Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.     

Quantitative estimation of 3'5' cyclic AMP phosphodiesterase using anion exchange resin in a batch process.

An improved method for the preparation of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) is described which includes: enzymes from Chlorella for PAPS synthesis; conversion of ATP to AMP after PAPS formation with hexokinase (EC 2.7.1.1) and myokinase (EC 2.7.4.3); and separation of PAPS on DEAE-Sephadex using triethylammonium bicarbonate buffers. Any specific activity can be obtained by using appropriate concentrations of carrier-free ³⁵S and nonradioactive sulfate in the incubations. Between 300 and 2000 μmol of PAPS per batch can be obtained depending on the scale of the preparation. The PAPS is over 95% pure radiochemically and shows only one ultraviolet-absorbing spot on paper electrophoresis at pH 5.8. Adenosine 5′-phosphosulfate (APS) is prepared by incubating PAPS with a 3′-nucleotidase (EC 3.1.3.6) from rye grass. Quantitative conversion of PAPS to APS is obtained, and the APS is purified by column chromatography in the same manner as for PAPS. The APS obtained is better than 95% pure radiochemically and shows only one uv-absorbing spot on paper electrophoresis at pH 5.8.     

Constituents of Plant Leaf Wax Contained in Rice Callus Tissues

Two enzyme preparations having both nuclease and 3′-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography.

The enzyme activities for RNA, native DNA, heat-denatured DNA and 3′-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymes were shown to be endonucleases (EC 3.1.30.2) which liberated 5′-mononucleotides and oligonucleotides from both RNA and DNA with the following relative rate of hydrolysis: RNA > native DNA = heat-denatured DNA.     

Biosynthesis of Riboflavin Glycosides by Plant Grains

An enzyme preparation from glutinous millet grains has been found to synthesize various riboflavin glycosides from riboflavin and disaccharides other than maltose (such as cellobiose, melibiose and lactose). Each of these riboflavin glycosides has been isolated in crystalline form and shown to have the structure, 5′-D-riboflavin-β-d-glucopyranoside, 5′-d-riboflavin-α-d-galactopyranoside and 5′-d-riboflavin-β-D-galactopyranoside.     

Synthesis and spectral studies of 2-alkoxystearic acids — I

A series of 2-alkoxystearic acids was synthesized during dehydrohalogenation of the 2-iodostearic acid with potassium hydroxide in different alcohols. Besides the formation of trans-2-enoic- and 2-hydroxyacids, the reaction provides a convenient method for the preparation of long-chain 2-alkoxyacids in high yield. With (?)-2-butanol and (?)-2-methylbutanol the formation of mixtures of diastereoisomeric 2,1′-methylpropoxy- and 2,2′-methylbutoxystearic acids is evident by gas chromatographic analysis of their methyl esters. The preparation of 2-alkoxystearic acids (I–XI), their separation by column chromatography and structure determination by elementary analysis, thin-layer chromatography (TLC), infrared (IR) and proton magnetic resonance spectroscopy as well as gas chromatography/mass spectrometry are described.     

Amplified luminometric assays of alkaline phosphatase using riboflavin phosphates

An asasy for alkaline phosphatase is described which is based on the hydrolysis of riboflavin phosphates (5′FMN or 4′FMN) to produce riboflavin. This is converted to 5′FMN using riboflavin kinase, and then asayed using the bacterial bioluminescent system from Vibrio harveyi or V. Fischeri. The most sensitive assay is obtained using 4′FMN, which can measure less than 20 amol after a 1-hour incubation.     

链霉菌FIM-080014产生的核苷类代谢产物研究

本文采用大孔吸附树脂、硅胶柱色谱、反相柱色谱、凝胶sephadex LH-20及HPLC等方法对链霉菌FIM-080014发酵液及菌丝体中的代谢产物进行分离,得到7个核苷类化合物。通过NMR及MS等方法鉴定了上述化合物的结构,分别为尿嘧啶(1)、尿嘧啶核苷(2)、2'-脱氧尿嘧啶核苷(3)、5'-C-甲基尿嘧啶核苷(4)、2'-脱氧胸腺嘧啶核苷(5)、2'-脱氧鸟嘌呤核苷(6)和次黄嘌呤(7),其中化合物4首次从微生物代谢产物中分离得到。活性分析表明化合物1～7对神经氨酸酶具有一定的抑制活性,其IC50值分别为3.7、2.1、4.4、1.4、3.6、2.5和2.4 mM。     

Determination of riboflavin by high-performance liquid chromatography with riboflavin-depleted urine as calibration and control matrix

