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We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and ⊃125 -labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacryllamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3. [ABSTRACT FROM AUTHOR]

1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharide of Escherichia coli U41/14 (serotype 03 :K2ab(L) : H2) an unusual amino sugar was isolated. By sequential oxidation with periodate and hypoiodite and by oxidation with periodate, followed by detection of acetaldehyde formed, the ammo sugar was characterized as a 2-amino- dideoxyaldose. The hydrochloride of the amino sugar, in comparison to those of 2-amino-2, 6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2, 6-dideoxy-D-mannose, 2-amino-2, 6-di- deoxy-D-allose, and 2-amino-2, 6-dideoxy-D-gulose, was studied with aid of ion exchange column chromatography. The N-acetylated amino sugar was compared with the N-acetylated reference amino sugars with regard to their relative mobihties in paper chromatography. The ammo sugar alcohols and reference amino sugar alcohols were studied using paper electrophoresis in molybdate buffer. On the basis of the results obtained the unknown amino sugar was identified as 2-amino- 2,6-dideoxymannose (rhamnosamine). Its optical rotation showed it to be of the L-configuration. This is the first report on the natural occurrence of L-rhamnosamine. [ABSTRACT FROM AUTHOR]

In human breast cancer MCF-7 and MCF-7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor-2 (FGF-2) had opposite effects, stimulating MCF-7 and inhibiting MCF-7ras cell proliferation. The inhibitory signal induced by FGF-2 was related to sustained mitogen-activated protein kinase (MAPK) activation in MCF-7ras cells, while transient MAPK activation was associated with MCF-7 cell proliferation. FGF-2 was further used in combination with insulin or cAMP. In MCF-7 cells, insulin and cAMP reversed the mitogenic effect of FGF-2. In MCF-7ras cells, insulin did not modify the inhibitory effect of FGF-2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF-2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF-2 on cell proliferation is dependent on the activity of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway. [ABSTRACT FROM AUTHOR]

Carotenoid-depleted fruit flies, Drosophila melanogaster, were reared on yeast/glucose medium containing lipid-depleted white corn grits and cholesterol. After rearing for more than a year, the yield of flies remained constant and the content of 3-hydroxyretinal in a head was three logarithmic units less than that of normal flies reared on medium containing yellow corn grits. When all-trans retinal was supplied as the sole source of retinoids, the flies formed and accumulated all-trans 3-hydroxyretinal in the dark. To examine the metabolic pathway to produce (3S)-3-hydroxyretinal in Drosophila, all-trans retinal was supplemented for two hours to carotenoid-depleted flies in the dark, and the subsequent changes in the composition of 3-hydroxyretinal enantiomers were analyzed using a chiral column on HPLC. The results indicated initial formation of (3R)-3-hydroxyretinal followed by isomerization into the 3S enantiomer. In another set of experiments, the membrane fraction was obtained from the head homogenate of retinoid-depleted flies and an in vitro assay of 3-hydroxyretinal formation from retinal was performed. The 3-hydroxyretinal produced was the 3R enantiomer, supporting the result obtained from the in vivo experiment whereby (3S)-3-hydroxyretinal is produced from retinal via (3R)-3-hydroxyretinal. Addition of NADPH enhanced 3-hydroxyretinal formation and the presence of carbon monoxide inhibited it, suggesting that hydroxylation at the C3 position of retinal occurred via the monooxygenase activity of cytochrome P-450. [ABSTRACT FROM AUTHOR]

