Identification of an N-Glucoside of cis-Zeatin from Potato Tuber Sprouts

A compound was isolated from potato (Solanum tuberosum L. cv Bintje) tuber sprouts by immunoaffinity chromatography with antibodies against the cytokinins zeatin riboside and isopentenyladenosine. Analysis by ultraviolet spectroscopy and gas chromatography-mass spectrometry of derivatives identified the compound as a 9-glucoside of 6-[(Z)-4-hydroxy-3-methyl-2-butenylamino]purine (cis-zeatin). N-glucosides have often been reported as metabolites of other cytokinins, but to our knowledge, they have never before been found for cis-zeatin. The finding gives proof that cis-zeatin, a modified base in tRNA, also exists as a free substance in plants, since the glucoside, unlike other tRNA-free cis-zeatins described earlier by others, cannot arise by enzymatic degradation of tRNA during plant extraction.     

O-glucosylation of cis-zeatin in maize. Characterization of genes,enzymes, and endogenous cytokinins

trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize (Zea mays) gene (cisZOG1) encoding an O-glucosyltransferase specific to cis-zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922-5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression of cisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1 at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K(m) values of 46 and 96 microM, respectively, for cis-zeatin and a pH optimum of 7.5. Other cytokinins, including N(6)-(delta(2)-isopentenyl)adenine, trans-zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and their O-glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.     

N-acetyl-beta-D-hexosaminidase A from rat urine: partial purification and characterization

N-Acetyl-beta-D-hexosaminidase A was purified from rat urine by ion-exchange chromatography on DEAE-cellulose, followed by concanavalin A chromatography, and finally by chromatography on 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-glucosylamine-Se pharose 4B. The enzyme was purified 482-fold with a yield of about 7%. The optimal pH was 4.5 for N-acetyl-glucosaminidase activity and 4.0-4.5 for N-acetylgalactosaminidase activity. The enzyme was heat-labile and stable from pH 4.5 to pH 7.0 but it was very unstable at lower pH values. Km values were 0.55 mM and 0.059 mM, respectively. The glycoprotein nature of the enzyme was deduced from its behavior on concanavalin A. The effect of some carbohydrates and ionic compounds on the activities of the enzyme was studied. When N-acetyl-D-glucosaminolactone and N-acetyl-D-galactosaminolactone were used as inhibitors, Ki values were also calculated.     

Lectin-binding domains on laminin

Chicken erythrocytes have been found to have at least two kinds of phospholipase A2. The first is a soluble enzyme from the cytosole fraction and has no calcium sensitivity. The second can be extracted from the plasma membrane fraction with the nonionic detergent Triton X-100. In this study the membrane-bound enzyme was partially purified by affinity chromatography on phosphatidylcholine-Sepharose, and its specific activity was increased 1100-fold compared with that of the cell homogenate without nuclei. It has an optimum pH of 8.5 and required calcium for maximum activity. It showed the specificity for both phosphatidylcholine and phosphatidylethanolamine, but reacted preferentially on the former substrate. Analysis by concanavalin A-Sepharose affinity chromatography revealed that the membrane-bound phospholipase A2 was retained on the resin and could be eluted specifically with a haptenic sugar, methyl alpha-D-mannopyranoside. The enzyme seems to be either a concanavalin A-binding glycoprotein or a part of a complex with certain concanavalin A-binding glycoproteins.     

The interaction of N-acetylhexosaminidase with insolubilized concanavalin A.

The specific interaction between human N-acetylhexosaminidase and concanavalin A was evaluated with respect to temperature, time, pH and concentration of specific ligand in incubation mixtures containing the enzyme and insolubilized lectin. Elution of the enzyme from insolubilized concanavalin A is dependent on both temperature and concentration of alpha-methyl mannoside. Conditions for a high yield of the enzyme from chromatography on insolubilized concanavalin A are described.     

Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes.

The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Purification of normal human urinary N-acetyl-beta-hexosaminidase A by affinity chromatography.

N-Acetyl-beta-hexosaminidase A was purified 1000-fold from human urine by chromatography on DEAE-Sephadex followed by concanavalin A--Sepharose affinity chromatography. The optimal pH range was 4.4--4.5 for both the N-acetylglucosamine and N-acetylgalactosamine derivatives. The Km values were 0.51 mM and 0.28 mM respectively for the N-acetylglucosamine and N-acetylgalactosamine derivatives. The glycoprotein nature of the urinary enzyme was established by its affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the molecule.     

High-molecular-weight renin converting enzyme from dog kidney: detection and fractionation

Renal cortical high-molecular-weight renin (Mw:60,000) of the dog is a complex of renin (low-molecular-weight renin; Mw:40,000) and a renin binding protein. We detected an enzyme-like substance that catalyzes the conversion from high- into low-molecular-weight renin. When the renal cortical extract was added to the high-molecular-weight renin and the preparation incubated at 37 degrees C for 30 min, the high-molecular-weight renin was converted into the low-molecular-weight form. No such conversion occurred in the case of renal medullary extract. This converting substance was fractionated using concanavalin A Sepharose, 70% ammonium sulfate saturation and DEAE-cellulose chromatography. The converting activity was inhibited by potassium tetrathionate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). These events suggest that this substance is an enzyme possessing sulfhydryl moieties. However, a cathepsin B inhibitor leupeptin did not affect the activity. Accordingly, the high-molecular-weight renin converting enzyme, which is sensitive to sulfhydryl oxidation, may explain the mechanism of interconversion between high- and low-molecular-weight renin involving the oxidation-reduction of tissue sulfhydryl groups.     

