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J Kellermann

Publications and source records attributed to J Kellermann.

16 recordsLinked to original sources
PubMed1992

Impaired intracellular transport and cell surface expression of nonpolymorphic HLA-E: evidence for inefficient peptide binding.

The assembly of the classical, polymorphic major histocompatibility complex class I molecules in the endoplasmic reticulum requires the presence of peptide ligands and beta 2-microglobulin (beta 2m). Formation of this trimolecular complex is a prerequisite for efficient transport to the cell surface, where presented peptides are scanned by T lymphocytes. The function of the other class I molecules is in dispute. The human, nonclassical class I gene, HLA-E, was found to be ubiquitously transcribed, whereas cell surface expression was difficult to detect upon transfection. Pulse chase experiments revealed that the HLA-E heavy chain in transfectants, obtained with the murine myeloma cell line P3X63-Ag8.653 (X63), displays a significant reduction in oligosaccharide maturation and intracellular transport compared with HLA-B27 in corresponding transfectants. The accordingly low HLA-E cell surface expression could be significantly enhanced by either reducing the culture temperature or by supplementing the medium with human beta 2m, suggesting inefficient binding of endogenous peptides to HLA-E. To analyze whether HLA-E binds peptides and to identify the corresponding ligands, fractions of acid-extracted material from HLA-E/X63 transfectants were separated by reverse phase HPLC and were tested for their ability to enhance HLA-E cell surface expression. Two fractions specifically increased the HLA class I expression on the HLA-E transfectant clone.

Animals↗
PubMed1992

Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli.

Maltodextrin glycosyltransferase (4-alpha-glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4-alpha-glucanotransferase, an approximately 53-kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55 degrees C up to 80 degrees C. The proposed action modus is the transfer of 1,4-alpha-glucanosyl chains, thus resulting in the disproportionation of 1,4-alpha-glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low-molecular-mass malto-oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto-oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4-alpha-glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues. Comparison of the N-terminal amino acid sequence of 4-alpha-glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N-termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch- and maltodextrin-converting enzymes.

Amino Acid Sequence↗
PubMed1992

Exercise dependent complete left bundle branch block.

Eleven patients with an exercise dependent complete left bundle branch block (CLBBB) were followed-up over a period of 2-13 years (mean 6.5 +/- 3.8). Their ages ranged from 19 to 62 years (mean 48). Four patients complained of chest pain on effort and one of palpitations. All patients underwent a clinical examination, 12 lead ECG, routine blood tests, chest X-ray, a multistage exercise test, echo Doppler, radionuclide ventriculography with TC99 and 48-h Holter monitoring. Ten were submitted to a coronary angiography with left ventriculography. The ECG at rest displayed a normal ECG in seven patients and an incomplete left bundle branch block (ILBBB) in four patients. The onset heart rate (HR) of CLBBB ranged from 95-146 beats.min-1 (mean 123) and the offset HR75-135 (mean 102 beats.min-1). Coronary angiography showed three-vessel disease in two patients and an obstruction of the left anterior descending coronary artery (LAD) in the third. In the other seven patients all the investigations (including coronary angiography) were normal. During the follow-up period the HR at onset of CLBBB decreased from 145 beats.min-1 to 100 beats.min-1 in four patients but no coronary artery disease (CAD) could be proven at coronary angiography. In our series chest pain did not always signify the presence of CAD. We conclude, that in patients with exercise-dependent CLBBB the prognosis is good if no underlying heart disease can be detected. It appears from our limited experience that an exercise-dependent CLBBB at heart rate below 125 beats.min-1 does not by itself constitute a sign of CAD.

Bundle-Branch Block↗
PubMed1992

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.

Amino Acid Sequence↗
PubMed1991

Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae.

Poly(A) polymerase is essential for the maturation of messenger RNA, adding tracts of adenosine residues to the 3' end of precursor RNA generated by endonucleolytic cleavage. This mechanism of mRNA 3' processing seems to be similar in yeast and in higher eucaryotes, although there are differences in the recognition signals in the pre-mRNA. Here we describe the cloning of the gene for yeast poly(A) polymerase. The enzyme is encoded by a single and essential gene located near the centromere on the left arm of chromosome 11. Poly(A) polymerase purified from recombinant Escherichia coli has the same physical and biochemical properties as the yeast enzyme. The yeast poly(A) polymerase shares features of sequence with its mammalian homologue.

