Presence Of Divalent Metal Ions Research Articles

Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

l-Arabinose isomerase (AI) catalyzes the isomerization of l-arabinose to l-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 °C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K m values are 36 mM for l-arabinose and 167 mM for d-galactose, and the catalytic efficiencies ( k cat/ K m) of the enzyme were 51.4 mM −1 min −1 ( l-arabinose) and 0.4 mM −1 min −1 ( d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65–70 °C, and is strongly activated by Mn 2+. It also has a much higher catalytic efficiency of 4.3 mM −1 min −1 for d-galactose and 32.5 mM −1 min −1for l-arabinose, with apparent K m values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI ( T m = 65–67 °C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T m (72 °C) than the holoenzyme (78 °C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.

A Peptide Region from Factor Va Increases Factor Xa Efficiency for Cleavage of Prothrombin.

The procoagulant enzymatic complex, prothrombinase, which is required for normal hemostasis, is composed of the enzyme, factor Xa, the protein cofactor, factor Va, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa increases the catalytic efficiency of the enzyme by five orders of magnitude as compared to factor Xa alone. While the importance of the contribution of factor Va to the activity of factor Xa for rapid thrombin formation by prothrombinase at the place of vascular injury has been long established, the consequence of the interaction of the cofactor with the members of prothrombinase and the molecular mechanism by which factor Va accelerates prothrombin activation remains an enigma. Prothrombin is activated following two cleavages (Arg271/Arg320). Depending on the order of peptide bond cleavage different intermediates are formed. Factor Xa alone cleaves prothrombin sequentially, first at Arg271 to produce fragment 1•2 and prethrombin-2, followed by cleavage at Arg320 to produce fragment 1•2 and thrombin. The prothrombinase complex catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271), resulting in a formation of an active intermediate (meizothrombin) and a 300,000-fold increase in the rate of the overall reaction compared with the rate of prothrombin activation observed with factor Xa alone. We have shown that amino acid region 307–348 of factor Va heavy chain is critical for cofactor activity. A peptide containing this amino acid sequence (42 amino acids, N42R) was found to interact with fluorescently labeled factor Xa and to inhibit prothrombinase activity. Our present data show that N42R can be cross-linked to the heavy chain of membrane-bound factor Xa in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have also demonstrated that amino acid region 323–331 from N42R (AP4′) contains a binding site for factor Xa of factor Va heavy chain. Our present data show that a peptide containing amino acid residues 317–326 (AP3) inhibited both prothrombinase activity and the high affinity interaction of factor Va with factor Xa on the membrane surface. Moreover, we have found using site directed mutagenesis and recombinant factor Va that amino acids at the NH2-terminal end of AP4′ (i.e. residues 323–325, Glu-Tyr-Phe) are responsible for the inhibitory effect of AP3 and AP4′ and are crucial for the interaction of factor Va with factor Xa. A tripeptide with this sequence inhibited prothrombinase activity in an assay using a fluorescent thrombin inhibitor. To identify the effect of these peptides on factor Xa's ability to cleave and activate prothrombin, we studied prothrombin activation by gel electrophoresis. The data demonstrated that several peptides that inhibited both the factor Va-factor Xa interaction on the membrane surface and prothrombinase activity, had the ability to accelerate cleavage of prothrombin by factor Xa alone, in the absence of factor Va. Specifically, N42R and AP3 were found to increase the rate of prothrombin consumption by factor Xa by approximately four-fold when compared to factor Xa acting alone. Both peptides induced acceleration in prethrombin-2 formation suggesting an increased in the rate of cleavage of prothrombin at Arg271. These data suggest that the binding of factor Va to factor Xa through amino acid region 323–331 alone produces an effect on factor Xa that increases its potency for cleavage at Arg271.