A simple method, exposure to natural-light, was developed to remove riboflavin from urine to enhance its use as the biological matrix for the preparation of calibration and control samples. Riboflavin-depleted urine containing less than 1 ng/ml of riboflavin was used to validate a high-performance liquid chromatography with fluorescence detection method for the determination of urinary riboflavin. The linearity of the assay (r2=0.999) was acceptable over the range of 10-5000 ng/ml. The intra-assay and inter-assay CVs were 3.3% and 9%, respectively. Subsequent stability studies found that urine riboflavin was stable for up to 6 months at 4 or -20 degrees C.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Flavin affinity chromatography: General methods for purification of proteins that bind riboflavin

Analogs of riboflavin that were altered at positions N(3), 8α, and N(10) of the 7,8-dimethylisoalloxazine ring were immobilized by covalent attachment to aminoalkylated agarose and polyacrylamide beads. These materials were used for affinity chromatographic purification of the riboflavin-carrier protein from egg white, egg yolk, and blood from laying hens, of flavokinase from rat liver, and of partially purified flavodoxin from Azotobacter vinelandii (FMN). The apo-carrier protein, which tightly complexes riboflavin (K_d ≈ 2 nm), was bound by the N(3)-, 8α-, and N(10)-flavinyl beads and was selectively displaced in moderate to high yield by 10 μm riboflavin or 1 m NaCl at pH 3.5. Flavokinase, which complexes less tightly with riboflavin (K_m ≈ 12 μm), was bound by the 8α- and N(10)-flavinyl beads. Binding to the latter was sufficiently tight that the addition of riboflavin was needed to displace flavokinase from the beads. The A. vinelandii flavodoxin, which normally complexes riboflavin 5′-phosphate (K₃ ≈ 5 nm) but less avidly complexes riboflavin (K_d ≈ 0.6 μm), was bound by the N(10)-flavinyl beads and eluted in low yield upon addition of FMN; most of the apoprotein denatured on the column despite the inclusion of thiol-protecting reagents. These flavin affinity materials may be generally useful for isolating a variety of other proteins that bind riboflavin.     

Preparation and properties of immobilized flavokinase.

Flavokinase (ATP: riboflavin 5'-phosphotransferase, EC 2.7.1.26) purified from rat liver by affinity chromatography, has been immobilized by amide linkage to omega-aminoalkyl-agarose beads. The immobilized enzyme differs from the soluble enzyme in having greater stability, slightly higher Km for the substrates, riboflavin and ATP, a broader pH optimum, and a lower energy of activation. These results suggest that the immobilized enzyme is influenced by the microenvironment of the bead and is subject to some degree of internal diffusional limitation. A small (3 ml), continuous, plug-flow reactor prepared with immobilized flavokinase effects 50% conversion of riboflavin to riboflavin 5'-phosphate (FMN) with a flow rate of 0.16 ml/min, which corresponds to an output of 5 nmol FMN/min. Immobilized flavokinase is effective for phosphorylating riboflavin and numerous riboflavin analogs and provides a facile method for preparing exclusively, unlike other synthetic methods, the 5'-phosphates.     

Identification of possible light emitters in the gills of a bioluminescent fungus Mycena chlorophos

The pileus of Mycena chlorophos actively, spontaneously, and continuously emits green light. Molecular mechanisms underlying this bioluminescence remain unclear. We investigated light emitters in the pileus of M. chlorophos to determine the underlying mechanisms. High‐performance liquid chromatography–fluorescence–photodiode array–mass detection analyses showed that actively luminescent gills in the pileus exclusively and abundantly possessed riboflavin, riboflavin 5′‐monophosphate, and flavin adenine dinucleotide as green‐fluorescent components. These components were localized in the bioluminescent region of the gills at the microscopic level. Fluorescence spectra of these green‐fluorescent components and the gills were identical with the spectrum of gill bioluminescence (maximum emission wavelength, 525 nm). Thus, our results indicated that the possible light emitters in the pileus of M. chlorophos were riboflavin, riboflavin 5′‐monophosphate, and/or flavin adenine dinucleotide. Copyright © 2016 John Wiley & Sons, Ltd.     

Fluorometric assay of pyridoxal and pyridoxal 5'-phosphate.

A new and very sensitive fluorometric method for the determination of pyridoxal and pyridoxal 5′-phosphate is reported. The specificity is based on the reductive amination of pyridoxal and its 5′-phosphate with methyl anthranilate and sodium cyanoborohydride at pH 4,5 to 5,0. Separation of the highly fluorescent methyl-N-pyridoxyl anthranilate was achieved by a combination of column and thin-layer chromatography on silica gel. This method has been applied to the assay of pyridoxal and pyridoxal 5′-phosphate in seruum.     

