inhibition of the enzyme and reveal that seryl, carboxyl and tyroysl residues are essential for the enzyme catalysis. The metal ion requirement of PAL was investigated and Mg 2+ particularly fulfilled this requirement for divalent cation. Ca 2+ decreased PAL activity below the control value.

Molecular Mechanism of Aminoglycoside Antibiotic Kinase APH(3′)-IIIa: ROLES OF CONSERVED ACTIVE SITE RESIDUES

The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3')-IIIa: Lys(44) and Glu(60) involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp(190), and the Mg(2+) ligands Asn(195) and Glu(208), which coordinate two Mg(2+) ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys(44) interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu(60), which forms a salt bridge with the epsilon-amino group of Lys(44), demonstrated that this residue does not play a critical role in ATP recognition or catalysis. Results of mutagenesis of Asp(190) were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp(208) was found to be absolutely required for enzyme activity and the Mg2 ligand Asn(195) is important for Mg.ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp(208) likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.

Enzymatic activation of autotaxin by divalent cations without EF-hand loop region involvement

Autotaxin (ATX) is a recently described member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins with potent tumor cell motility-stimulating activity. Like other NPPs, ATX is a glycoprotein with peptide sequences homologous to the catalytic site of bovine intestinal alkaline phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca 2+ or Mg 2+ was each demonstrated to increase the PDE activity of ATX in a concentration-dependent manner, whereas incubation of ATX with chelating agents abolished this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effect on V max. Three mutant proteins, Ala 740-, Ala 742-, and Ala 751-ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to that of the wild-type protein. A deletion mutation of the entire loop region resulted in slightly reduced PDE activity but normal motility-stimulating activity. However, the PDE activity of this same deletion mutant remained sensitive to augmentation by cations, strongly implying that cations exert their effect by interactions outside of the EF-hand loop region.

Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688

Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschizomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5′-GAAGA-3′/3′-CTTCT-5′), and cleaves the DNA 8 and 7 nucleotides downstream from the recognition site leaving a single 3′-protruding nucleotide. We have purified this enzyme to electrophoretic homogenity using a four-step chromatographic procedure. NcuI endonuclease is a monomeric protein with a M r=48 000±1000 under denaturing conditions. The properties of NcuI are consistent with those for MboII, the position of the cleavage site being identical and the pH profile and divalent cation requirements being similar. Moreover, NcuI cross-reacts strongly with anti- MboII serum suggesting the presence of similar antigenic determinants. We have determined the sequence of 20 N-terminal amino acids for NcuI and concluded that this sequence is identical to the N-terminal portion of the MboII enzyme.

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions.

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.

Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688.

Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschizomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5'-GAAGA-3'/3'-CTTCT-5'), and cleaves the DNA 8 and 7 nucleotides downstream from the recognition site leaving a single 3'-protruding nucleotide. We have purified this enzyme to electrophoretic homogeneity using a four-step chromatographic procedure. NcuI endonuclease is a monomeric protein with a M(r)=48,000+/-1000 under denaturing conditions. The properties of NcuI are consistent with those for MboII, the position of the cleavage site being identical and the pH profile and divalent cation requirements being similar. Moreover, NcuI cross-reacts strongly with anti-MboII serum suggesting the presence of similar antigenic determinants. We have determined the sequence of 20 N-terminal amino acids for NcuI and concluded that this sequence is identical to the N-terminal portion of the MboII enzyme.

Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations.

Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator. Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity. The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme. In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk. When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column. Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions. Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound. The retained Csk enzyme was eluted with 1 M imidazole. The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column.

Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.

Critical amino acid residues of the α4 subunit for α4β7 integrin function

AbstractA characteristic feature of integrin–ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the α and β subunit of the α4 integrins, α4β1 and α4β7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM‐1 and MAdCAM‐1. The extracellular domain of the murine and human α4‐subunit contains three conserved LDV motifs, designated LDV‐1 to ‐3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for α4β7 integrins. We present evidence that LDV‐1 mutants (D489N) behave like α4 wt cells, but LDV‐3 mutants (D811N) are impaired in α4β7 integrin‐triggered homotypic cell aggregation and in adhesion and spreading on α4 specific ligands. Further characterization of LDV‐3 mutants revealed a defect in mAb‐induced α4β7‐cell surface cluster formation. Mutation of the LDV‐2 motif (D698N) caused loss of α4β7 integrin cell surface expression. Our results indicate: (i) that LDV‐3, located proximal to the cell membrane, is important for α4β7 integrin‐triggered functions and for lateral clustering and (ii) that LDV‐2 affects α4β7 heterodimer stability. J. Cell. Biochem. 83: 304–319, 2001. © 2001 Wiley‐Liss, Inc.

Isolation and partial characterization of a lectin from Bauhinia pentandra (bong) vog. Ex. Steua.

