2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Binds to Actin-Based Cytoskeletal Elements in an Isoprenylation-Independent Manner

Abstract: 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton.     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

Polarized Raman spectrum of single crystal uridylyl (3′–5′) adenosine: Local Raman tensors of some functional groups

Polarized Raman scattering measurements have been made of a single crystal of uridylyl(3′–5′)adenosine (UpA) by the use of a Raman microscope with 488.0 nm excitation. The UpA crystal belongs to space group P2₁ (monoclinic), and Raman intensities I_aa, I_bb, and I_c′c′, have been determined for each Raman band. These intensities correspond to the aa, bb, and c′c′ components of the crystal Raman tensor, where c′ is defined as an axis perpendicular to the crystallographic a axis in the ac plane. From these experimental data, and by taking the known crystal structure into account, anisotropic and isotropic molecular Raman tensors have been calculated for the following 11 normal modes: ring stretching modes of the adenine residue (protonated) at 1560, 1516, 1330, and 715 cm⁻¹; ring stretching modes of the uracil residue at 1696, 1657, 1615, 1228, and 790 cm⁻¹; PO⁻₂ symmetric stretching mode at 1080 cm⁻¹; P(—)O single bond stretching mode at 801 cm₋₁. These pieces of information of the Raman tensors are considered to be useful for estimating the orientations of the DNA and RNA strands in a biological complex from a polarized Raman spectroscopic measurement of such a complex. © 1998 John Wiley & Sons, Inc. Biopoly 45: 135–147, 1998     

Evidence for the Association of 2',3'-Cyclic-Nucleotide 3'-Phosphodiesterase with Myelin-Related Membranes in Peripheral Nervous System

Abstract: In PNS, the specific activity of 2′,3′-cyclic nucleotide 3′-phospho–diesterase (CNP) in myelin was not enriched over the starting homogenate. Nevertheless, most of the total activity was recovered in myelin. In myelin-deficient mutants, low CNP activities were measured in sciatic nerves. CNP specific activities were similar in myelinated and non-myelinated nerves but in non-nervous tissues, they were significantly lower than in nervous tissue. There was no indication for the presence of an isoenzyme of CNP in peripheral nerves. These results indicate that CNP is present in PNS myelin and preferentially localized in Schwann cell plasma membranes.     

Mutational tests of the NMR-docked structure of the staphylococcal nuclease–metal–3′,5′-pdTp complex

In the X-ray structure of the staphylococcal nuclease–Ca²⁺ ?3′,5′-pdTp complex, the conformation of the inhibitor 3′,5′-pdTp is distroteed Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3′,5′-pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR-docked structure, the 5′-phophate of 3′,5′-pdTp overlaps with that in the X-ray Structure. However the 3′-phosphate accepts a hydrogen bond from Lys-49 (2.89Å) rather than from Lys-84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 Å) which does not occur in the X-ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′-pdTp, Ca²⁺, and Mn²⁺ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca²⁺ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While thye K84A mutation did not significantly weaken 3′,5′-pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′-pdTp binding to the enzyme by the factor of 4.4 ± 1.8-fold. Similarly, the Y115A mutation weakened 3′,5′-pdTp binding to the enzyme 3.6 ± 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca²⁺-3′,5′-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3′,5′-pdTp than by the X-ray structure. © 1993 Wiley-Liss, Inc.     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Determination of 3′-azido-2′,3′-dideoxyuridine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

