Ammonium Bicarbonate Buffer Research Articles

Fibrillin molecules aggregate to form an extendible beaded structure which is a major component of “Elastin Associated” microfibrils

A mouse monoclonal antibody was used to identify beaded aggregates found in guanidine extracts of human amnion as assemblies of fibrillin molecules. These aggregates were also shown to be a major component of extracellular matrix microfibrils. We further demonstrated that the periodicity of these aggregates can be increased when subjected to mechanical stress.Human amnion was extracted with guanidine and the extracted material purified using ion exchange and molecular sieve chromatography. A high molecular weight fraction was precipitated by dialyzing against dilute acetic acid. Part of the precipitate was suspended in 0.2 M ammonium bicarbonate buffer and rotary shadowed. A second portion was resuspended in culture medium containing antibody which recognizes matrix microfibrils, diluted 1:5 in ammonium bicarbonate and reacted for 120 minutes at room temperature. Antibody labeled precipitate was washed by repeated pelleting and resuspension in buffer and then incubated in Janssen GAM 5 nm gold conjugate for 60 minutes at room temperature.

Liquid chromatographic separation of acidic phosphoserine peptides on macroporous copoly(styrene-divinyl-benzene) using amines to regulate retention

Using phosphoserine peptides as model substances, we have studied the separation of acidic peptides as substituted ammonium ion-pairs on a macroporous copoly(styrene-divinylbenzene) ( PLRP-S) chromatographic column. Various hydrophobic amines were examined, of which long-chain N-methyl- and N,N-dimethyl- n-alkylamines proved to be the best with respect to selectivity and peak symmetry. Excellent separations were obtained in ammonium bicarbonate buffer (p H 7.5) with ethanol as organic modifier under isocratic conditions using the corresponding heptylto nonylamines.

A simplified procedure for the fractionation of Tityus serrulatus venom: Isolation and partial characterization of TsTX-IV, a new neurotoxin

E. C. Arantes, W. A. Prado, S. V. Sampaio and J. R. Giglio. A simplified procedure for the fractionation of Tityus serrulatus venom: isolation and partial characterization of TsTX-IV, a new neurotoxin. Toxicon 27, 907–916, 1989.—Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX 3, IX 5, and X 4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX 3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. ld 50, in mice, of 826 ± 156 μg/kg and an intracisternal ld 50 of 11 ± 9 μg/kg, compared to 375 ± 45 and 4.9 ± 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX 5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.

A unique low molecular weight zinc-binding ligand in the kidney cytosol of the musselMytilus edulis, and its relationship to the inherent variability of zinc accumulation in this organism

Mussels,Mytilus edulis, are known to have a high degree of variability in their whole soft tissue zinc concentrations which cannot be explained by any known ecological or physiological factor. In the present study, 70 individual mussels collected from an uncontaminated site at Bellevue, Newfoundland, in Spring 1987 had kidney zinc concentrations ranging from 148 to 4 907 μg g-1 dry weight while 40 mussel exposed to 25 μg l-1 zinc for 18 d had kidney zinc concentrations ranging from 144 to 14 072 μg g-1. Pooled or individual kidneys were homogenized in 50 mM ammonium bicarbonate buffer (pH 8.0) and ultracentrifuged for 1 h at 105 000 g. On average, about 70% of the total kidney zinc load was found in the 105 000 g pellet. Cytosolic zinc was separated into two peaks on a column of Sephadex G-25. The first peak coincided with the void volume of the column representing molecules with molecular weights of at least 5 000 (exclusion limits of Sephadex G-25). This peak would include any metallothionein present since mussel metallothionein has a molecular weight of 10 000 to 20 000. Little variability was observed in this peak so it was concluded that zinc-thionein did not play a major role in the genesis of the inherent variability. The second peak represented zinc complexed to an unknown substance with an approximate molecular weight of 700 to 1 300. This very low molecular weight zinc showed an extremely high degree of inherent variability and a strong positive correlation with the whole kidney zinc concentration. It is concluded that this very low molecular weight zinc complex plays a major role with regard to kidney zinc variability. Some very low molecular weight zinc was also noted in the digestive gland and gills. It is speculated that this substance may aid in zinc transport as well as in the incorporation of zinc into granules.

Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase.

When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATP-Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001-1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high-performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min-1 at pH 8.3, 25 degrees C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.

Susceptibility of the myelin-associated glycoprotein and basic protein to a neutral protease in highly purified myelin from human and rat brain.

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin‐associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower‐molecular‐weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin‐related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.

Concentration and preservation of nuclear androgen receptor by lyophilisation

Nuclei from rat ventral prostate were disrupted by sonication and treated with micrococcal nuclease to precipitate nuclear proteins including the androgen receptor. The precipitate was dissolved in 0.6–1.2 M ammonium bicarbonate buffer, pH 7.6, with no loss of receptor when compared to the conventional Tes buffer, pH 7.0 containing 0.6–1.2 M NaCl. Lyophilisation of the solubilised protein did not produce any qualitative or quantitative differences in the recovery of receptor relative to a non-lyophilised control preparation, both of which were analysed for binding properties by Sephadex G-25/G-100 dual-column chromatography. Over longer periods of storage at −80°C, the rate of inactivation of receptor was found to be 6% per week. The stability of the lyophilised receptor was improved by the inclusion of MgCl 2 and SH-reducing agents in the ammonium bicarbonate buffer. Recovery improved also with increasing ionic strength of the buffer used to dissolve the lyophilised receptor.

A straightforward method for the simultaneous preparation of radiolabeled l-dihydroörotic and N-carbamyl- l-aspartic acids

A straightforward method is presented for the simultaneous enzymatic preparation of radiolabeled l-dihydroörotic and N-carbamyl- l-aspartic acids. [ carboxyl- 14C]Orotic acid is incubated in the presence of excess NADH to drive the reaction catalyzed by l-dihydroörotate dehydrogenase in the reverse direction, forming l-[ 14C]dihydroörotic acid. The contamination of the commercial reagent enzyme, l-dihydroörotate dehydrogenase from Zymobacterium oroticum, with l-dihydroörotase yields N-carbamyl- l-[ 14C]aspartate as a second product of the reaction. The products are purified by ion-exchange chromatography using ammonium bicarbonate buffer and desalted by repeated lyophilization. Over 95% of the starting radioactivity is recovered in the two products which, in a typical preparation, were present in a ratio of 4:6, l-[ 14C]dihydroörotic acid: N-carbamyl- l-[ 14C]aspartic acid. The identities of the reaction products are established by chromatographic, electrophoretic, and enzymic means.

Germ Tube Growth Inhibitor from Cronartium comandrae Aeciospores

Two compounds showing self-inhibitory action during germination of aeciospores of the comandra blister rust fungus (Cronartium comandrae Pk.) were extracted from these aeciospores by shaking with 0.2 M NH(4)HCO(3) (pH 7.8) for 4 h. One of these, the germination self inhibitor (D. A. Eppstein and F. H. Tainter, Phytopathology 66:1395-1397, 1976), was removed from the ammonium bicarbonate buffer by using chloroform. The water layer which remained contained a substance which, at ca. 10 M concentration, had no apparent effect on germ tube emergence but which inhibited normal germ tube growth. Linear germ tube growth ceased or a dendritic or vesicular pattern of growth resulted, depending on the concentration of inhibitor added to extracted germinating spores. The germ tube growth inhibitor appears to be a peptide with a molecular weight of ca. 2,000.

