Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

Summary Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K⁺ transport activity of these vesicles was assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted system was found to contain a K⁺ transporting protein, which is sensitive to Ba²⁺ like the K⁺ channel previously demonstrated to be activated in intact cells after cell swelling.     

Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol: acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.     

Increases in cytokinin levels in Scots pine in relation to chilling and bud burst

Levels of isopentenyladenosine and zeatin riboside were monitored in buds and needles of Scots pine ( Pinus sylvestris L.) seedlings grown under controlled climatic conditions and in field-grown trees. Extracts were purified by immunoaffinity chromatography and high-performance liquid chromatography. Cytokinin levels were quantified with an enzyme-linked immunosorbent assay. The cytokinin content was low in buds and needles of dormant seedlings but increased during dormancy release, reaching peak values in buds just before resumption of shoot growth. Samples collected in the field also showed a marked increase in the levels of cytokinins just prior to bud burst. Further, the biological activity of applied cytokinins in activating the dormant buds of Scots pine is shown. The results indicate a probable role of cytokinins in seedlings during dormancy release.     

Studies on zinc- and cadmium-bound proteins in bovine kidneys by biochemical and neutron activation techniques

Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd-protein complexes were observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results showed the presence of at least four Zn-binding proteins with mol wt>300,000, 260,000, 89,000, and 27,000 and at least three Cd-binding proteins of mol wt>300,000, 32,000, and 13,000.     

白百利烟草愈伤组织细胞细胞分裂素结合蛋白的研究

利用BA-Sepharose 4B亲和层析技术丛白百利烟草(Nicotiana tabacum Baibaili)愈伤组织细胞分离提纯了分子量为4400±100道尔顿的细胞分裂素结合蛋白(CB-蛋白)。在细胞表面、核糖体、线粒体、叶绿体和细胞核上以及在细胞液中都有CB-蛋白存在,而核糖体上的CB-蛋白含量量高。探讨了CB-蛋白的功能。     

Identification of heparin-binding proteins in bovine seminal plasma

A group of four similar proteins, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP-A1, -A2, -A3, and -30-kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin-Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin-Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well-characterized BSP proteins indicated the presence of BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Purification and characterization of Kurloff cell sialoglycoproteins with acid phosphatase activity

The majora2–6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange andSambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1)S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the majora2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4–3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylatedN-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but alsoa2-6-linked sialic acid residues themselves may participate in natural killer activity.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.