The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

The use of high-pressure liquid chromatography in the preparation of tritium-labeled chloramphenicol and its analogs.

The 1-hydroxy epimers of chloramphenicol and thiamphenicol formed from the reduction of the respective 1-oxo derivatives with [³H]NaBH₄ have been separated preparatively by high-pressure liquid chromatography on a μBondapak C₁₈ column. This separation procedure permits the facile and rapid preparation of the 1-³H-labeled derivatives of chloramphenicol and its analogs.     

High resolution gel chromatography of proteins

An assay for the determination of l-glutaminase in extracts and intact cells is described. The method is based on the stereospecific release of ³H₂O from l-[2-³H]glutamine when l-glutaminase is coupled to l-aspartate:2-oxoglutarate amino transferase. The substrate, glutamine, and intermediate product, glutamate, are separated from the final reaction product, tritiated water, on a mixed-bed ion-exchange column which retains amino acids and organic acids but not water. The method has been adapted to determine the activity of l-glutaminase in cultured human diploid fibroblasts.     

Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA

We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.     

Deoxynucleoside composition of DNAs and modified nucleoside composition of tRNAs determined at nanomole sensitivity by reversed-phase liquid chromatography

Selected purified tRNA and DNA samples were digested by standard enzymatic methods, and the nucleosides were separated by high-pressure liquid chromatography on reversedphase columns. Nanomole sensitivity was obtained by use of an integrator. The nucleosides were detected at wavelengths near their uv-absorption maxima, including 220 nm for dihydrouridine, and 330 nm for 4-thiouridine. Recovery values for the individual nucleosides were in the range of 94–100%. The nucleoside composition of the DNA and tRNA digests were in accurate and precise agreement with published values.     

High hydrostatic pressure and the dissection of fertilization responses : I. The relationship between cortical granule exocytosis and proton efflux during fertilization of the sea urchin egg

High hydrostatic pressure applied between sperm attachment and the onset of cortical granule exocytosis will inhibit this exocytotic event in sea urchin eggs. Such pressure-treated zygotes, nevertheless, are activated and capable of development. Thus, this technique can be used as a tool to study the relationship between cortical granule breakdown and other fertilization-related responses. We have studied whether the exocytosis of cortical granules is necessary for proton efflux (acid release) to occur. Our results indicate that although Ca²⁺ is released while the eggs are under pressure (a prerequisite for the following events to take place), cortical granule exocytosis and acid release are pressure-sensitive and completely inhibited at pressures above 400 atm (6000 psi) and 275 atm (4000 psi), respectively. However, upon decompression, acid release is initiated which amounts to 65–70% of that seen in the unpressurized controls, suggesting that the efflux mechanism does not require cortical granule exocytosis and must result from some modification of the original plasma membrane of the egg. The remaining 30–35% of the acid release is related to cortical granule exocytosis, since it can be obtained upon induction of the cortical granule fusion 30 min later under atmospheric pressure. The initiation of acid release after decompression indicates that the efflux mechanism is not transiently turned on at fertilization, but undergoing long-term modification; the recovery of the ability to induce cortical granule fusion after fertilization under pressure suggests a refilling of cytoplasmic Ca²⁺ stores within this time course.     

Characterization of proteins and peptides by high-performance liquid chromatography and fluorescence monitoring of their tryptic digests.

Proteins and peptides can be characterized and compared at the subnanomole level by treatment with trypsin followed by high-performance liquid chromatography on reverse-phase partition columns. A fluorescamine monitoring system automatically analyzes a portion of the column effluent while the remainder can be collected for further studies. The method has already been used for characterization of rat β-endorphin and a protein which cross-reacts with antiserum prepared against prolyl hydroxylase.     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Specificity of the dopamine sensitive adenylate cyclase for antipsychotic antagonists

