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21.
Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral high-performance liquid chromatography 总被引:1,自引:0,他引:1
Leucovorin (5-formyltetrahydrofolate, LV) is a reduced folate that has been in clinical use for many years as a rescue agent following methotrexate (MTX) therapy. Commercially available LV is a 1:1 mixture of [6R]-and [6S]-isomers. Due to the lack of a specific method for directly separating and quantitating the stereoisomers of LV, it has been difficult to precisely define the pharmacokinetic and biological characteristics of each stereoisomer. We have now developed a novel HPLC method to completely separate [6S]-LV and [6S]-5-methyltetrahydrofolate (MeTHF) from their respective [6R]-isomers using bovine serum albumin (BSA)-bonded silica as the chiral stationary phase. Baseline separation was achieved using 5 and 25 mM sodium phosphate buffers (pH 7.4) as the mobile phase with resolution factors of 1.65 for LV and 2.31 for MeTHF, respectively. The purity of each isomer prepared by this HPLC method is greater than 99%. The stereoisomers were identified by examining their ability to protect CEM cells from MTX (0.04 microM)-induced inhibition of growth. In the LV chromatogram, the first eluted peak provided complete protection from MTX growth inhibition when LV concentrations of 0.1 microM and above were used, whereas the last eluted peak failed to reverse MTX toxicity at concentrations up to 1.0 microM. Chemically pure synthetic [6R]-and [6S]-LV standards confirmed that the first eluted, biologically active peak is the [6S]-isomer. For MeTHF, only the last eluted peak effectively protects cells from MTX growth inhibition and is therefore believed to be the [6S]-isomer. This new HPLC method will serve as a useful tool to elucidate the clinical and cellular pharmacology of the stereoisomers of LV and MeTHF. 相似文献
22.
Structure of the horseradish peroxidase isozyme C genes 总被引:13,自引:0,他引:13
K Fujiyama H Takemura S Shibayama K Kobayashi J K Choi A Shinmyo M Takano Y Yamada H Okada 《European journal of biochemistry》1988,173(3):681-687
We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus. 相似文献
23.
Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9. 相似文献
24.
Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries 总被引:5,自引:0,他引:5
The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier. 相似文献
25.
A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the killer factor also inhibited the growth of killer-sensitive cells satisfactorily when it was grown in grape juice. 相似文献
26.
Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography 总被引:2,自引:0,他引:2
H U Choi L H Tang T L Johnson L Rosenberg 《The Journal of biological chemistry》1985,260(24):13370-13376
Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation. 相似文献
27.
28.
A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells. 总被引:18,自引:5,他引:13
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A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence. 相似文献
29.
The nucleotide sequence of the embryonic chicken beta-type globin genes 总被引:11,自引:0,他引:11
J B Dodgson S J Stadt O R Choi M Dolan H D Fischer J D Engel 《The Journal of biological chemistry》1983,258(20):12685-12692
30.
Low-molecular-weight nuclear RNAs 总被引:2,自引:0,他引:2