Showing papers by "Friedrich Lottspeich published in 1988"
TL;DR: A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level.
Abstract: A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.
292 citations
TL;DR: Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which glesolin has been derived by a duplication.
171 citations
TL;DR: Two‐dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures.
Abstract: Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.
123 citations
TL;DR: Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA and two clones expressed sialidase activity when assayed with the fluorogenic substrate 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid.
86 citations
TL;DR: A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4), an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade.
Abstract: A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10(6)independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA we identified 9 other recombinants encoding a protein with considerable similarity (74%) TO PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.
76 citations
TL;DR: Drawing conclusions on the conformation of the phytochrome protein were drawn, sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are thought to be localized in the interior of the native phy tochrome.
Abstract: Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543-8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.
52 citations
TL;DR: The membrane portion of H + -ATPase from R. capsulatus was found to be composed of four polypeptides, and the ATP-synthase of this photosynthetic bacterium is in this respect similar to that of chloroplasts.
22 citations
TL;DR: The isolation of a major portion of the gene for nuclear factor I (NFI) including its 5′‐flanking region with transcriptional start sites is described including its5′‐ Flanking Region with transcriptionAL start sites.
21 citations
18 citations
TL;DR: It is demonstrated that TSP‐1 mRNA is only expressed in activated T lymphocytes and is absent from all mouse tissues tested including those containing resting mature T lymphocyte populations treated with alloantigen and/or lectin, and suggested that T SP‐1 gene transcription is a useful marker to characterize T effector cells in vitro and in vivo.
Abstract: An oligonucleotide probe corresponding to nucleotides of a cDNA encoding the T cell-associated proteinase 1 (TSP-1) was chosen to study the induction and expression of TSP-1-specific transcripts in mouse T lymphocytes and tissues. We demonstrate that TSP-1 mRNA is only expressed in activated T lymphocytes and is absent from all mouse tissues tested including those containing resting mature T lymphocytes. Expression of the TSP-1 gene was observed in T lymphocytes in vitro in response to either phorbolester (phorbol 12-myristate 13-acetate), Ca2+ ionophore (A23187), lectin or alloantigen. In general, TSP-1 mRNA appeared and peaked later compared to interleukin 2 transcripts. Furthermore, TSP-1 mRNA was inducible in vitro in both Ly-2+ and L3T4+ lymphocyte populations treated with alloantigen and/or lectin. The transcription of the TSP-1 gene was always accompanied by the expression of proteinase activity. High expression of TSP-1 transcripts was also observed in in vivo derived T effector cells specific for lymphocytic choriomeningitis virus. However, TSP-1 mRNA was predominantly associated with virus-specific Ly-2+ T cells and correlated with their proteinase and cytolytic activities. The data suggest that TSP-1 gene transcription is a useful marker to characterize T effector cells in vitro and in vivo.
15 citations
TL;DR: A potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins, comparable to recognition site DNA affinity chromatography for enrichment of nuclear factor I from porcine liver.
TL;DR: The dimeric structure of HuTSP, together with its extensive sequence homologies with the murine T cell specific proteinase, MTSP‐1, suggests phylogenetic conservation of this serine proteinase family.
01 Jan 1988
TL;DR: In this paper, the primary structure of the S-layer polypeptide of Deinococcus radiodurans has been determined using a 3D reconstruction of the cell envelope.
Abstract: Deinococcus radiodurans possesses a complex cell envelope which is quite unique regarding both structure and composition (Thompson et al. 1982, Brooks et al. 1980). The structure determination of its S-layer (HPI-layer) is particularly advanced (Baumeister et al. 1986, Rachel et al. 1986). As part of our objective to obtain a high-resultion 3-D structure of the HPI-layer we have determined the primary structure of the HPI-layer polypeptide.