Bauhinia pentandra (Bong) Vog. ex. Steua seeds were investigated with respect to phenologic aspects (size, mass, hilum and length) and with respect to their chemical composition. The total nitrogen content of the seed flour was determined, and the flour was extracted in different pH values. A lectin was isolated from the seeds by Sepharose-4B affinity chromatography. The homogeneity of the lectin was demonstrated by SDS-PAGE in the presence of beta-mercaptoethanol. Only one protein band with an apparent molecular mass of 30 kDa was found. The B. pentandra lectin showed a carbohydrate specificity for D-galactose, a requirement for divalent metal cations (Ca2+ and Mn2+) for full activity and amino acid composition with a high content of aspartic acid, glutamic acid and alanine and low levels of methionine, cysteine and tryptophan. The lectin agglutinated rabbit and human A group erythrocytes and was relatively stable to heat treatment, retaining half of its original activity after 60 min at 70 ºC.

Human intestinal epithelial cells express a novel receptor for IgA.

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.

Inhibition of Enzymes of the Glycolytic Pathway and Hexose Monophosphate Bypass by Phosphonate

Previous studies have suggested that the phosphonate ion (Phi), an isostere of phosphate, might be a general inhibitor of enzymes which are allosterically regulated by phosphate or which have a requirement for divalent cations. In this paper, the capacity of Phi to inhibit selected enzymes of this type from Phytophthora palmivora is compared with its effects on the same enzymes isolated from other organisms, in particular from Saccharomyces cerevisiae, which are widely used in linked assays of many enzymes. It was found that Phi inhibited the activity of the enzymes from P. palmivora investigated, though to different degrees. IC50 values (concentration required to inhibit enzyme activity by 50%) for Phi ranged from 0.74 ± 0.07 mM (NAD-dependent glyceraldehyde-3-phosphate dehydrogenase) to 116.1 ± 7.3 mM (6-phosphogluconate dehydrogenase). Among the activities tested glucose-6-phosphate dehydrogenase activity from P. palmivora was significantly more sensitive to Phi than the same enzyme from yeast, although its absolute IC50 value (29.0 ± 3.4 mM) was high in comparison to most fungicides. It was also found that the auxiliary enzymes from rabbit muscle (aldolase, glycerophosphate dehydrogenase, and triosephosphate isomerase) and yeast (glucose-6-phosphate dehydrogenase) used in enzyme-linked assays were all sensitive to Phi, giving IC50 values between 7.7 ± 0.4 and 73.6 ± 2.0 mM, a sensitivity comparable to the other enzymes under investigation. Inorganic phosphate also inhibited the activity of the enzyme glucose-6-phosphate dehydrogenase and the aldolase/triosephosphate isomerase/glycerophosphate dehydrogenase mixture with IC50 values of 108.3 ± 7.7 and 13.0 ± 0.6 mM, respectively. In conclusion, Phi inhibition was found to be widespread, supporting the hypothesis that Phi may inhibit several enzymes rather than acting at a single unique site. It was also found that the coupling enzymes used in many of the assays for these enzymes were themselves susceptible to Phi and phosphate inhibition, which must be taken into account in the interpretation of the results obtained with this type of assay.

Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin ( Lupinus luteus) seeds

A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin ( Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (p I=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism.

Purification, Cloning, and Characterization of an Acidic Ectoprotein Phosphatase Differentially Expressed in the Infectious Bloodstream Form of Trypanosoma brucei

We purified an ecto-phosphatase of 115 kDa (TryAcP115) specifically expressed by bloodstream forms of Trypanosoma brucei. The corresponding gene coded for a 45-kDa protein potentially including a signal peptide, a membrane-spanning domain and an N-terminal domain containing 8 N-glycosylation sites. There was no significant sequence homology with other phosphatases. Antiserum to the Escherichia coli recombinant N-terminal domain, Petase7, recognized a protein of 55 kDa in Western blots after deglycosylation of the TryAcP115 protein by N-glycosidase F. Immunofluorescence and trypsin treatment of living parasites showed that TryAcP115 was localized to the surface of the parasite and that its N-terminal domain was oriented extracellularly. The recombinant N-terminal domains, expressed in E. coli and Leishmania amazonensis, harbored phosphatase activity against Tyr(P)-Raytide, Ser(P)-neurogranin, and ATP. The enzymatic properties of native TryAcP115 and the recombinant proteins for the substrate Tyr(P)-Raytide were virtually identical and included: (i) K(m) and V(max) values of 15 nM and 200 pmol/min/mg, (ii) no requirement for divalent cations, and (iii) sensitivity to vanadate, sodium fluoride, and tartrate, but insensitivity to okadaic acid and tetramisole. Although the function of TryAcP115 remains unknown, a differentially expressed, unique ecto-phosphatase could regulate growth or influence parasite-host interactions and might provide a useful target for chemotherapy.