518 CYSTIC FIBROSIS SERUM FACTORS SEPARATED BY HIGH-PRESSURE VACUUM DIALYSIS AND COLUMN CHROMATOGRAPHY

A limitation to the biochemical separation and subsequent isolation of the ciliary dyskinesia factor (CDF) from sera of Cystic Fibrosis (CF) patients is the sample size restriction. Treatment of CF serum IgG fractions, with high molarity solutions of guanidine hydrochloride(GHCl) has been reported necessary to dissociate low molecular weight substances. This treatment renders concentration of these substances difficult by usual methods. We have employed a reservoir apparatus to which is attached dialysis tubing, enclosed in a vacuum bottle. This permits the initial use of large volumes of CF samples for further chromatography. These CF samples are treated with 5M GHC1, added to the reservoir of the above apparatus and a pressure of .010 Torr applied. The dialyzed material is collected and desalted on G-15 Sephadex or applied to G-50 Sephadex in either a GHCl or an ammonium bicarbonate buffer system for subsequent fractionation and bioassay. We have isolated two major fractions by these methods whose biological activity does not agree with that of comparable fractions obtained by chromatography of PM-10 Amicon ultrafiltration of similar CF samples. It is our contention that treatment of CF serum-derived IgG with chemicals such as guanidine hydrochloride not only alters molecular stoichiometry but also affects the biological activity of the dissociated moieties.

The oxidative cleavage of folates: A critical study

Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C 9N 10 bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N 5-formyl-tetrahydrofolic acid are cleaved at the C 9N 10 bond to produce p-aminobenzoylglutamic acid. N 5, N 10-methenyl-tetrahydrofolic acid, N 5, N 10-methylene-tetrahydrofolic acid, and N 10-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N 10-formyl-folic acid which is completely stable to the oxidative treatment employed. N 5-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N 5-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5- 3H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2- 14C]folic acid to label the folates in vivo is presented.

Regulation of human gonadotropins. XIV. Gel filtration and electrophoretic analysis of endogenous and extracted immunoreactive human follicle-stimulating hormone of pituitary, serum and urinary origin.

To obtain further information regarding the nature of the immunoreactive material measured as FSH with one radioimmunoassay method, a number of different preparations were subjected to electrophoresis (7.5% polyacrylamide gel, boric acid-EDTA-tris buffer, pH 8.9) or gel filtration (Sephadex G-100, 0.05m ammonium bicarbonate buffer, pH 8.0) and resulting elution profiles of immunoreactive material measured by radioimmunoassay. Preparations studied included sera from non-pregnant and pregnant subjects, serum from a patient with choriocarcinoma, urinary extract and pituitary extract. With the exception of activity in sera from pregnant subjects, immunoreactive material was eluted as a single symmetrical peak, nearly coincident with that observed for highly purified, radioiodinated human FSH of pituitary origin. In contrast, no discrete peak of activity was located when sera from women at all stages of pregnancy were examined in a similar fashion. Instead, low amounts of “FSH” activity were identified over a ...

Controlled ductal infusion and absorption from the interstitium of canine pancreas.

Infusion of 0.1M ammonium bicarbonate buffer solution into the pancreatic duct in dogs with and without obstruction of the pancreatic veins or portal vein and with or without obstruction or collection of lymph from the thoracic duct revealed that, in the normal canine pancreas, ductal pressures above 40 cm resulted in passage of fluid into the interstitium of the gland. Large volumes of innocuous fluid, such as ammonium bicarbonate buffer, were absorbed by the pancreatic microcirculation, which appears to have extensive collateral outflow pathways. The portal venous bed appeared to be a major route for absorption of fluid from the pancreatic interstitium, while the lymphatic system played a secondary role, and increased in importance when flow in the venous outflow was compromised.

Studies on nucleotide sequences in ribonucleic acids: I. Separation of oligonucleotides on DEAE-cellulose

A method for the separation of mono- and oligonucleotides on DEAE-Cellulose is described using an ammonium bicarbonate buffer for elution. All mono-, di- and trinucleotides in a pancreatic RNase digest of ribonucleic acid could be identified. The separation of the mono-, di- and triphosphates of adenosine, guanosine, cytidine and uridine as well as that of uridylic and pseudouridylic acids is described. The various conditions and the dependence of the elution on the salt concentration are discussed.