Reversibility of monensin inhibition of oligosaccharide processing of human fibronectin

Monensin impairs oligosaccharide processing in fibronectin primarily by inhibiting the conversion of oligosaccharides from the high mannose type to the complex type. The separate effects of monensin and cations on alpha-mannosidase activity in fibroblasts were examined using an in vitro assay system. The results indicated that monensin did not directly inhibit alpha-mannosidase activity in vitro, although prior treatment of fibroblasts with monensin caused an irreversible suppression of enzyme activity. The reversibility of monensin action on oligosaccharide processing was also examined. Analyses using concanavalin A (ConA) Sepharose affinity chromatography showed that the inhibitory action of monensin on oligosaccharide processing was biologically reversible. A progressive return to complex type oligosaccharides began about 11 h after the removal of the monensin. These composite results indicate that the reversibility of monensin action on oligosaccharide processing in fibronectin may be attributed to the restoration of enzyme activity, although the mechanism by which restoration occurs remains to be deciphered.     

Isolation and properties of a glycohydrolase specific for nicotinamide mononucleotide from Azotobacter vinelandii.

A glycohydrolase that catalyzes the irreversible conversion of NMN to nicotinamide and ribose 5-phosphate has been partially purified from a sonic extract of Azotobacter vinelandii. The enzyme is highly specific for NMN. NAD, NADP, nicotinic acid-adenine dinucleotide, nicotinamide riboside and alpha-NMN are not significantly hydrolyzed by this enzyme, nor do they compete with NMN. The enzyme also exhibits an absolute dependence on guanylic acid derivatives with following order of relative effectiveness: GTP, guanosine 5'-tetraphosphate greater than dGTP, GDP, 2'-GMP, 3'-GMP greater than GMP, dGMP. A heat-resistant, nondialyzable factor which could replace the GTP requirement was found in the sonic extract. The Ka for GTP and the Km for NMN in the presence of GTP at 1mm were calculated to be 0.025 mM and 4.5 mM respectively. GMP, dGMP, and dCMP were found to be effective inhibitors of the enzyme when 1 mM GTP was also present. The kinetic data suggest that the binding site for these mononucleotides is distinct from the active site or the GTP binding site. The ability of this enzyme to cleave NMN is suggestive of a metabolic role of the enzyme in selective conversion of NMN to nicotinamide, which, in turn, would be re-utilized by the cell as a precursor of NAD via nicotinic acid.     

Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.     

Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin O-glucosyltransferase with position specificity related to receptor recognition

Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.     

Purification and properties of beta-N-acetylhexosaminidase A from pig brain

1. Adult pig brain beta-N-acetylhexosaminidase was separated into four different forms by ion exchange chromatography on diethylaminoethyl-cellulose. 2. Form A was purified 1300-1500 fold by an unusual procedure, the technique of ampholyte displacement, followed by chromatography on concanavalin A Sepharose. 3. The enzyme catalyses the hydrolysis of both beta-N-acetylglucosaminides and beta-N-acetylgalactosaminides. 4. The kinetic studies support the evidence of the association of both activities to a single protein, and at the same active site. 5. A natural substrate, N,N'-diacetylchitobiose, is also hydrolyzed, but not ovalbumin. 6. This enzyme may be considered as an exoglycosidase.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Mass spectrometry and chromatography of t-butyldimethylsilyl derivatives of cytokinin bases

Di-(t-butyldimethylsilyl) derivatives of the cytokinin bases zeatin, cis-zeatin, and dihydrozeatin may be prepared quantitatively in the presence of dimethylaminopyridine. These derivatives have good gas chromatographic properties and are very suitable for gas chromatography-mass spectrometry analysis of cytokinin bases. The t-butyldimethylsilyl (tBuDMS) group at N-9 may be selectively hydrolyzed and the resulting mono-O-silyl derivatives are sufficiently stable to be subjected to thin-layer chromatography and high-performance liquid chromatography. The mass spectral fragmentation of the mono- and di-tBuDMS derivatives of adenine, zeatin, cis-zeatin, and dihydrozeatin and also of the mono-tBuDMS derivatives of N6-isopentenyladenine and 6-benzylaminopurine have been rationalized. The 9-tBuDMS moiety was characterized by an elimination of isobutene (M-56) and of isobutene plus a methyl radical (M-56-15).     

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.     

A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.     

Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to cis-zeatin

Genes for cytokinin-responsive His-protein kinases (ZmHK1, ZmHK2, and ZmHK3a) were isolated from maize (Zea mays). Heterologous expression of each of the ZmHKs in Escherichia coli having the DeltarcsC and cpslacZ genetic background conferred cytokinin-inducibility of lacZ expression on the bacteria. In the recombinant E. coli system, ZmHK1 and ZmHK3a were more sensitive to free-base cytokinins than to the corresponding nucleosides; isopentenyladenine was most effective for ZmHK1, while ZmHK2 tended to be most sensitive to trans-zeatin and the riboside. In contrast to a known cytokinin receptor of Arabidopsis (AHK4/CRE1/WOL), all ZmHKs responded to cis-zeatin (cZ), which generally is believed to be inactive or only weakly active. In cultured maize cells, expression of ZmRR1, a cytokinin-inducible response regulator, was induced by cZ as well as by trans-zeatin. These results strongly suggest that maize cytokinin receptors differ in ligand preference, and that cZ is an active cytokinin at least in maize.     

PARTIAL PURIFICATION, PROPERTIES AND GLYCOPROTEIN NATURE OF ARYLSULPHATASE B FROM SHEEP BRAIN

Abstract— A simple method has been developed for the partial purification of arylsulphatase B from sheep brain. This includes concanavalin A-Sepharose affinity chromatography and ionic strength-dependent binding and dissociation of the enzyme with Dextran Blue; by these methods the enzyme was purified 1344-fold with 10% recovery. The partially purified enzyme was shown to be a glycoprotein and its kinetic properties were compared with that of purified arylsulphatase A from the same source.