One-step Column Research Articles

Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase).

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).

Improved separation of isomeric gangliosides by anion-exchange high-performance liquid chromatography.

A new anion-exchange HPLC method for separation of gangliosides has been developed using TMAE (trimethylaminoethyl)-Fractogel as the stationary phase and a gradient system of ammonium acetate in methanol for elution. Chromatography of mouse brain gangliosides resulted in separation according not only to their degree of sialylation into mono-, di-, tri- and tetrasialogangliosides, but also in separation of positional isomers within the four elution domains, e.g. GD1a (IV3Neu5Ac,II3Neu5Ac-GgOse4Cer) and GD1b (II3(Neu5Ac)2-GgOse4Cer) of the disialoganglioside fraction. Furthermore, base-line separation of alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides e.g. IV3Neu5Ac-nLcOse4Cer and IV6Neu5Ac-nLcOse4Cer, isolated from human granulocytes, was achieved and pure fractions of each ganglioside were obtained. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the isolation of pure gangliosides on a preparative scale by one-step column chromatography.

Methionyl-tRNA synthetase gene from an extreme thermophile, Thermus thermophilus HB8. Molecular cloning, primary-structure analysis, expression in Escherichia coli, and site-directed mutagenesis.

The gene for the methionyl-tRNA synthetase (MetRS) from an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. By expression of the T. thermophilus MetRS gene in Escherichia coli cells, thermostable MetRS was overproduced and purified to homogeneity by heat treatment and one-step column chromatography. The amino acid sequence of T. thermophilus MetRS showed low identities (approximately 25%) with those of MetRSs from E. coli, and cytoplasm and mitochondria of Saccharomyces cerevisiae. However, the amino acid residues in the binding sites for ATP and the anticodon and the 3' terminus of tRNA(Met) are highly conserved among the four MetRSs. T. thermophilus MetRS has a zinc finger-like sequence with all the three cysteine residues and a histidine residue. By site-directed mutagenesis of one of the cysteine residues (Cys127) of T. thermophilus MetRS, the SH group was found to be important for methionyl-tRNA synthesis. Just upstream of the structural gene for T. thermophilus MetRS there is a short open reading frame which codes for a methionine-rich peptide and is partly overlapped with an alternative terminator/antiterminator structure, suggesting that transcription of this gene is regulated by attenuation. Further upstream a region contains a nucleotide sequence homologous to that of the 5' half of T. thermophilus initiator tRNA(Met).

Macrophage uptake and retention of radiolabeled glycopeptidolipid antigens associated with the superficial L1 layer of Mycobacterium intracellulare serovar 20.

Glycopeptidolipid (GPL) antigens which are associated with the superficial L1 layer of Mycobacterium intracellulare serovar 20 were labeled with radioisotopes by means of internal labeling techniques and used in macrophage uptake and retention studies. The use of tritiated alanine and phenylalanine allowed the incorporation of label into the GPL invariant fatty acyl peptide core, which is common to all members of the Mycobacterium avium-M. intracellulare complex. Radiolabeled GPL antigens were then purified by a one-step column chromatographic procedure and subsequently used to determine the maximum uptake and retention in peritoneal macrophages isolated from C57BL/6 and CBA/J mice. Maximum uptake for peritoneal macrophages from both strains of mice occurred at a concentration between 200 and 250 micrograms of antigen per ml of medium when 3.4 X 10(5) cells were pulsed. Timed experiments demonstrated that approximately 20% of the antigens remained associated with the macrophages up to 4 days after a pulse of 200 micrograms of GPL, and examination of chloroform-extractable components from both macrophages and spent medium revealed that 98% or more of the radioactivity corresponded to intact GPL components. The ability of the GPL antigens to become associated with macrophages is demonstrated by these results, which strongly suggest that these potentially important mycobacterial antigens are inert to degradation by those cells.

One-step column chromatographic procedure for purification of mycobacterial glycopeptidolipid antigens

One-step column chromatographic procedure for purification of mycobacterial glycopeptidolipid antigens

Quantitation of radiolabeled mucous glycoproteins secreted by tracheal explants

An agarose gel column filtration method has been developed to quantitate labeled mucous glycoprotein secreted into culture medium by explants of tracheal epithelium. A multichannel pump is employed to simultaneously analyze 15 samples. Compared to other methods, such as dialysis and trichloroacetic acid/phosphotungstic acid precipitation methods, the column method not only quantitates high-molecular-weight mucous glycoprotein specifically but also offers the advantages of rapid processing of samples, low-background radioactivity, linearity and reproducibility even with small amounts of labeled mucous glycoproteins, and recovery of samples for chemical analysis. This method can be used to quantitate any substances that can be isolated by a one-step column chromatographic technique.

Separation of mannosylretinylphosphate from dolichylmannosylphosphate by chromatography on columns of DEAE-Sephacel

A one-step column chromatographic procedure on DEAE-Sephacel allows the separation of mannosylretinylphosphate from dolichylmannosylphosphate with minimal breakdown of the mannosylretinylphosphate. Using this procedure, subcellular fractions of rat liver were shown to be active in synthesizing both mannolipids from GDP-[ 14C]mannose in the absence or presence of exogenous retinylphosphate.

The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA Tyr appears to be a competitive inhibitor of tRNA Tyr in the aminoacylation reaction (tRNA Tyr K m = 0.42 μM, mercury derivative of tRNA Tyr K i = 0.11 μM). The mercury derivative of Tyr-tRNA Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.