The role of Β-glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by the phosphoglucomutase for Β-glucose 1-phosphate (Β-phosphoglucomutase) has been examined in purified extracts of Euglena gracilis var. bacillaris. 1. The incubation of Β-glucose 1-[32P]phosphate with Β-phosphoglucomutase in presence of high concentrations (0.1 mM) of a commercial preparation of α-D(+)-glucose 1,6-bisphosphate (prepared synthetically) yielded a labelled compound running electrophoretically and chromatographically as the sugar bisphosphate. Specificity studies with Β-phosphoglucomutase and muscle phosphoglucomutase (α-phosphoglucomutase) strongly suggest that the compound formed is Β-glucose 1,6-[32P]bisphosphate. The results would also indicate that the commercial preparation of α-D(+)-glucose 1,6-bisphosphate contains Β-glucose 1,6-bisphosphate as contaminant. 2. When synthetic Β-glucose 1-phosphate preparations were purified by paper chromatography and then incubated with Β-phosphoglucomutase, no glucose 6-phosphate could be detected. On the other hand the reaction readily took place when challenged with the chromatographic fraction that runs as sugar bisphosphate. Upon analysis with etectrophoresis, chromatography and weak acid hydrolysis, and experiments with Β-phosphoglucomutase and α-phosphoglucomutase it was concluded that this compound is Β-glucose 1,6-bisphosphate. 3. It was established that the natural α-D(+)-glucose 1,6-bisphosphate not only fails to sustain the reaction catalyzed by Β-phosphoglucomutase but rather inhibits the reaction when added to the whole Β-phosphoglucomutase system, it was also demonstrated that Β-glucose 1,6-bisphosphate acts as inhibitor of the α-phosphoglucomutase system. 4. These results show for the first time that Β-glucose 1,6-bisphosphate is an active and necessary participant in the reaction catalyzed by Β-phosphoglucomutase. The role played by Β-glucose 1,6-bisphosphate in this reaction would be essentially the same as that of the α-anomer in the reaction catalyzed by α-phosphoglucomutase. [ABSTRACT FROM AUTHOR]

This article presents a list of several research papers to be published in the European Journal of Biochemistry. Some of listed papers are "Changes in the Insulin-Sensitive Glycosyl-Phosphatidyl-Inositol Signalling System With Aging in Rat Hepatocytes," "Localization of the Tightly Bound Divalent-Cation-Dependcnt and Nucleotidc-Dependent Conformation Changes m G-Actin Using Limited Proteolytic Digestion" and "Structure of the Promoter Region of the Gene Encoding Cytochrome c Oxidase Subunit V in Dictyostelium."

Succinate dehydrogenase flavocoenzyme (‘SD-flavin’), previously shown to be an 8α-substituted riboflavin derivative containing a tertiary nitrogen homoconjugated to the flavin nucleus, was subjected to further hydrolysis and to reduction under acid conditions. Both conditions resulted in the liberation of 1 mole of histidine per mole of flavin. This proves histidine to be the covalent link between flavin and peptide backbone in succinate dehydrogenase and imidazole to be the tertiary nitrogen function homoconjugated to the flavin. 8α-Histidyl-riboflavin has been synthesized starting from riboflavin chemically and shown to be completely identical with the natural product in optical, ESR and NMR spectra, pH-fluorescence curve and behavior on thin-layer and paper chromatography, as well as paper electrophoresis. [This equation cannot be represented into ASCII Text] 3. Both the natural compound isolated by acid hydrolysis of flavin peptide and the synthetic one contain two isomers, which may be separated by high voltage electrophoresis. The isomers appear to be the N(1)- and N(3)-imidazole substituted compounds. Digestion of the flavin peptide with aminopeptidase M yields only one isomer but on treatment with 6-N HCl this is gradually converted to a mixture of the two isomers. The absolute assignment of the natural isomer is suggested as 8α-[N(3)-histidyl]-riboflavin on the basis of imidazole quaternization with CH3I, reductive cleavage of the flavin-imidazole bond and identification of the methyl-histidine liberated as 1-methyl-histidine. [ABSTRACT FROM AUTHOR]

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojidmycin and A'-methyl-1-deoxynojidmycin [Hughes, R, C, Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544], Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [³H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean α-mannosidase. The data show that Glc3Man8GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2, Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosacchaddes to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases. [ABSTRACT FROM AUTHOR]

Several studies indicate that in human choriogonadotropin the N-linked oligosaccharide at position 52 of the α-subunit is important for bioactivity. We have generated choriogonadotropin mutants in which the α52 glycosylation site is removed and the α and β subunits are covalently linked by intersubunit disulfide bonds. These mutants display wild-type receptor binding and bioactivity. Furthermore, we show that removal of the α52 sugar leads to instability of heterodimeric choriogonadotropin. Therefore, we conclude that the α52 oligosaccharide of choriogonadotropin is not involved in signal transduction, but in the stability of the heterodimer. [ABSTRACT FROM AUTHOR]