Robert N. Dreyer

A fluorometric assay for HIV-protease activity using high-performance liquid chromatography

A rapid sensitive method for the quantification of in vitro HIV-protease activity has been developed on the basis of the endoproteolytic conversion of N-Dns-SQ-NYPIV to N-Dns-SQNY. The use of the N-dansyl group as a fluorescence label was shown to not significantly alter the apparent kinetic parameters for the peptide-enzyme interaction. Using fluorescence detection, the dansylated product and unconverted substrate are detected in a single rapid (3 min) isocratic reverse-phase HPLC separation in quantities as low as 0.2 pmol. The method is highly reproducible and suited to a variety of applications including the analysis of large sample numbers and rigorous enzymological studies.

Partial characterization of an analog of pyridinoline isolated from human skin

The most abundant amine in acid hydrolysates of human skin, eluting in the crosslink region of a reversed-phase HPLC chromatogram, has the same retention time as pyridinoline standard. This amine is not pyridinoline, since it is a weak fluorophore and its U/V spectrum does not agree with that of pyridinoline. The unknown amine was isolated and characterized by fast atom bombardment mass spectrometry and its structure is consistent with a deoxy-analogue of pyridinoline. It may be a crosslink component of some biological importance, since it is not detectable in skin from a patient with Marfans Syndrome.

Synthesis of sialoglycoconjugates during dimethylsufoxide-induced erythrodifferentiation of friend leukemia cells

We have previously shown that erythroid differentiation of Friend murine leukemia cells by dimethylsulfoxide results in a decrease in sialic acid content and net negative surface charge. The mechanism responsible for the decrease in sialic acid content was examined by measuring the synthesis of sialic acid from N-acetylmannosamine and its catabolic removal from sialoconjugates during the maturation process. A decrease in the incorporation of N-[3H]acetylmannosamine into sialoglycoconjugates occurred as early as 12 h after exposure to dimethylsulfoxide. Radioactivity incorporated into sialoglycoconjugates was relatively stable in untreated and dimethyl-sulfoxide-treated cells, implying that catabolic removal of sialic acid residues was not a factor in the decreased surface sialic acid content of differentiated erythroleukemia cells. In addition, no difference existed between control and treated cells in sialyltransferase activity. Significant decreases occurred, however, in the incorporation of radioactivity from N-[3H]acetylmannosamine into N-acetylneuraminic acid, CMP-N-acetylneuraminic acid and a material tentatively identified as N-acetylmannosamine-6-phosphate, 48 h after the addition of dimethylsulfoxide. The decrease in sialic acid biosynthesis in differentiated erythroleukemia cells was reflected by an 83% decrease in the amount of radioactively-labeled sialic acid released by neuraminidase treatment of cells exposed to dimethylsulfoxide. These findings are consistent with a cellular aging phenomenon triggered by the polar solvent-induced differentiation of the leukemic cells into more mature forms.

Sedimentation field flow fractionation of fused unilamellar vesicles: Comparison with electron microscopy and gel filtration

Sedimentation field flow fractionation (SF3), a relatively new instrumentation methodology for separating particles according to size, has been used to monitor changes in vesicle size during the formation of fused unilamellar vesicles of dipalmitoylphosphatidylcholine. The fusion of 500-A small unilamellar vesicles to 700-A large unilamellar vesicles (LUVs) and the slower conversion to 950-A LUVs at 4 degrees C was readily monitored by SF3 over the time course of a month. Changes in the physical state of these vesicles as a result of structural reorganizations were characterized by high sensitivity differential scanning calorimetry. The advantages and limitations of SF3 are discussed and correlated with electron microscopy and gel filtration.

Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells

SummaryMethionine-auxotrophic L1210 cells were used to study the effect of methotrexate (MTX) on methionine utake and metabolism. MTX was shown to inhibit amino acid transport systems and cause a decrease of methionine uptake into L1210 cells. Conversely, a nonmetabolizable amino acid analogue reduced MTX uptake into L1210 cells. MTX also blocked the transfer of the beta carbon from serine into methionine. Therefore, methionine deprivation may be an additional mechanism of action for MTX in methionine-auxotrophic tumor cells.

Conversion of an analytical high-performance liquid chromatographic system into an automated semi-preparative unit and its application to the separation of a mixture of benzyl α- and β-D-glucofuranosides and glucopyranosides

Abstract The conversion of an analytical high-performance liquid chromatograph to an automated semi-preparative unit capable of continuous 24-h operation is described. By the use of this technique a mixture of α- and β- D -glueofuranosides and glycopyranosides was separated into their individual components. The continuous operating capability of this system allowed 2.5–3 g of processed material to be collected over a 24-h period.

Determination of 5-hydroxytryptamine metabolites in isolated perfused rabbit lung by high-performance liquid chromatography

Abstract An analytical procedure, utilizing high-performance liquid chromatography (HPLC) hasbeen developed for the separation of radiolabeled metabolites of 5-hydroxytryptamine (5-HT) in biological fluids. Four different chromatographic systems are described, which enable the separation of groups of metabolites possessing similar organic functionality to be achieved. As an example of this general analytical method, it is demonstrated that no methylation of 5-HT occurs in perfused rabbit lung, the principal metabolites being 5-hydroxyindoleacetic acid and 5-hydroxytryptophol.

Pyrimidine nucleoside phosphorylase assay by automated high-performance liquid chromatography.

A new assay for pyrimidine nucleoside phosphorylase is reported. This method utilizes an isocratic reversed-phase high-performance liquid chromatographic system for separation of nucleosides and bases. Product detection is accompanied by ultraviolet monitoring and radioactive flow detection. Use of an automated sample injector allows for the analysis of a series of samples, with data recorded onto a microprocessor-based cassette recorder. Data can then be downloaded into computer memory. The velocity of uridine phosphorylase (E.C. 2.4.2.3) was a linear function of enzyme concentration. The Michaelis constant for uridine at pH 8.0 was found to be in close agreement with the value obtained by a thin-layer chromatographic assay method.