Separation and partial characterization of isolectins with different subunit compositions from Datura stramonium seeds

The lectin from Datura stramonium seeds was separated into three individual isolectins by hydrophobic-interaction chromatography on phenyl-Sepharose. Two of these isolectins are homodimers made up of two A- or two B-subunits, whereas the third is a heterodimer composed of one A- and one B-subunit. Analysis of the homodimeric AA- and BB-isolectins revealed that the A- and B-subunits have similar but not identical M_r values (32 000 and 28 000, respectively), amino acid and carbohydrate compositions. The A-subunit has a higher affinity for N-acetyl-D-glucosamine oligomers than the B-subunit, whereas the latter is more specific for the carbohydrate determinants of some animal glycoproteins such as fetuin, asialofetuin and ovomucoid.     

6′-substituted and 6′,6″-disubstituted derivatives of raffinose

The reaction of 6′-chloro-6′-deoxyraffinose deca-acetate with a variety of nucleophilic anions (Br^-,I^-,N^-₃) gave the corresponding 6′-substituted raffinoses. The 6′-azide was further converted into 6′-amino-6′-deoxyraffinose, isolated as its N-acetyl derivative, and silver fluoride-induced elimination of hydrogen iodide from the 6′-iodide gave the 6′-deoxy-5′-ene. Treatment of 6′-chloro-6′-deoxyraffinose and 6′,6″-dichloro-6′,6″-dideoxyraffinose with base afforded, respectively, 3′,6′-anhydro-and 3′,6′;3″,6″-dianhydro-raffinose in high yields. In addition, the dichloride was converted into 6′,6″-diazido-6′,6″-dideoxyraffinose.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.     

N-(Phenoxyalkyl)amides as MT1 and MT2 ligands: Antioxidant properties and inhibition of Ca2+/CaM-dependent kinase II

Recently a series of chiral N-(phenoxyalkyl)amides have been reported as potent MT₁ and MT₂ melatonergic ligands. Some of these compounds were selected and tested for their antioxidant properties by measuring their reducing effect against oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) in the DCFH-diacetate (DCFH-DA) assay. Among the tested compounds, N-[2-(3-methoxyphenoxy)propyl]butanamide displayed potent antioxidant activity that was stereoselective, the (R)-enantiomer performing as the eutomer. This compound displayed strong cytoprotective activity against H₂O₂-induced cytotoxicity resulting slightly more active than melatonin, and performed as Ca²⁺/calmodulin-dependent kinase II (CaMKII) inhibitor, too.     

Fast protein liquid chromatographic purification and some properties of a partially O-methylated flavonol glucoside 2′-/5′-O-methyltransferase

A flavonol O-methyltransferase was partially purified from Chrysosplenium americanum by fractional precipitation with ammonium sulphate followed by gel filtration and chromatofocusing using an FPLC system. The enzyme which was purified 420-fold catalysed the transfer of the methyl group of SAM to the 2′- or 5′-positions of partially methylated flavonol glucosides, the two terminal methylation steps in the biosynthesis of Chrysosplenium flavonoids. The enzyme had a pH optimum of 7 in Pi buffer, a pI of < 5, an M, of 57 000, no Mg²⁺ requirement and was inhibited by both N-ethylmaleimide and phenylmercuriacetate. The K_m value for the flavonol substrate was 2 μM and that for SAM was 100 μM. The role of this enzyme is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.     

A New Lectin from Meadow Saffron (Colchicum automnale)

A lectin has been isolated from tubers of the meadow saffron (Colchicum autumnale). It is an octameric protein (M_r 100,000) composed of 4A- and 4B-subunits of M_r 15,000 and 10,000, respectively. It is a glycoprotein with 4.4% carbohydrate, the main sugars are (N-acetyl-) glucosamine, mannose, fucose, and xylose. Although the Colchicum autumnale agglutinin (CAA) agglutinates human red blood cells, it has a much higher activity with rabbit erythrocytes. With respect to its carbohydrate-binding specificity CAA behaves rather unusually as it is inhibited by lactose, galactose, N-acetylgalactosamine and related sugars when assayed with human red blood cells but not in assays with rabbit erythrocytes.     