Identification and Characterization of a Novel Hepatic Canalicular ATP Diphosphohydrolase

We have identified and characterized a novel ATP diphosphohydrolase (ATPDase) with features of E-type ATPases from porcine liver. Immunoblotting with a specific monoclonal antibody to this ectoenzyme revealed high expression in liver with lesser amounts in kidney and duodenum. This ATPDase was localized by immunohistochemistry to the bile canalicular domain of hepatocytes and to the luminal side of the renal ductular epithelium. In contrast, ATPDase/cd39 was detected in vascular endothelium and smooth muscle in these organs. We purified the putative ATPDase from liver by immunoaffinity techniques and obtained a heavily glycosylated protein with a molecular mass estimated at 75 kDa. This enzyme hydrolyzed all tri- and diphosphonucleosides but not AMP or diadenosine polyphosphates. There was an absolute requirement for divalent cations (Ca(2+) > Mg(2+)). Biochemical activity was unaffected by sodium azide or other inhibitors of ATPases. Kinetic parameters derived from purified preparations of hepatic ATPDase indicated V(max) of 8.5 units/mg of protein with apparent K(m) of 100 microM for both ATP or ADP as substrates. NH(2)-terminal amino acid sequencing revealed near 50% identity with rat liver lysosomal (Ca(2+)-Mg(2+))-ATPase. The different biochemical properties and localization of the hepatic ATPDase suggest pathophysiological functions that are distinct from the vascular ATPDase/cd39.

Identification of a β-like DNA Polymerase Activity in Bovine Heart Mitochondria

A new DNA polymerase activity, distinct from DNA polymerase γ, has been identified in bovine heart mitochondria. First detected among proteins isolated in a complex with mitochondrial DNA, the DNA polymerase activity has been partially purified 47,000-fold. Enzyme activity separates from DNA polymerase γ on several chromatographic columns and appears to copurify with a 38 ± 2-kDa polypeptide. Unlike DNA polymerase γ, this enzyme is relatively resistant to inhibition by N-ethylmaleimide and dideoxynucleotides, has moderately low monovalent and high divalent cation requirements, and possesses 20-fold-higher apparent Km values for deoxynucleotides. The enzyme polymerizes deoxynucleotides onto a primed template DNA in a relatively nonprocessive fashion and lacks a detectable 3′ to 5′ exonuclease activity. Many of these characteristics resemble a β-like mitochondrial DNA polymerase previously identified in, and considered unique to, trypanosomes. We propose that the bovine and trypanosomal enzymes are related and represent a new class of ubiquitous mitochondrial DNA polymerases.

The effect of divalent cations on bovine retinal NOS activity.

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca2+ 0.25, Mg2+ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 microM) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe2+, Zn2+ or Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba2+, Zn2+, Co2+, Mn2+, Mg2+, Ca2+), as well as La3+, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca2+.

Monoterpene Synthases of Loblolly Pine (Pinus taeda) Produce Pinene Isomers and Enantiomers

The turpentine fraction of conifer oleoresin is a complex mixture of monoterpene olefins and plays important roles in defense and in the mediation of chemical communication between conifer hosts and insect predators. The stereochemistry of the turpentine monoterpenes is critical in these interactions, influencing host recognition, toxicity, and potency of derived pheromones, and the stereochemical composition of these compounds lends insight into their biogenetic origin, with implications for the numbers and types of enzymes responsible and their corresponding genes. Analysis of the oleoresin from several tissues of loblolly pine (Pinus taeda) showed the derived turpentine to consist mainly of (+)-(3R:5R)-α-pinene and (−)-(3S:5S)-β-pinene. Cell-free extracts from xylem tissue yielded three monoterpene synthases which together account for the monoterpene isomer and enantiomer content of the turpentine of this tissue. The major products of these enzymes, produced from the universal precursor of monoterpenes, geranyl diphosphate, were shown to be (+)-α-pinene, (−)-α-pinene, and (−)-β-pinene, respectively. In most properties (molecular mass of ∼60 kDa, Km for geranyl diphosphate of 3 μM, requirement for monovalent and divalent cations), these enzymes resemble other monoterpene synthases from conifer species.

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of – single strand preferential – deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M r of the purified enzyme determined by native PAGE was 67 000 and it is a tetramer and each protomer consists of two unidentical subunits of M r 21 000 and 13 000. It is an acidic protein with a p I of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg 2+, Mn 2+ and Co 2+ for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg 2+ was 40°C, it showed higher optimum temperature (45°C) for dsDNA hydrolysis in presence of Mn 2+ and Co 2+. Nuclease Rsn was inhibited by divalent cations like Zn 2+, Cu 2+ and Hg 2+, inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3′- or 5′-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3′-hydroxyl and 5′-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.

S-adenosylmethionine synthesis in Leishmania infantum promastigotes.

Methionine adenosyltransferase (MAT), S -adenosylmethionine (AdoMet), and S -adenosylhomocysteine (AdoHcy), have been analysed at different time-points during the growth curve of Leishmania infantum. MAT activity and AdoMet content peaked in the lag and early log phases, whereas higher levels of AdoHcy were found in stationary phase cells. MAT activity of cell extracts displayed hyperbolic kinetics for both its substrates, l -methionine and ATP, with km values of 35 microm and 5 m m, respectively. MAT has an absolute requirement for divalent cations, and is dependent on sulfydryl protective agents. Unlike other sources, L. infantum MAT activity seems to be transcriptionally regulated, with an accumulation of MAT-mRNA during rapid growth periods of promastigotes.