Separation of glutathione and its related amino acids by nanofiltration

Nanofiltration experiments of glutathione and its related amino acids ( l-glutamate, l-cysteine, glycine, and l-glutamine) were carried out using NTR-7450 nanofiltration membrane in order to determine the potential use of nanofiltration in the enzymatic glutathione synthesis, in which the reaction equilibria are need to be driven to production by separating glutathione from starting substrates. In the experiments with a single component system, the rejection of NTR-7450 membrane for the electrolytes corresponded well to the ratio of their anionic species varying with pH. Difference in the rejection between glutathione and some amino acids was maximal in the neutral pH region: at pH 7.4, the rejection for glutathione and l-glutamate was almost 100%, whereas that for l-cysteine, glycine, and l-glutamine was below 30%. In the presence of divalent metal ions, the rejection for glutathione and l-glutamate decreased with increasing the metal concentration, probably due to partial neutralization of negative charges of the electrolytes forming complexes with the metal ions. In the experiments with a multi-component system, glutathione was completely blocked again by the membrane in a range of glutathione concentration from 0.1 to 5.0 mM in the presence of 1.0, 2.0, or 5.0 mM each of l-glutamine, glycine, and l-cysteine. The amino acid rejection decreased with increasing the amino acid concentrations. These results suggest that nanofiltration would be applicable to the separation of glutathione from substrates, l-cysteine, glycine, and l-glutamine, in the enzymatic glutathione synthesis.

Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Drastic Improvements in Trapping Efficiency and Dispersibility for Phosphatidylcholine Liposomes in the Presence of Divalent Metal Ions

The effects of divalent metal ions (Ca2+, Mg2+) on the physicochemical properties and dispersibility of phosphatidylcholine (PC) liposomes were examined by using an optical microscopy, and the measurements of trapping efficiency, lamellarity, zeta potential. The trapping efficiency of PC liposomes was markedly improved (ca. 8 times) in the presence of an equimolar amount of a divalent metal ion, and that bearing Ca2+ ions was then higher than that of liposomes bearing Mg2+ ions. Optical microscopy on PC liposomes showed that the formation of liposomes is markedly promoted by the addition of divalent metal ions. The lamellarity measurements revealed that liposome bearing divalent metal ions is a unilamellar structure; this gives a higher trapping efficiency of liposomes compared with a control. The lamellarity of liposomes bearing Ca2+ ions was then lower than that of those bearing Mg2+ ions. Liposomal solutions containing equimolar amount of divalent metal ions remained well dispersed for more than 1 month, whereas that without divalent metal ions exhibited a significant phase separation. The liposomes bearing Ca2+ ions then exhibited a better dispersibility than that of those bearing Mg2+ ions, because of their higher zeta potential. Finally, we concluded that the unilamellar liposomes formation with a high dispersibility is promoted by not only electrostatic repulsion between bilayers but also the spontaneous curvature of liposomal bilayers induced by bearing of divalent metal ions.

Identification of the putative mannose 6-phosphate receptor (MPR 46) protein in the invertebrate mollusc.

Mannose 6-phosphate receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403-409). In the present study we demonstrate that the unio also contains the putative mannose 6-phosphate receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & PhysioL 123B:261-265). The authenticity of the receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [35S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with mannose 6-phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same receptor protein.

Sticky DNA, a Long GAA·GAA·TTC Triplex That Is Formed Intramolecularly, in the Sequence of Intron 1 of the Frataxin Gene

Friedreich's ataxia is caused by the massive expansion of GAA.TTC repeats in intron 1 of the frataxin (X25) gene. Our prior investigations showed that long GAA.TTC repeats formed very stable triplex structures which caused two repeat tracts to adhere to each other (sticky DNA). This process was dependent on negative supercoiling and the presence of divalent metal ions. Herein, we have investigated the formation of sticky DNA from plasmid monomers and dimers; sticky DNA is formed only when two tracts of sufficiently long (GAA.TTC)(n) (n = 59-270) are present in a single plasmid DNA and are in the direct repeat orientation. If the inserts are in the indirect (inverted) repeat orientation, no sticky DNA was observed. Furthermore, kinetic studies support the intramolecular nature of sticky DNA formation. Electron microscopy investigations also provide strong data for sticky DNA as a single long triplex. Hence, these results give new insights into our understanding of the capacity of sticky DNA to inhibit transcription and thereby reduce the level of frataxin protein as related to the etiology of Friedreich's ataxia.

Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change.

Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans-acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site. We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3' cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5'-terminal cytidine residue prevents this conformational change and its absence stabilizes the product-ribozyme complex in the presence of Mg2+. The structure of the Mg2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.

The divalent cation-induced DNA condensation studied by atomic force microscopy and spectra analysis

Three types of DNA condensates in the presence of divalent metal ion (Mg, Mn and Cu) and EB simultaneously have been observed by atomic force microscopy (AFM). In the presence of Mg (II) and EB, DNA molecular exhibits three or four maggot-like branch crossing structures at the same point, which were constructed by some bead-like particles in each branch. However, in the presence of Cu (II) or Mn (II)–EB, DNA molecular forms a network or a semicircular structure, respectively. Their average height was 0.95±0.5 nm (Cu (II)–DNA–EB), 17±5 nm (Mg (II)–DNA–EB) and 12±3 nm (Mn (II)–DNA–EB), respectively. However, DNA molecular or DNA–EB complex still remained a line double-stranded structure. The fluorescence, melting point and CD spectra indicate that the different morphologies induced by divalent metal ion (Mg (II), and Mn (II) and Cu (II)) are related to its binding position in molecular DNA.

Dissecting the metal ion dependence of DNA binding by PvuII endonuclease.

Divalent cations can provide an effective means of modulating the behavior of nucleic acid binding proteins. As a result, there is strong interest in understanding the role of metal ions in the function of both nucleic acid binding proteins and their enzymes. We have applied complementary fluorescence spectroscopic and nitrocellulose filter binding assays to quantitate the role of metal ions in mediating DNA binding and sequence specificity by the representative PvuII endonuclease. At pH 7.5 in the presence of the catalytically nonsupportive Ca(II), this enzyme binds the PvuII target sequence with a K(d) of 50 pM. Under strict metal-free conditions, the enzyme exhibits a K(d) of only 300 nM for the cognate sequence, an affinity which is weak relative to those measured for other systems in the absence of metal ions. This represents a 6000-fold increase in PvuII affinity for cognate DNA upon the addition of Ca(II). The pH dependences of both metal ion-dependent and metal ion-independent DNA binding are remarkably shallow throughout the physiological range; other characterized restriction enzymes exhibit more pronounced pH dependences of DNA binding even in the absence of metal ions. Similar measurements with noncognate sequences indicate that divalent metal ions are not important to nonspecific DNA binding; K(d) values are approximately equal to 200 nM throughout the physiological pH range, a behavior shared with other endonucleases. While some of these results extend somewhat the range of expected behavior for restriction enzymes, these results indicate that PvuII endonuclease shares with other characterized systems a mechanism by which cognate affinity and sequence discrimination are most effectively achieved in the presence of divalent metal ions.

The contribution of metal ions to the conformational stability of ribonuclease T1: crystal versus solution.

In the crystalline state, ribonuclease T1 binds calcium ions at different lattice-dependent positions. In solution, its conformational stability is also remarkably increased in the presence of divalent metal ions. Combining urea unfolding studies and X-ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution. We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution.

FokI requires two specific DNA sites for cleavage

FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5′ overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity.

Ordered association of tobacco mosaic virus in the presence of divalent metal ions.

The formation of ordered aggregates of tobacco mosaic virus (TMV) in the presence of divalent metal ions has been studied in concentrated (1-25 mg/ml) solutions of the virus. The divalent metal cations Cd2+, Zn2+, Pb2+, Cu2+, and Ni2+ have been found to promote TMV precipitation from solution at a critical concentration Ccrit, which for a given metal depends on the pH and the ionic strength of the solution, but is largely independent of the virus concentration. The TMV precipitate behaves as a nematic liquid crystal and on drying at a glass surface produces highly ordered, optically birefringent films. However, precipitation is not observed with alkali-earth metals such as Ca2+ and Mg2+. The experimental data suggest that, apart from two 'internal' metal-binding sites in each TMV subunit, the virus contains metal-binding sites of a lower affinity which promote cross-linking of TMV rods via metal bridges. The latter seem to be responsible for the precipitation of TMV in the presence of divalent cations at neutral pH. We propose that the metal-induced cross-linking may be the predominant mechanism to account for the limited solubility of a variety of proteins in solution containing metal cations with valence 2 and higher.