3′-Azido-2′,3′-dideoxyuridine (AZDU, Azddu, CS-87) is a nucleoside analog of 3′-azido-3′-deoxythymidine (zidovudine, AZT) that has been shown to inhibit human immunodeficiency virus (HIV-1). AZDU is a potential candidate for treatment of pregnant mothers to prevent prenatal transmission of HIV/AIDS to their unborn children. A rapid and efficient high-performance liquid chromatography (HPLC) method for the determination of AZDU concentrations in rat maternal plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in distilled water, protein precipitated and extracted using a C-18 solid-phase extraction (SPE) method prior to analysis. Plasma and amniotic fluid samples were protein precipitated with 2 M perchloric acid prior to analysis. Baseline resolution was achieved using a 4.5% acetonitrile in 40 mM sodium acetate (pH 7) buffer mobile phase for amniotic fluid, placenta and fetus samples and with a 5.5% acetonitrile in buffer solution for plasma at flow-rates of 2.0 ml/min. The HPLC system consists of a Hypersil ODS column (150×4.6 mm) with a Nova-Pak C-18 guard column with detection at 263 nm. The method yields retention times of 6.2 and 12.2 min for AZDU and AZT in plasma and 8.3 and 17.6 min for AZDU and AZT in amniotic fluid, fetal and placental tissues. Limits of detection ranged from 0.01 to 0.075 μg/ml. Recoveries ranged from 81 to 96% for AZDU and from 82 to 96% for AZT in the different matrices. Intra-day (n=6) and inter-day (n=9) precision (% RSD) and accuracy (% Error) ranged from 1.48 to 6.25% and from 0.50 to 10.07%, respectively.     

Dual mechanism of laminin modulation of ecto-5′-nucleotidase activity

The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′₁ and E₈ modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E₈, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′₁, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E₈ on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′₁. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

The anticarcinogen 3,3′-diindolylmethane is an inhibitor of cytochrome P-450

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3′-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with K_i values in the low micromolar range. We now report a similar potent inhibition by 3,3′-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3′-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B₁. There was no inhibition of cytochrome c reductase. In addition, we found 3,3′-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3′-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans. © 1995 John Wiley & Sons, Inc.     

Structural studies on KijD1, a sugar C‐3′‐methyltransferase

Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d ‐kijanose, and a tetrasaccharide chain composed of l ‐digitoxose units. d ‐Kijanose is a highly unusual nitro‐containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C‐3′‐methyltransferase using S‐adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S‐adenosylhomocysteine (SAH) and dTDP or SAH and dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐d ‐glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C‐3′‐methyltransferase isolated from Micromonospora chalcea. By the judicious use of site‐directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Elevated specific activity of 5′-nucleotidase in a spinal cord myelin fraction from shiverer mice comparison with other myelin-associated enzymes and myelin proteins

Meylin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5′-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2′,3′-cyclic nucleotide-3′-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiveres, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hhypothetically, the 5′-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.     

Studies on the Wolfgram High Molecular Weight CNS Myelin Proteins: Relationship to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.     

(1′R,2′S,5′R)-Menthyl-(5R)-acetoxy-1,3-oxathiolan-(2R)-carboxylate: Synthesis and isomeric purity determination by HPLC

Chemoselective reduction of one isomer of the 1-menthylester of 1,3-oxathiolan-5-one-2-carboxylic acid produces a mixture of four lactol diastereomers from which the title compound was isolated after acylation. The isomeric purity and absolute stereochemistry were determined by spectroscopic methods, chiral HPLC techniques, and conversion to (?)-2′-deoxy-3′-thiacytidine (Lamivudine, 3TC^TM). © 1994 Wiley-Liss, Inc.     

Cyclic AMP Induction of the Myelin Enzyme 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in Rat Oligodendrocytes

Cyclic AMP (cAMP) is known to induce the activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37) in C6 rat glioma cells. This report shows that CNP is also inducible in oligodendrocytes explanted from 1-day-old rat cerebrum and grown in tissue culture. Induction was observed after a 1-day treatment with 1 mM N6, O2-dibutyryl cyclic AMP (dbcAMP) and was maximal after 5 days, reaching 200-240% of control. Induction was observed both in mixed cerebral cell cultures containing oligodendrocytes and astrocytes, and in purified cultures of oligodendrocytes prepared by a differential shakeoff procedure. Addition of dbcAMP to the cultures 3-9 days after the cells were explanted from rat brain induced CNP activity, but no induction was observed when dbcAMP treatment was begun 13 or more days after explanation. These results demonstrate that one component of myelin, CNP, is inducible in oligodendrocytes by a cAMP-mediated mechanism, and suggest a role for cAMP in the regulation of the myelin-associated functions of oligodendrocytes.