Chlorpromazine, haloperidol and clozapine are approximately equipotent in antagonizing dopamine sensitive adenylate cyclase activity in homogenates of rat brain striatum, in contrast to the differences in clinical antipsychotic potencies reported by others. The antagonism appeared to occur at a structurally specific dopamine site, as inhibition by a series of chlorpromazine analogues of similar hydrophobicity exhibited a structural specificity similar to that found for their neuroleptic and cataleptic activities. Sulpiride, a dopamine antagonist with antipsychotic activity, and metoclopramide, a structurally related central dopamine antagonist, failed to inhibit the dopamine sensitive adenylate cyclase. Pre-treatment of rats with haloperidol (3 mg/kg per day) for 6 or 28 days did not induce a supersensitive response of the adenylate cyclase to stimulation by dopamine or apomorphine or inhibition by clozapine. It was concluded that the dopamine sensitive adenylate cyclase may not be the site of action of all anti-psychotic agents.     

Sea urchin embryo chromatin and nuclear ribonucleoproteins fractionated by anion exchange chromatography.

Chromatin and ribonucleoproteins released from sea urchin embryo nuclei were characterized on the basis of sedimentation properties, buoyant densities and fractionation by anion exchange chromatography. DEAE- and ECTEOLA-cellulose chromatography was used to assay nuclear purity, insofar as ribosomes and polyribosomes could be readily distinguished from ribonucleoproteins released from nuclei. This chromatography was used to separate chromatin fragments on the basis of DNA size, to prepare chromatin fragments substantially enriched in nonhistone proteins, and to analyze nuclear ribonucleoproteins. Solubilized chromatin is fractionated into major and minor components by ECTEOLA-cellulose chromatography. The DNA of these chromatin fractions was analyzed with respect to buoyant density and hybridization with nuclear RNA.     

Biochemical analysis of pleiotropy in Dictyostelium.

The patterns of accumulation of five developmentally regulated enzymes were determined in four aggregateless mutant strains of Dictyostelium. Previous analyses of these strains had predicted that they should not accumulate these markers. The results are consistent with the concept that a linear dependent pathway controls the temporal expression of development in this system.     

Simple and sensitive procedure for the assay of serotonin and catecholamines in brain by high-performance liquid chromatography using fluorescence detection

A simple, rapid and specific method for the determination of serotonin and catecholamines in brain is described. After tissue homogenisation, catecholamines are isolated by adsorption onto alumina and elution with perchloric acid. Serotonin is isolated by extraction into n-heptanol and back-extraction into acid. High-performance liquid chromatography of the acid extracts is performed with a C₁₈ reversed-phase column and simple mobile phases. Detection is by the intrinsic fluorescence of the amines on excitation at 200 nm. Detection limits are 100 pg for norepinephrine, 300 pg for dopamine and 20 pg for serotonin. The results are found to correlate well with a catechol O-methyl transferase radioenzymatic assay for catecholamines and a ninhydrin derivatisation procedure for serotonin.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

High-performance liquid chromatographic separation and quantification of the four biliverdin dimethyl ester isomers of the IX series

High-performance liquid chromatography (hplc) has been used to separate and quantificate the dimethyl ester (DME) derivatives of the four biliverdin isomers of the IX series: biliverdin-IXα, -IXβ, -IXγ, and -IXδ. Samples of 0.5 to 10.0 nmol of biliverdin DME were detected quantitatively upon elution by monitoring the absorbance at 375 nm. A technique was developed in which p-bromoacetanilide (Dupont's recommended test compound for their Zorbax column) is used as a marker for biliverdin-IXα DME. To facilitate quantification of biliverdin-IXβ DME, its extinction coefficient was determined. This method has been used to study biliverdin isomers in various biological species. High-resolution NMR (360 MHz) was used to further characterize the isomers.     

Assay of plasma catecholamines by liquid chromatography with electrochemical detection

A method was developed for the simultaneous assay of noradrenaline and adrenaline in 2 ml of human plasma. The method involves adsorption of the catechols onto alumina, desorption, lyophilizing, reconstitution, and injection into a reverse-phase ion-pairing liquid chromatography system. Sensitivity and selectivity are introduced using direct electrochemical detection of the column eluant.