Fractionation of transfer RNA on Sepharose 4B. Effects of Sepharose batch differences.
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Chromatography, Gel↗
Biomedical subjects
U Hellman
Publications and source records attributed to U Hellman.
Isolation of tryptic fragments of human C4 expressing Chido and Rodgers antigens.
It has been shown previously that methylamine is incorporated into the alpha-chain of human C4, resulting in a loss of haemolytic function and the appearance of a free thiol group in the molecule. In the present study it was demonstrated that a fragment resembling C4d is liberated from C4 by trypsin. The fragment--Try-C4d--contains both the methylamine binding site and the free thiol group. When separated on DEAE-Sepharose, four types of Try-C4d, differing in charge and size, could be defined. The size difference was found to parallel the presence of Chido and Rodgers blood group antigens. Fragments of Mr 30,000 carried the Rodgers antigen and the Chido antigen was expressed on fragments of Mr 28,000.
Amino Acid Sequence↗
Interaction between the labile binding site of human C4 and methylamine.
Human complement component C4 was irreversibly inactivated by low concentrations of methylamine at slightly alkaline pH. The inactivated C4 molecules lost the ability to bind to EAC1 cells but retained th capacity to participate in the formation of classical pathway C3 convertase in the fluid phase. 14C-methylamine was incorporated into the alpha-chain at a ratio of 1 mol methylamine per mol C4.
Complement C4↗
Preparative scale fractionation of Escherichai coli robosomal RNA and transfer RNA on Sjepharose 6B.
Total Escherichia coli RNA has been fractionated on Sepharose 6B in 0.1 M ammonium acetate, pH 5.0. The elution order was 23S rRNA, 16S rRNA,, 5S rRNA, and tRNA, which is in contrast with the reported elution order of eukaryotic RNA, chromatographed under similar conditions. tRNA was obtained in two regions, well separated from the high molecular weight rRNA but with a slight contamination of 5S rRNA. The capacity is at least 40 A260 units of RNA per ml of gel.
Chromatography, Agarose↗
Chromatographic fractionation of Escherichia coli transfer RNA on a new support, naphthoyl-Sepharose.
The noncharged naphthoyl-Sepharose CL-6B has been prepared. Escherichia coli tRNA binds to this new adsorbent in 0.75 M ammonium sulphate at neutral pH at room temperature. Using a negative salt gradient, the tRNAs are eluted in a defined order. The chromatographic pattern is clearly different from those of other commonly used tRNA separation techniques.
Chromatography↗
Two transfer RNA (1-methylguanine) methylases from yeast.
Two distinct tRNA (m-1G) methylases have been found in the yeast Saccharomyces cerevisiae. They differ in their chromatographic properties on hydroxyapatite, in their response to spermine, and in their site specificity. Only one of the methylases is active against normal tRNA from Escherichia coli.
Chromatography↗
Micropreparative ion exchange high performance liquid chromatography: applications to microsequence analysis.
Data on the characterization of anion and cation micropreparative (50 x 1.6 mm i.d.) HPLC columns is presented. It is shown how subnanomole quantities of protein can be efficiently recovered from such columns, rendering them compatible for use in multidimensional chromatographic strategies for the purification of trace biological samples. By selection of appropriate solvent systems (e.g., buffer-free sodium chloride solutions), the small eluant peak volumes can be loaded directly onto the gas phase sequencer, and N-terminal sequence data obtained. The potential of the technique is illustrated for the purification of a GTPase activating protein (GAP-3).
Amino Acid Sequence↗
Proteinergic profiles in cerebrospinal fluid from alcoholic subjects.
Minute amounts of cerebrospinal fluid samples from alcoholics were subjected to separation by HPLC-molecular sieving, combined with multispectral UV analysis of the acquired data. A significant difference in the protein/polypeptide pattern within the molecular weight range of 7-10 kDa has been observed between samples, taken directly after detoxification and 2 weeks later. Spectral analysis of the results suggests that the components are of peptidergic nature. On the other hand, albumin content did not differ significantly, suggesting that the blood-brain barrier was not affected. An enzyme marker, dynorphin converting enzyme, remained unchanged in both groups.
Adult↗
Micropurification and amino acid sequence of beta-casomorphin-8 in milk from a woman with postpartum psychosis.
Milk was obtained from a woman with acute postpartum psychosis and with ongoing lactation. Defatted samples were subjected to micropurification and collected fractions were analyzed by means of their beta-casomorphin-8 immunoreactivity. Immunoreactive material with the same chromatographic properties as synthetic human beta-casomorphin-8 was determined by amino acid sequence analysis to be Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro. Its molecular mass was determined by fast atom bombardment-mass spectrometry to be 962.3 Da. These determinations, which ultimately identify the immunoreactive material as human beta-casomorphin-8, represent the first structural identification of a beta-casomorphin peptide from a body fluid.
Acute Disease↗
Mapping of IgE binding regions in the major rat urinary protein, alpha 2u-globulin, using overlapping peptides.
Exposure to laboratory animals poses a hazard for development of occupational allergy. Identification of antigenic determinants of allergenic proteins may be valuable for immunotherapeutic purposes. Overlapping peptides of the major allergen in rat urine, Rat n 1.02, corresponding to the protein alpha2u-globulin were synthesised on solid support and screened simultaneously to locate IgE binding linear epitopes using a simple modified ELISA procedure. Thirty-nine peptides were synthesised, each 8 amino acids long with 4 amino acids overlaps. Sera from fifteen rat-sensitized subjects were analyzed and as controls sera from 7 non-rat-sensitized individuals were used. In general low binding and a great individual variation between sera from rat allergic individuals were seen. Some peptides were more frequently recognized by IgE antibodies in sera from rat allergics. These peptides were mainly clustered towards the N-terminal and C-terminal parts of the protein. Taken together our data suggest the existence of linear IgE binding epitopes in the rat urine allergen, Rat n 1.02. However, the role of these sequences in the allergic reaction needs further investigation.
Alpha-Globulins↗
Microanalysis of proteins and peptides.
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Chromatography, Liquid↗