Gold Nanoparticle-Based Sensing of “Spectroscopically Silent” Heavy Metal Ions

A simple colorimetric technique for the detection of small concentrations of aqueous heavy metal ions, including toxic metals such as lead, cadmium, and mercury, is described. Functionalized gold nanoparticles are aggregated in solution in the presence of divalent metal ions by an ion-templated chelation process; this causes an easily measurable change in the absorption spectrum of the particles. The aggregation also enhances the hyper-Rayleigh scattering (HRS) response from the nanoparticle solutions, providing an inherently more sensitive method of detection. The chelation/aggregation process is reversible via addition of a strong metal ion chelator such as EDTA. Suggestions for improving the sensitivity and selectivity of the technique are given.

Oxidation-active flavin models: oxidation of alpha-hydroxy acids by benzo-dipteridine bearing metal-binding site in the presence of divalent metal ion and base in organic solvents.

The oxidizing ability of benzo-dipteridine bearing a bipyridin-6-ylmethyl moiety (4) was found to be increased with Zn(2+) by approximately 10(3)-fold for sulfite addition in MeOH and approximately 10(2)-fold for oxidation of an NADH model in MeCN. It was found for the first time that 4 is able to oxidize alpha-hydroxy acids to alpha-keto acids in the presence of a divalent metal ion such as Zn(2+), Co(2+), and Ni(2+) and an amine base in MeCN or t-BuOH, whereas benzo-dipteridine having a bipyridin-5-ylmethyl moiety (3) is unable to oxidize them under the same conditions. The oxidation reaction was kinetically investigated including the kinetic isotope effect for deuterated mandelic acids (k(H)/k(D) = 2.1-3.7) and the Hammett plots for substituted mandelic acids (V-shaped plots). In the reaction of alpha-substituted alpha-hydroxy acids such as alpha-methyl mandelic and benzylic acids with 4, novel oxidative decarboxylation was found to take place, giving acetophenone and benzophenone, respectively. The oxidation mechanism for mandelic acid was proposed to proceed via a ternary complex of 4.Zn(2+).PhCH(OH)CO(2)(-), in which alpha-oxyanion of mandelate attacks C(4a)-position of 4 to form an adduct followed by 1,2-elimination to afford benzoyl formate and 2e-reduced 4. The roles of the metal ion were proposed as follows; (i) activation of 4, (ii) substrate-binding site, and (iii) activation of the bound alpha-hydroxy acid by lowering pK(a)'s of alpha-OH and alpha-CH. This is a first example that a flavin model oxidizes alpha-hydroxy acids in the presence of a metal ion.

Factor V: Dr. Jeckyll and Mr. Hyde.

The regulation of the delicate balance between the procoagulant and anticoagulant mechanisms is of extreme importance for survival. The procoagulant enzymatic complexes (i.e. prothrombinase, intrinsic tenase and extrinsic tenase) are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. Factor Va and factor VIIIa, which are very similar in structure and function, are required for prothrombinase and intrinsic tenase activities respectively because both cofactors express a dual function in their respective complexes, acting as an enzyme receptor and catalytic effector on the cell surface. The cofactors derive from inactive plasma precursors by regulatory proteolytic events, which involve alpha-thrombin. In general bleeding tendencies are usually associated with defects in the activation of one of the zymogens or the cofactors of the procoagulant complexes. a-Thrombin, participates in its own down-regulation by binding to the endothelial cell receptor thrombomodulin, and initiating the protein C pathway, which in turn leads to the formation of activated protein C (APC). APC is required for efficient neutralization of factor Va cofactor activity which results in the inactivation of the prothrombin-activating complex. This inactivation can only occur in the presence of the appropriate membrane surface. APC down-regulates the prothrombinase complex by cleaving specific peptide bonds on the heavy chain of factor Va which results in the dissociation of the A2 domain of factor Va from the rest of the molecule. Irregularities in the mechanism of inactivation of factor Va by APC, are associated with thrombotic risk, presumably due to sustained prothrombin activation.