Amino Acid Sequence↗
PubMed1991

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.

Amino Acid Sequence↗
PubMed1991

Differential expression of two cadherins in Xenopus laevis.

Using a cadherin fraction from Xenopus tissue culture cells as an immunogen, two monoclonal antibodies were obtained that allowed the characterization of two distinct cadherins in the Xenopus embryo. The two cadherins differ in molecular weight, in their time of appearance during development and in their spatial pattern of expression. One of the antigens was identified as E-cadherin. It appears in the embryonic ectoderm during gastrulation when epidermal differentiation commences and it disappears from the neural plate area upon neural induction. The second antigen could not be allocated to any of the known cadherin subtypes and was termed U-cadherin. It is present in the egg and becomes deposited in newly formed inner cell membranes during cleavage, the outer apical membranes of the embryo remaining devoid of the cadherin throughout development. U-cadherin is found on membranes of all cells up to the late neurula stages. A conspicuous polarized expression of the antigen on the membranes of individual inner cells suggests its participation in the segregation of cell layers and organ anlagen. These findings are discussed in the context of current hypotheses on the role of cadherins in establishing the spatial structure of the embryo.

Animals↗
PubMed1991

Biosynthesis of tetrahydrofolate. Sequence of GTP cyclohydrolase I from Escherichia coli.

The sequence of the gene coding for GTP cyclohydrolase I of Escherichia coli and of the adjacent regions was determined. The open reading frame contains 669 nucleotides. The deduced amino-acid sequence represents a protein consisting of 223 amino-acid residues with a molecular mass of 24,873 Da. Partial amino-acid sequences of the N-terminal region and of 5 peptides obtained by trypsin and BrCN cleavage were determined by Edman degradation and were in full agreement with the sequence deduced from the nucleotide sequence. The starting methionine is removed by posttranslational modification. The protein shows extensive homology to the recently reported GTP cyclohydrolase from rats.

Amino Acid Sequence↗
PubMed1990

Mapping of the prekallikrein-binding site of human H-kininogen by ligand screening of lambda gt11 expression libraries. Mimicking of the predicted binding site by anti-idiotypic antibodies.

High molecular weight (H-)kininogen, a non-enzymatic cofactor of the contact activation system, has on the COOH-terminal part of its light chain a unique binding site which complexes prekallikrein or factor XI with high affinity and specificity. In a conventional protein fragmentation approach, the prekallikrein-binding site was mapped to positions 556-595 of the human H-kininogen sequence (Tait, J. F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). To gain more insight into the minimum structural requirements of the prekallikrein-binding site, we have developed an alternative strategy employing the lambda gt11 expression cloning system. A ligand assay was established which probes for the binding site in H-kininogen or recombinant fusion proteins thereof by complexation with prekallikrein, followed by a specific antibody against prekallikrein and a secondary labeled antibody. A cDNA library constructed in lambda gt11 from random fragments of a cDNA clone encoding the COOH-terminal part of the kininogen light chain was screened by the ligand assay, and 17 positive clones were identified. Analysis of their inserted cDNA sequences revealed a consensus sequence of 119 nucleotides which maps to the extreme 3' end (positions 1759-1877) of the coding part of the prekininogen mRNA. The consensus sequence encodes positions 569-607 of the kininogen light chain and overlaps by 27 residues (positions 569-595) with the binding segment identified previously by the fragment approach. Analysis of successively shortened peptides revealed that the common segment of 27 residues but not truncated versions thereof contains the essential structural elements for prekallikrein binding. This conclusion was corroborated by the finding that anti-idiotypic antibodies toward a monoclonal antibody directed to the binding segment of 27 residues bear internal image(s) of the binding site of H-kininogen. It is pointed out that the methodology described in this study may prove generally useful in the cloning and mapping of high affinity binding sites of proteins.

Amino Acid Sequence↗
PubMed1990

Riboflavin synthases of Bacillus subtilis. Purification and amino acid sequence of the alpha subunit.