Comparison of cytokinin activities of the base,ribonucleoside and 5′- and cyclic-3′,5′-monophosphate ribonucleotides of N6-isopentenyl-, N6-benzyl or 8-bromo-adenine

The cytokinin activities of adenosine 3′,5′-monophosphate, N⁶,O^2″-dibutyryladenosine 3′,5−'monophosphate, 8-bromoadenosine 3′,5′-monophosphate, N⁶-(Δ²-isopentenyl)adenosine 3′,5′-monophosphate, and N⁶-benzyladenosine 3′,5′-monophosphate were determined in the tobacco bioassay and compared with the activities of the corresponding non-cyclic nucleotides, nucleosides and bases of the N⁶-isopentenyl-substituted, N⁶-benzyl-substituted, 8-bromo-substituted, and unsubstituted adenine series. In each of these series the cytokinin activities in decreasing order were: bases ⪢ nucleosides ⪖ nucleotides > cyclic nucleotides. All members of the N⁶-isopentenyl- substituted and N⁶-benzyl-substituted series were highly active cytokinins, reaching maximum activity at concentrations of 1 μM or less, whereas, as expected, all members of the unmodified adenine series were inactive in the tested concentration ranges of up to 180 and 200 μM for adenosine and adenine, and 40 μM for the adenine nucleotides. Members of the 8-bromo-substituted adenine series were much weaker cytokinins than the N⁶-substituted adenine derivatives but showed activity in the same sequence starting at a concentration of about 5 μM. Thus, in the cases of 8-bromoadenosine 3′,5′-monophosphate and N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate, both of which have been reported to promote cell division and growth of plant tissues, the cytokinin activity is related to the 8-bromo substituent and to the N⁶-butyryl substituent, respectively, rather than to the 3′,5′-cyclic monophosphate moiety.     

Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress

The dynamic modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked β-N-acetyl-d-glucosamine (O-GlcNAc) has been shown to regulate over 3000 proteins in a manner analogous to protein phosphorylation. O-GlcNAcylation regulates the cellular stress response and the cell cycle, and is implicated in the etiology of neurodegeneration, type II diabetes, and cancer. The antibody CTD110.6 is often used to detect changes in the O-GlcNAc modification. Recently, it has been demonstrated that CTD110.6 recognizes N-linked N,N′-diacetylchitobiose, which is thought to accumulate in cells experiencing severe glucose deprivation. In this study, we have addressed two questions: (1) Which other antibodies used to detect O-GlcNAc cross-react with N-linked N,N′-diacetylchitobiose? (2) Does N-linked N,N′-diacetylchitobiose accumulate in response to other cellular stressors? To delineate between O-GlcNAc and N-linked N,N′-diacetylchitobiose, we developed a workflow that has been used to confirm the specificity of a variety of O-GlcNAc-specific antibodies. Using this workflow we demonstrated that heat shock, osmotic stress, endoplasmic reticulum stress, oxidative stress, DNA damage, proteasomal inhibition, and ATP depletion induce O-GlcNAcylation but not N-linked N,N′-diacetylchitobiose. Moreover, we demonstrated that while glucose deprivation results in an induction in both O-GlcNAc and N-linked N,N′-diacetylchitobiose, the induction of N-linked N,N′-diacetylchitobiose is exacerbated by the removal of fetal bovine serum.     

Dibutyryl cyclic adenosine monophosphate enhancement of α-methyl-d-glucoside accumulation by kidney cortex slices

Cyclic adenosine 3′,5′-monophosphate and N⁶-2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate increase the accumulation of α-methyl-d-glucoside by cortical slices from rat, rabbit, dog and human kidney. The characteristics of the effect have been studied in rat tissue. At least 90 min of exposure of the tissue to cyclic nucleotide prior to onset of glucoside accumulation is required as well as presence of the cyclic nucleotide during the accumulation phase. Inhibition of protein synthesis does not abolish the effect of N⁶-2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate. The cyclic nucleotide causes an increase in the initial entry rate of α-methyl-d-glucoside into cells and an increase in the intracellular steady state concentration. The cyclic nucleotide does not affect the apparent K_m of the glucoside entry process but increases the maximum velocity of accumulation.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling

The conjugation of 4-N-(3-aminopropanyl)-2′-deoxy-2′,2′-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1?M Na₂CO₃ buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl₃) (0.6?N AcONa/H₂O, pH?=?9.3) over 15?min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [⁶⁸Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%–96%) with the radioligand almost exclusively found in the aqueous layer (～95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the ⁶⁸Ga-labelled radioligand in BALB/c mice.     

Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon tetrachloride-induced hepatic fibrosis.

