Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

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.     

An analytical study of catecholamine metabolites by phosphorimetry

Phosphorescence excitation and emission spectra, lifetimes, phosphorimetric analytical curves, and limits of detection have been determined at 77°K in methanol: water 10:90 solution for tyrosine and 11 catecholamine metabolites. The influence of pH on the phosphorescence efficiency is shown to be valuable for the identification of phenolate species and enhancement of sensitivity of the method. Strongly alkaline solution (pH ≥ 10) are the most suitable solvent for the phosphorimetric studies of nondegradable catechnolamine metabolites (3-methoxy-4-hydroxy derivatives). Low limits of detection between 0.2 and 0.02 μg/ml are obtained. For most of the compounds, phosphorimetry is shown to give better sensitivity and accuracy than the classical fluorometric assays of catecholamines.     

Determination of protein synthesis initiation factor levels in crude lysates of Escherichia coli by a sensitive radioimmune assay.

Antisera specific for protein synthesis initiation factors IF1, IF2, and IF3 were prepared by immunizing rabbits. When crude cell lysates are analyzed by double immunodiffusion or by immunoelectrophoresis, each antiserum forms a single precipitin line antigenically identical to its cognate factor. The antisera do not crossreact with other initiation factors or with ribosomal proteins. A radioimmune assay was developed for each initiation factor by using the specific antisera and radioactive factors prepared by reductive alkylation with [¹⁴C]formaldehyde. The assays detect as little as 10 to 30 ng of factor. Initiation factor concentrations were measured in crude Escherichin coli MRE600 extracts prepared from cells grown exponentially in a rich medium. The three initiation factors are present in approximately stoichiometric amounts and comprise about 1% of the cell protein. The molar ratio of initiation factors to ribosomes is about 0.15, which corresponds to the concentration of native ribosomal subunits.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Modification of ribulose bisphosphate carboxylase from Rhodospirillum rubrum with tetranitromethane.

The synthesis of DL-5,5′-dihydroxyleucine, by diborane reduction of N-phaloyl-DL-γ-carboxyglutamic acid-α-methylester, and the chromatographic and spectral characteristics of this amino acid are reported.     

Hyaluronidase activity during thyroxine-induced tadpole metamorphosis.

Immersion of Rana catesbeiana tadpoles in 10^?7Ml-thyroxine gives rise to increases in brain and backskin hyaluronidase activity. After 10 days of immersion, there is a 1.9-fold increment in brain enzyme activity and a 2.5-fold increment in the backskin. The rise in activity occurs mainly between the seventh and tenth days of treatment. During the 10-day treatment, hyaluronate content in the backskin decreases to 22% of the control level while sulfated glycosaminoglycan increases markedly, but no significant change in brain glycosaminoglycan composition occurs. The onset of major metamorphic events was observed between the seventh and tenth days of immersion in thyroxine.     

Analysis of leukotrienes by high-pressure liquid chromatography

A method is described for the partial synthesis of saturated mixed-chain phosphatidylcholines of a high degree (typically 99 mol%) of purity. This procedure has been designed to eliminate the contamination of the mixed-chain product by symmetric chain phosphatidylcholine and the mixed-chain isomer of the desired product, the two principal impurities introduced by previous techniques. This high degree of purity is obtained by employing a method designed for the complete enzymatic hydrolysis of the C-2 fatty acyl moiety in saturated symmetric phosphatidylcholines and a new technique for the acylation of lysophosphatidylcholines employing the catalyst 4-pyrrolidinopyridine. The versatility of this new procedure is illustrated with the synthesis of several saturated mixed-chain phosphatidylcholines.     

Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution.

Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg²⁺, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution.     

Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from E1 and E2, respectively). Amino acid composition data and NH₂-terminal sequence analysis established their location at the extreme COOH-terminal end of each glycoprotein. RE1 and RE2 are rich in hydrophobia amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with E1 and E2 was found to be clustered in their hydrophobic, membrane-associated roots. Beginning six to seven residues from their NH₂ termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), E1 and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, E1 probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOH-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOH-terminal end of E2, which is protected by the virion envelope from digestion by a-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.     