Bacillus subtilis has two different riboflavin synthases characterized by the subunit structures alpha3 (light enzyme) and alpha3beta60 (heavy enzyme). The light enzyme was purified by a novel procedure with increased yield and excellent reproducibility. The proposed trimer structure was confirmed by cross-linking experiments with dimethyl suberimidate. Fragments of alpha subunits were prepared by cleavage with cyanogen bromide, trypsin, protease Lys-C, and Staphylococcus aureus protease V8, respectively. Sequences were determined by automated liquid or gas phase Edman degradation. The complete sequence (202 amino acids) was established by direct sequencing of the N terminus and sequencing of overlapping peptides. The sequence shows marked internal homology between the NH2-terminal and COOH-terminal half encompassing 26 identical positions and 23 conservative replacements. This suggests that the protomer forms two structurally similar domains. Since it is known that the enzyme has two binding sites per subunit for the substrate 6,7-dimethyl-8-ribityllumazine, it appears likely that each of the homologous protein domains provides one binding site. The stereochemical features of the enzyme mechanism and the structural relation of the alpha trimer to the beta60 capsid of heavy riboflavin synthase suggest that the six domains corresponding to the alpha subunit trimer are related by pseudo 32 symmetry.

Amino Acid Sequence↗
PubMed1990

Evidence of the existence of structurally distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the rabbit.

The flavin-containing monooxygenase has been purified from rabbit liver and lung microsomes. SDS-PAGE analysis shows that both enzyme forms migrate as a single band with an apparent Mr of 59000. The NH2-terminus of both forms is blocked. The liver oxidase contains a lower percentage of glutamine/glutamate and a greater amount of phenylalanine than does the lung flavoprotein. Polyclonal antibodies to a 14-amino-acid peptide obtained after CNBr cleavage of the liver oxidase cross-react with the microsomal and purified liver enzyme, but do not recognize the lung oxidase. HPLC profiles of tryptic digests of the liver and lung enzymes exhibit different patterns. Sequence alignment of selected peptides from the liver and lung oxidases reveals aberrant residues within homologous segments. These findings are interpreted to mean that both enzymes represent distinct gene products.

Amino Acid Sequence↗
PubMed1990

Ile-Ser-bradykinin is an aberrant permeability factor in various human malignant effusions.

In this study we provide evidence for the presence of the aberrant peptide, Ile-Ser-bradykinin, in various human malignant exudates. The peptide was detected by deproteinisation of the effusion, application to reversed-phase HPLC, collection of the fractions containing Ile-Ser-bradykinin (retention time 6.90 min), degradation with carboxypeptidase B, and rechromatography of the resulting des-Arg-Ile-Ser-bradykinin (des-Arg-ISB) (retention time 13.5 min). In addition, all positive samples were confirmed by amino acid analysis and most of them (7/8) by amino-acid sequencing. In malignant effusions from 8 patients out of a group of 113 patients, Ile-Ser-bradykinin was found in concentrations between 12 and 520 mumol. In 44 malignant effusions, Ile-Ser-bradykinin was suspected, but could not be confirmed by the required additional methods (amino-acid analysis, sequencing) because of its low concentration. Sixty eight benign effusions were negative for Ile-Ser-bradykinin.

Amino Acid Sequence↗
PubMed1989

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens.

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.

Amino Acid Sequence↗
PubMed1985

Limited proteolysis of human low-molecular-mass kininogen by tissue kallikrein. Isolation and characterization of the heavy and the light chains.

The limited proteolysis of human low-molecular-mass kininogen by kallikrein from tissue sources has been studied. Porcine pancreatic kallikrein applied in catalytic amounts split the kininogen molecule (apparent mass 68 kDa) with the release of lysyl-bradykinin (1 kDa). This generated a nicked kininogen molecule with a heavy chain and light chain interconnected via disulfide bridging. Following reductive cleavage of the disulfide bonds, the heavy chain of apparent mass 62 kDa was isolated by preparative sodium dodecyl sulfate electrophoresis, and the light chain of 5 kDa by reversed-phase high-performance liquid chromatography. The light chain was found to be composed of 38 amino acids with a single half-cystine residue. Amino-terminal sequence analysis revealed that the light chain is derived from the carboxy terminus of the kininogen molecule [Lottspeich et al. (1984) Eur. J. Biochem. 142, 227-232]. Immunological characterization of the isolated L chain indicated that it harbours antigenic site(s) unique for low-Mr kininogen as well as sites common to high-Mr and low-Mr kininogen.

Amino Acids↗
PubMed1985

Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data.

Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.

Amino Acid Sequence↗