Ion-exchange thin-layer chromatography and paper ionophoresis of dinucleoside monophosphates

The thin-layer chromatography of dinucleoside monophosphates on plates coated with DEAE-cellulose powder and on plates coated with cellulose impregnated with polyethyleneimine, together with their ionophoretic mobilities on DEAE-cellulose paper, is described. It is shown that the 2′→5′ dinucleoside monophosphates can be separated from the corresponding 3′→5′ isomers by ion-exchange thin-layer chromatography and paper ionophoresis. The method is very sensitive and can replace the commonly used enzymic hydrolysis for analyzing the nature of the phosphodiester linkage of a given dinucleoside monophosphate.     

Nucleotide sequence analysis with polynucleotide kinase and nucleotide `mapping'' methods. 5′-Terminal sequence of deoxyribonucleic acid from bacteriophages λ and 424

The polynucleotide kinase reaction was used in analyses of complex mixtures of oligodeoxynucleotides which were fractionated by various two-dimensional nucleotide ;mapping' procedures. Parallel ionophoretic analyses on DEAE-cellulose paper, pH2, and AE-cellulose paper, pH3.5, of venom phosphodiesterase partial digests of 5'-terminally labelled oligonucleotides enabled the sequence of the nucleotides to be deduced uniquely. A ;diagonal ionophoresis' method has been used with mixtures of nucleotides. Application of these methods to 5'-terminally labelled DNA from bacteriophage lambda gave the terminal sequences pA-G-G-T-C-G and pG-G-G-C-G. Identical 5'-terminal sequences were found with DNA from bacteriophage 424.     

Sequence of 6S RNA of <Emphasis Type="Italic">E. coli</Emphasis>

THE principal technical difficulty in sequencing uniformly ³²P-labelled RNA is the fractionation of the complicated mixture of products present in partial enzyme digests. We have recently improved the methods for the separation of oligonucleotides larger than decanucleotides by using thin-layer chromatography on DEAE-cellulose in conjunction with ionophoresis on cellulose acetate at pH 3.5 (ref. 1). We have used this technique to establish the nucleotide sequence of the 6S RNA of Escherichia coli.     

Simple method for the preparation of haptoglobin]

The present paper deals with a method permitting the isolation of haptoglobin 2-2 from human serum or plasma. The haptoglobin is adsorbed to DEAE-cellulose at pH 5.1 by batching. The loaded cellulose is given into a column and the haptoglobin eluted by a 0.1 M to 0.15 M acetate buffer. The last step is a gel-chromatography on Sephadex G-200. Disc- and immunoelectrophoresis were used to test the purity. As by-product acid alpha1-glycoprotein can be obtained.     

Rapid assays for adenylate cyclase and 3',5' cyclic AMP phosphodiesterase activities. Simultaneous measurements of other pathways of ATP catabolism

This paper deals with a rapid assay of adenylate cyclase activity and 3′,5′ cyclic AMP phosphodiesterase activity which permits simultaneous measurements of other pathways of ATP catabolism. The separation of all the adenylic nucleotides was obtained by an electrophoresis on cellulose acetate 15 min at 50 V/cm with a fluorescent buffer pH 8.6. Before electrophoresis, the incubation sample was added with a carrier solution of nucleotides to allow their localization under uv light, by fluorescence inhibition. Each fraction was cut and dissolved in Bray's liquid for scintillation counting. This assay method is rapid, reliable, and sensitive, it is suitable either for research or for routine and clinical purposes.     

A simple preparative method for the isolation of amylose and amylopectin from potato starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8,0.1 M) + urea (2 M) + 1-butanol (8.5%, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and beta -amylolysis limit.     

A Simple Preparative Method for the Isolation of Amylose and Amylopectin from Potato Starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8, 0.1 M) ± urea (2 M) ± 1-butanol (8.5 %, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and β -amylolysis limit.     

Bismuth chemistry and the glutaraldehyde insensitive staining reaction

The free metal ion, Bi3+, is the only chemical species of bismuth that stains a strongly bismuth-reactive molecule, polyarginine, in vitro. The bismuth solution specifically requires tartrate as a chelating agent for the reaction to occur between pH 7.4 and 8.0. Since Bi3+ reacts strongly with polyarginine, creatine and ATP fixed to cellulose acetate strips and DEAE-cellulose and P-cellulose, the free metal ion (Bi3+) may bind to phosphate or guanidyl groups, or both, after glutaraldehyde fixation.     