Localization of the cyclic adenosine 3' : 5' -monophosphate phosphodiesterase activator protein in rat heart.

A cyclic adenosine 3′ : 5′ — monophosphate phosphodiesterase activator protein has been partially purified from rat heart by a procedure involving ammonium sulfate fractionation and affinity column chromatography with cyclic AMP phosphodiesterase bound to Sepharose 4B. Freezing and thawing of the rat heart was essential for solubilization of the activator protein in the crude homogenate. Activator activity was localized on sarcoplasmic reticulum isolated from fresh heart which could be solubilized with a low yield that resulted in a labile product. Maximal activation of cyclic AMP phosphodiesterase with excess protein activator was 100%.     

Guanosine 3′:5′-cyclic monophosphate changes during germination of Hordeum vulgare

Guanosine 3′: 5′-cyclic monophosphate (cGMP) isolated from barley seeds and seedlings was purified using neutral alumina and anion-exchange column chromatography, then descending paper chromatography, and finally estimated by means of radioimmunoassay. The putative compound was identified on cellulose chromatography in three solvent systems.During the early phase of the germination, the cGMP content decreased steadily from 30 fmol/g of dry seeds to undetectable amounts in seeds after 18 h of germination. The process of seedling growth was associated with the increase of cGMP concentration. Nine-day-old seedlings contained 147 and 200 fmol/g of fresh weight in the roots and in the coleoptiles plus leaves, respectively.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]

Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.     

Studies on Metabolic Pathway of NAD in Yeast Cells

A new method for preparing NMN (nicotinamide mononucleotide) by the use of yeast 5′-nucleotidase is presented. After hydrolysis of NAD into NMN, adenosine and P_i by yeast 5′-nucleotidase which is a single protein having nucleotide pyrophosphatase activity, NMN in the hydrolysate of NAD was purified on active carbon and subsequently on Amberlite IRC-50.

In the typical experiment, 0.74 g of NMN (88% purity) was obtained from 2g of NAD preparation, giving 76% recovery on the basis of the theoretical value.

The NMN preparation was identified as NMN by IR spectra, UV spectra, paper chromatography, and also by component analysis.     

Large scale synthesis of dinucleoside monophosphates catalyzed by ribonuclease from Aspergillus clavatus

A gram scale enzymatic synthesis of eight, dinucleoside monophosphates (ApC, ApU, CpC, CpU, GpC, GpU, UpC, and UpU) is described. The synthesis involves a reaction between the appropriate ribonucleoside-2′,3′-cyclie phosphates and cytidine or uridine in the presence of ribonuelease from Aspergillus clavatus at 30°C. The enzyme is removed from the reaction mixture by chromatography on Bio-Gel P–4, and the dinucleoside monophosphate is further purified by chromatography on a DEAE-Sephadex A–25, column. A procedure for the large scale preparation of the ribonuclease from Aspergillus clavatus is also described.     

飞机草地上部分化学成分及其抑菌活性研究

为探究飞机草地上部分化学成分及其抑菌作用。采用正相硅胶、Sephadex LH-20等色谱方法,从飞机草乙酸乙酯提取物中得到10个化合物,根据1H NMR、13 C NMR、MS波谱数据,分别鉴定为:5,6,7,4′-四甲氧基黄烷酮(1)、圣草素-7,4′-二甲醚(2)、5,7-二羟基-6,4′-二甲氧基黄酮(3)、二氢山柰素(4)、异野樱素(5)、4′-羟基-5,6,7-三甲氧基黄烷酮(6)、5,6,7,3′,4′-五甲氧基黄烷酮(7)、3′-羟基-5,7,8,4′-四甲氧基黄烷酮(8)、香叶木素(9)、山柰酚(10),其中,化合物2和8均首次从飞机草中分离得到。采用生长速率法测定了飞机草乙醇提取物、不同萃取物、乙酸乙酯萃取物柱层析不同馏分对4种核桃病原真菌的抑制活性,确定其活性成分集中于馏分2(石油醚∶丙酮=5∶1),选择该部分所得化合物4、5、7对4种病原菌进行毒力测定,结果表明,化合物5对拟茎点霉菌丝生长抑制作用最强,EC 50为0.1062 mg/mL。研究结果为利用飞机草开发植物源农药提供理论依据。