9] Human ribonuclease P

Publisher Summary Ribonuclease P is a ribonucleoprotein nuclease required for the site-specific cleavage of the 5′ leader sequence of precursor tRNAs. In eubacteria, the RNA subunit of RNase P is the catalytic moiety and is capable of processing precursor tRNA in the presence of divalent metal ions. The RNA subunits of eukaryotic RNase P (human and yeast) do not exhibit enzymatic activity In Vitro, an indication that their protein components are required for the catalytic reaction. This chapter describes a purification procedure for human RNase P from HeLa cells that facilitated the characterization of nine of its protein subunits. This procedure also allows the separation of RNase P from the structurally related RNase MRP nuclease. The purification procedure shows that nuclear RNase P from HeLa cells has at least nine protein subunits in association with a single RNA species, H1 RNA. Nuclear RNase P in other eukaryotes, such as S. cerevisiae and Aspergillus nidulans, also consists of multiple protein subunits, some of which are evolutionarily conserved and an RNA subunit. The precise function of these subunits in tRNA processing is not yet understood. Reconstitution of the activity of RNase P in vitro may reveal the role of these subunits in catalysis.

Purification and some properties of UDP-xylosyltransferase of rat ear cartilage.

UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase EC 2.4.2.26) initiates the formation of chondroitin sulfate in the course of proteoglycan biosynthesis. The enzyme catalyzes the transfer of D-xylose from UDP-D-xylose to specific serine residues in the core protein. A procedure for purification of xylosyltransferase from rat ear cartilage was developed which includes ammonium sulfate fractionation, chromatography on heparin-agarose, on Sephacryl S300 and finally a substrate affinity chromatography applying the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G. The specific activity of the purified enzyme was about 420 mU per mg protein. The purification factor was about 26.000 with 27% yield. In SDS-polyacrylamide gel electrophoresis, the highly purified enzyme is homogeneous and yields only a single distinct band of 78 kDa. An apparent molecular mass of 71 kDa was determined for the native enzyme. These data suggest a monomeric structure for the enzyme. Xylosyltransferase activity was found to depend essentially on the presence of divalent metal ions. The K(m) value for UDP-D-xylose was determined to 6.5 micromol/l and for the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G as xylose acceptor to 8 micromol/l.

Role of mg2+ in chromomycin a3 - DNA interaction: a molecular modeling study.

Chromomycin A(3) (CHR) is an antitumor antibiotic that inhibits macromolecular biosynthesis by reversibly binding to double stranded DNA via the minor groove, with GC-base specificity. At and above physiological pH when CHR is anionic, interaction of CHR with DNA requires the presence of divalent metal ions like Mg(2+). However, at acidic pHthe molecule is neutral and it binds DNA even in absence of Mg(2+). Molecular dynamics simulation studies at 300K of neutral CHR and 1:1 CHR:Mg(2+) complexes formed at pH 5.2 and 8.0 show that hydrophobicity of CHR:Mg(2+) complex formed with the neutral drug is greater than that of the two other species. Interactions of CHR with DNA in presence and absence of Mg(2+) have been studied by simulated annealing to understand the role of Mg(2+) in the DNA binding potential of CHR. This shows that the antibiotic has the structural potential to bind to DNA even in the absence of metal ion. Evaluation of the direct interaction energy between the ligand and DNA does not explain the observed GC-base specificity of the antibiotic. When energy contributions from structural alteration of the interacting ligand and DNA as a sequel to complex formation are taken into account, atrue picture of the theoretical binding propensity emerges. This implies that DNA and/or the ligand undergo significant structural alterations during the process of association, particularly in presence of Mg(2+). Accessible surface area calculations give idea about the entropy contribution to the binding free energy which is found to be different depending upon the presence and absence of Mg(2+).