The lectin from Euonymus europeus seeds was purified by adsorption onto insoluble polyleucyl hog A + H blood group substance and subsequent elution with lactose. The isolated lectin formed three lines in immunoelectrophoresis against rabbit antisera to the crude seed extract and showed three components on electrophoresis in acrylamide gel at pH 9.4. In analytical isoelectric focusing the purified lectin had six closely spaced bands with pI from 4.3 to 4.7. It sedimented as two peaks: a big symmetrical peak with s_20,w⁰ of 7.8 and another small, diffuse moving peak. The intrinsic viscosity was 0.057 dl/g and the M_r calculated from the sedimentation coefficients, intrinsic viscosity, and V? of 0.71 was about 166,000. In sodium dodecyl sulfate, it gives subunits of M_r 17,000 and 35,000; 20% of the 35,000 subunit resists reduction by dithiothreitol in 7 m guanidine-HCl. The Euonymus lectin is a glycoprotein containing 4.8% d-galactose, 2.9% d-glucose, and 2.8% N-acetyl-d-glucosamine. The purified lectin precipitated well with B and H blood group substances and with the P1 fraction of blood group B substance but not with A₁ substances. It precipitated poorly with Le^a and Le^b and precursor I blood group substances. Inhibition of precipitation with milk and blood group oligosaccharides showed the lectin to be most specific for blood group B oligosaccharides having the structure: dGalα1 → 3[lFucα1 → 2]dGalβ1 → 3 or 4dGlcNAcβ→. It is also inhibited by blood group H oligosaccharides but to a lesser degree. For 50% inhibition of precipitation, 3.5, 850, and 290,000 nmol of B and H oligosaccharides and lactose, respectively are required. The B and H specificities are an intrinsic property of a single lectin site since absorption and elution from an H immunoadsorbent gave material with B as well as H specificity. Millipore-filtered crude extracts of Euonymus europeus preserved with 0.02% sodium azide are stable in the refrigerator for many months and can be used for quantitative precipitin and for quantitative inhibition assays, results being the same as with purified lectin.     

Terminal N-Acetylgalactosamine-Specific Leguminous Lectin from Wisteria japonica as a Probe for Human Lung Squamous Cell Carcinoma

Millettia japonica was recently reclassified into the genus Wisteria japonica based on chloroplast and nuclear DNA sequences. Because the seed of Wisteria floribunda expresses leguminous lectins with unique N-acetylgalactosamine-binding specificity, we purified lectin from Wisteria japonica seeds using ion exchange and gel filtration chromatography. Glycan microarray analysis demonstrated that unlike Wisteria floribunda and Wisteria brachybotrys lectins, which bind to both terminal N-acetylgalactosamine and galactose residues, Wisteria japonica lectin (WJA) specifically bound to both α- and β-linked terminal N-acetylgalactosamine, but not galactose residues on oligosaccharides and glycoproteins. Further, frontal affinity chromatography using more than 100 2-aminopyridine-labeled and p-nitrophenyl-derivatized oligosaccharides demonstrated that the ligands with the highest affinity for Wisteria japonica lectin were GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc, with K _a values of 9.5 × 10⁴ and 1.4 × 10⁵ M^-1, respectively. In addition, when binding was assessed in a variety of cell lines, Wisteria japonica lectin bound specifically to EBC-1 and HEK293 cells while other Wisteria lectins bound equally to all of the cell lines tested. Wisteria japonica lectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence of N-acetylgalactosamine, but not galactose, mannose, or N-acetylglucosamine, and was completely abrogated by β-hexosaminidase-digestion of these cells. These results clearly demonstrate that Wisteria japonica lectin binds to terminal N-acetylgalactosamine but not galactose. In addition, histochemical analysis of human squamous cell carcinoma tissue sections demonstrated that Wisteria japonica lectin specifically bound to differentiated cancer tissues but not normal tissue. This novel binding characteristic of Wisteria japonica lectin has the potential to become a powerful tool for clinical applications.     

The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes

The synthesis and degradation of 5′-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24–36 h in culture. After a 30 h lag period 5′-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5′-nucleotidase in the cultured hepatocytes using [³⁵S]methionine showed that the 5′-nucleotidase monomer was synthesised as an M_r 67 000 form which was converted to the mature M_r 72 000 form. [³⁵S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an M_r 57 000 form. The immature M_r 67 000 form of 5′-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5′-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.     

Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine,N,N′-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N′-diacetylchitobiose [(GlcNAc)₂] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.     

Metabolism of kaurenoids by Gibberella fujikuroi in the presence of the plant growth retardant,N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide

The plant growth retardant, N,N,N-trimethyl-1-methyl-(2′,6′,6′-trimethylcyclohex-2′-en-1′-yl)prop-2-enylammonium iodide, is shown to block gibberellin biosynthesis in Gibberella fujikuroi between mevalonate and ent-kaur-16-ene, probably by inhibiting ent-kaur-16-ene synthetase A-activity. In the presence of the plant growth retardant, cultures of the fungus incorporate (26.5%) added ent-[¹⁴C]-kaur-16-ene into gibberellin A₃. Under the same conditions kaur-16-ene, 13β-kaur-16-ene, and ent-kaur-15-ene are not metabolised to gibberellin analogues.