Some Observations on the Isolation and Estimation of the Soluble Nucleotides of Wheat Seedlings

Extracts of germinating wheat seedlings contain substances whichabsorb ultraviolet light strongly but are not nucleotides. Inparticular, two glucosides seriously interfere with the isolationand estimation of soluble nucleotides and these compounds arenot separated from the nucleotides by a charcoal adsorptionprocedure. A new extraction method is now described which avoidsthe use of charcoal. Freeze-dried tissue powders placed abovea column of cellulose powder are extracted first with methanol/5per cent, potassium acetate buffer pH 6.0 (90:10), then absoluteethanol, and finally with 30 per cent, ethanol containing aceticacid (0.01 M). The effluent containing the nucleotides is subjectedeither to dialysis or to ion-exchange resin absorption. Duringgermination soluble nucleotides in the endosperm remain at alow level, but in the embryo there is a thirtyfold increasein 2 days. No evidence for the presence of a cytosine compoundin the soluble nucleotide fraction of wheat embryos has beenobtained. Guanine, adenine, and uridylic acid were the onlydetectable products of acid hydrolysis of the wheat seedlingsoluble nucleotides.     

High resolution microanalysis for phosphorus in Golgi complex beads of insect fat body tissue by electron spectroscopic imaging

Golgi complex beads are 10 nm particles arranged in rings on the smooth forming face of the Golgi complex that stain specifically with bismuth in arthropod cells. In vitro experiments with biological molecules spotted on to cellulose acetate strips indicated that bismuth bound to the beads through phosphate groups. We could detect a weak phosphorus signal from the beads using a new technique called electron spectroscopic imaging that is capable of very high spatial resolution (0.3–0.5 nm) and sensitivity (50 atoms of phosphorus). Detection was not obscured by tissue staining with bismuth or uranyl acetate or by using an inorganic buffer (Na cacodylate). Localization of phosphorus was greatly improved by using colour-enhanced computer pictures of the electron spectroscopic images and quantitating the images. The results indicate that the phosphorus content of the beads is large enough to account for their bismuth reactivity.     

Two-dimensional electrophoresis of yeast histones on cellogel strips and polyacrylamide gels

For a quick analytical identification of histones a two-dimensional electrophoretic system has been developed. First the proteins are separated on cellulose acetate strips in alkaline buffer. Then they are reelectrophoresed in a second dimension on polyacrylamide gels either with sodium dodecyl sulfate or in the presence of urea at pH 2.7. The technique is demonstrated with yeast and calf thymus histones.     

Two-dimensional electrophoresis on the layers of cellulose acetate membrane

A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.     

Mononucleotides of the cell nucleus

1. It has been demonstrated by ion exchange chromatography that the cell nucleus contains mononucleotides of adenine, guanine, cytosine, uracil, together with diphosphopyridine nucleotide, and several uridine diphosphate derivatives; the adenine nucleotides predominating in amount. Nucleotide components in the cell nucleus are in close agreement both quantitatively and qualitatively with those found in the cytoplasm. 2. In calf thymus sucrose nuclei, nucleotide monophosphates can be phosphorylated to the energy-rich triphosphate form without participation of cytoplasmic components. As to the nature of the phosphorylation, it has been shown that there exist certain differences as well as resemblances between nuclei and mitochondria. A distinctive feature of nuclear phosphorylation is that only intranuclear monophosphates seem to be phosphorylated. The process is completely inhibited by cyanide, azide, and dinitrophenol. However, certain reagents which block oxidative phosphorylation of mitochondria, namely dicumarol, Janus green B, methylene blue, and calcium ions, have no effect on phosphorylation within the nucleus. 3. The bulk of mononucleotides is preserved within thymus nuclei after their isolation in sucrose. Nucleotides are surprisingly well retained by nuclei in a sucrose medium whether or not electrolytes are present and in buffers ranging from pH 3 to 10; under all conditions sucrose is required for retention. 4. Dilute acetate in sucrose releases nucleotides from the nucleus below pH 5.1. As to the effective pH of acetate, there is a sharp boundary between pH 5.1 and pH 5.9. At pH 5.9, and above, acetate does not remove nucleotides from the nucleus. The effects of propionate, formate, and monochloroacetate on the nuclei are the same as that of acetate. 5. When nuclei are exposed to a wide variety of conditions a close correlation is found between the retention in the nucleus of nucleotides and of potassium. This suggests that both substances are part of a common complex in the cell nucleus. 6. It has been shown that upon removal of nucleotides and potassium from calf thymus sucrose nuclei by acetate, the ability to incorporate C¹⁴-alanine into nuclear protein is greatly impaired.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

Rat haemoglobin heterogeneity. Two structurally distinct alpha chains and functional behaviour of selected components.