Two-dimensional electrophoretic analysis of major histone species and their variants from somatic and germ-line tissue

A two-dimensional electrophoretic procedure has been developed and applied to the analysis of histones from the mouse thymus, liver, and seminiferous epithelium. The technique uses acetic acid-urea polyacrylamide gel electrophoresis in the first dimension to provide a primary separation of major histone species. Separation of additional histone species and variants is achieved in the second dimension by adding 0.4% of the nonionic detergent Lubrol-WX to the polyacrylamide gel. The procedure is relatively simple and highly reproducible and enables the simultaneous resolution of 9 to 16 protein spots corresponding to the major histone species and their variants.     

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.     

Analysis of neutron distance data

Techniques are described for processing neutron distance data for the purpose of deriving information about the three-dimensional organization of macromolecular assemblies.     

Use of insoluble heparin for isolation of DNA polymerase enzymes from murine myeloma

Heparin was found to be a potent inhibitor of a DNA polymerase present in the murine myeloma tumor MGPC-21. By increasing the KCl concentration of the reaction mixture, the inhibition of this enzyme could be completely reversed, suggesting that insoluble heparin might be a useful tool in the isolation of DNA polymerases. When heparin covalently bound to Sepharose was used, some of the DNA polymerases present in MOPC-21 myeloma tumors were separated and partially purified.     

Failure of preparative flat bed electrofocusing to resolve rat liver chromosomal proteins

Rat liver nonhistone chromosomal (NHC) proteins were preparatively fractionated by isoelectrofocusing in 40 × 15 × 0.5-cm layer of Sephadex G75-8 m urea gel. The fractions were collected and analyzed by reclectrofocusing and by SDS-polyacrylamide gel electrophoresis. It is shown that most of the rat liver NHC proteins do not focus as single bands but rather within a wide pH interval. The conclusion is drawn that a protein mixture as complex as the total chromatin protein can not be completely resolved by isoelectrofocusing because the protein molecules form equilibrium complexes either with the ampholines or among themselves.     

Quantitation of chlordiazepoxide and its metabolites in biological fluids by thin-layer chromatography

Chlordiazepoxide and its 4 major metabolites were assayed after separation by thin-layer chromatography following extraction from biological fluids. The compounds become intensely fluorescent in the presence of red, fuming nitric acid. The resulting compounds are quantitated with a spectrodensitometer with a fluorescent attachment. The sensitivity varies between 0.05 and 0.1 μg. The coefficient of variation is 1.4% for assays in urine and 6.4% in serum.     

The atypical RNA components of cytoplasmic ribosomes from Crithidia fasciculata

RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit of the trypanosomatid flagellate Crithidia fasciculata and analyzed by polyacrylamide gel electrophoresis at 4 °C consisted of one species with a molecular weight of 1.3 × 10⁶ (relative to ribosomal RNA from E. coli MRE 600). When extracted with hot phenol (65 °C), the large ribosomal subunit gave rise to two components with molecular weights of 0.72 and 0.56 × 10⁶. On heating for 60 s, followed by rapid cooling, the single cold-phenol-extracted 1.30 × 10⁶-dalton species completely dissociated into two components of molecular weights 0.72 and 0.56 × 10⁶, present in equimolar amounts. When analyzed by polyacrylamide-agarose gel electrophoresis in the presence of SDS, RNA extracted by cold phenol from the large cytoplasmic ribosomal subunit consisted of three components of molecular weights 1.3, 0.72, and 0.56 × 10⁶, present in apparently equimolar amounts. RNA from the small cytoplasmic ribosomal subunit consisted of one species with a molecular weight of 0.84 × 10⁶, independent of extraction or analytical conditions. It is proposed that under high salt and low temperature conditions, the large ribosomal RNA molecule is held together by its secondary structure, and that denaturing extraction or analytical conditions reveal an otherwise “hidden” lesion present in the molecule in vivo.