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two alpha chains and three non-alpha chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor alpha chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.     

血红蛋白F-新疆[~Aγ~T25(B7)Gly-Arg]——一种新的慢速不稳定胎儿血红蛋白变异体

本文报告一种新的胎儿血红蛋白变异体。先证者为一健康汉族女性新生儿。变异体含量占全部Hb的7.7％。醋纤薄膜电泳(pH8.6)显示变异体区带稍慢于HbD组,但比HbA_2快。分离的变异体的热变性曲线显示其沉淀速度较HbA和HbF为快,说明它属于不稳定Hb。通过DEAE-纤维素柱层析,去除血红素、CM-纤维素柱层析、TPCK-胰蛋白酶消化、滤纸指纹图谱分析、氨基酸组成分析和微量顺序测定等手段,变异体被确定为Ar~T25(B7)Gly→Arg,根据发现地,命名为Hb F-新疆。     

Starch conversion by immobilized glucoamylase

Glucoamylase bound to DEAE-cellulose in 0.05 M sodium acetate, pH 4.0, is active in the conversion of starch to glucose. The activity of the DEAE-cellulose-bound enzyme ranges from 16 to 55% of the activity of the free enzyme. Binding of the enzyme narrows the pH optimum to approximately 4.0 and lowers the temperature optimum to 40–50°C as compared to a 60°C temperature optimum for the free enzyme. Concentrations of acetate buffer above 0.1 M disrupt the DEAE-cellulose-enzyme complex. Columns were used with some success for the continuous conversion of starch. Pretreatment of the starch with α-amylase and clarification were necessary to prevent blocking of the column. Columns maintained activity for more than 3 weeks of continuous operation.     

Bidimensional electrophoresis of factor VIII antigen on cellulose acetate

It is described two-dimensional immunoelectrophoresis using cellulose acetate as supporting medium compared with agarose gel method. The results show that the determination of the FVIIIR: Ag on cellulose acetate is a technique more simple and rapid than agarose gel method and many free from the technical failure.     

Influence of blood proteins on biomedical analysis. VIII. Attempts at purification of gliclazide-degrading factor in human serum

Gliclazide(hypoglycemic drug having sulfonylurea structure)-degrading activity was found in fraction M(macroglobulin, Fr. M) obtained from pooled human serum by gel filtration using a Sephadex G-150 column. The main degrading activity was in the fraction eluted from the Fr. M-subjected DEAE-cellulose column with 0.4 M phosphate buffer (pH 5.2), and the gliclazide-degrading protein localized around alpha 2 to beta-globulin on an electrophoretic pattern using a cellulose acetate membrane. The degrading activity was enhanced about two-fold by lyophilizing Fr. M solution containing a higher sodium phosphate (Na2HPO4-NaH2PO4), over 0.27 M. This indicates that the appearance and enhancement of the degrading activity required the combination of the lyophilization of the sample solution and a certain initial concentration of sodium phosphate prior to lyophilization.     

Maturation pathway for Novikoff ascites hepatoma 5.8 S ribosomal ribonucleic acid. Evidence for its presence in 32 S nuclear ribonucleic acid.

Evidence that 32 S nRNA contains 5.8 S rRNA was provided by studies on specific oligonucleotide sequences of these RNA species. Purified 32P-labeled 5.8 and 28 S rRNA and 32 S RNA were digested with T-1 ribonuclease, and the products were fractionated according to chain length by chromatography on DEAE-Sephadex A-25 at neutral pH. The oligonucleotides in Peak 8 were treated with alkaline phosphatase and the products were separated by two-dimensional electrophoresis on cellulose acetate at pH 3.5 and DEAE-paper in 7% formic acid. Seven unique oligonucleotide markers for 5.8 S rRNA including the methylated octanucleotide A-A-U-U-Gm-G-A-Gp were present in 32 S RNA but were not found in 28 S rRNA, indicating that 5.8 S rRNA is directly derived from the 32 S nucleolar precursor. These studies confirm a maturation pathway for rRNA species in which 32 S nucleolar RNA is a precursor of 5.8 S rRNA as well as 28 S rRNA.