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A Otto

Publications and source records attributed to A Otto.

At least 109 records · Page 6Linked to original sources
PubMed1994

Protein composition of the human heart: the construction of a myocardial two-dimensional electrophoresis database.

Molecular changes occurring in myocardial diseases are not well understood. Proteins, as regulatory molecules, should play an important role in the etiology of these diseases. The method of two-dimensional electrophoresis (2-DE) allows the analysis of some thousand proteins with one experiment. An important prerequisite for this kind of investigation is the possibility of identifying the proteins separated by 2-DE. We resolve 3239 proteins of the human myocardium and tried to identify 33 proteins by amino acid analysis and microsequencing. Twenty proteins were identified by search for the protein-chemical data obtained in the Martinsried Institute Protein Sequence Database. Comparisons of 2-DE patterns of different size, which were obtained in different laboratories, were performed with the result that proteins identified on a 2-DE map of one laboratory can be assigned to spots of 2-DE maps produced by another laboratory. Our results show the usefulness of a myocardial 2-DE database; they can be used in different laboratories and make it possible to generate a collection of important human myocardial proteins in a 2-DE database for comparative studies worldwide.

Amino Acid Sequence↗
PubMed1993

Bacterial protein disulfide isomerase: efficient catalysis of oxidative protein folding at acidic pH.

Periplasmic protein disulfide isomerase (DsbA) is essential for disulfide formation in Escherichia coli. The tryptophan fluorescence of DsbA measures the redox state of the enzyme during catalysis of the oxidative folding of hirudin, a thrombin inhibitor containing three disulfide bonds and lacking tryptophan. With stoichiometric amounts of DsbA, reduced hirudin is rapidly oxidized in a process initially leading to random disulfides. Disulfide reshuffling involving reduced DsbA yields completely native inhibitor within 1 h, even at pH 4. Catalytic amounts of DsbA become essential for hirudin folding in the presence of redox buffers at acidic pH. The second-order rate constants of disulfide exchange reactions involving DsbA are several orders of magnitude above the known values for alkyl dithiols and disulfide oxidoreductases. DsbA preferably reacts with reduced, unfolded polypeptides. The reduction of DsbA by hirudin is faster by 1 order of magnitude than its reduction by the strong reductant dithiothreitol. Together, unusually fast disulfide interchange reactions and a preference for folding polypeptides appear to be responsible for the catalytic efficiency of DsbA and for disulfide formation in vivo at acidic pH.

Amino Acid Sequence↗
PubMed1993

Recognition and separation of isoenzymes by metal chelates. Immobilized metal ion affinity partitioning of lactate dehydrogenase isoenzymes.

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites. It was demonstrated that the homotetramers (LDH-1)(H4) from rabbit, bovine and pig displayed weak binding to chelated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to this ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenzymes are bound to chelated copper in a reversed sequence. The comparison of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and PEG-dextran systems revealed that the discriminatory effect of copper is promoted by high salt concentrations. Resolution of isoenzymes by multiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk proteins. The efficacy of metal chelate affinity partitioning for the purification of LDH from tissue samples by batchwise extraction was also demonstrated.

Animals↗
PubMed1993

A tetrameric complex of membrane proteins in the endoplasmic reticulum.

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called 'signal sequence receptor' that is now renamed as 'translocon-associated protein' (TRAP). Two glycosylated subunits of the TRAP complex have been identified before (alpha and beta subunits). We now show that TRAP complex is actually comprised of four membrane proteins (alpha, beta, gamma, delta), present in a stoichiometric relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, non-glycosylated subunits were deduced from cloning of the corresponding cDNAs. The delta subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The gamma subunit is predicted to span the membrane four times.

Amino Acid Sequence↗
PubMed1993

Upregulation of mdm-2 expression in Meth A tumor cells tolerating wild-type p53.

Overexpression of mouse wild-type p53 (wt p53) in mouse Meth A tumor cells after transfection of wt p53 encoding vectors induced a strong growth-inhibitory response. Cells of only few of randomly selected surviving colonies contained and expressed the transfected wt p53 specific DNA. Despite expressing authentic wt p53, such cells (MethAp53wt) exhibited a similar phenotype as the parental Meth A cells. These cells overexpressed the mdm-2 (mouse double minute-2) gene, both at the RNA and at the protein level. Recently, the MDM-2 protein has been identified as a cellular target of p53, which can abolish its tumor suppressor activity. We, therefore, suggest that MDM-2 has mitigated the growth-inhibitory effect of wt p53 in MethAp53wt cells. Upregulation of mdm-2 expression in MethAp53wt cells was mediated by wt p53, as analysis of Meth A cells carrying a tsp53 (p53Val135) revealed a strict dependence of mdm-2 upregulation upon wt p53 expression. Our results propose that a balanced ratio of MDM-2 and p53 will allow cells to tolerate a limited expression of wt p53. This tolerance is not mediated by a direct inactivation of wt p53 via complex formation with MDM-2, as the majority of both MDM-2 and wt p53 in MethAp53wt cells was not complexed to each other.

Animals↗
PubMed1992

In vivo binding and clearance of circulating antigen by bispecific heteropolymer-mediated binding to primate erythrocyte complement receptor.

We have used the avidin/biotin system to construct soluble, cross-linked bispecific heteropolymers containing mAb to both the primate E C receptor and the DNP group. These heteropolymers facilitate in vitro binding of DNP-bovine gamma-globulin (DNP-BGG) to both human and squirrel monkey E. Intravenous injection in squirrel monkeys of DNP-BGG followed by heteropolymer leads to E binding and clearance from the circulation of a significant fraction of both heteropolymer and DNP-BGG, without lysis or clearance of the E. This methodology may potentially be used to treat a variety of infectious diseases and other syndromes associated with blood-borne pathogens.

Animals↗
PubMed1991

Characterization, stability and refolding of recombinant hirudin.

A recombinant variant of hirudin, the blood-clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide-bonded inhibitor from completely unfolded, reduced polypeptide chains. The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet ([theta]Mr,220 nm = -8 +/- 1 x 10(2) deg.cm2.dmol-1) indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent-exposed tyrosines, fluorescence emission and near-ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65 degrees C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two-state model with a transition enthalpy of delta Hvan't Hoff = 159 kJ/mol and a transition entropy of 464 J.mol-1.K-1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low-molecular-mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed-phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material.

Chromatography, High Pressure Liquid↗
PubMed1991

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model.

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.

Animals↗
PubMed1991

On the clinical importance of Dientamoeba fragilis infections in childhood.

Clinical and laboratory findings among 123 paediatric patients infected by intestinal protozoa were analysed. Dientamoeba fragilis (D. f) was found in 102 cases. The other patients proved to be carriers of Giardia lamblia or of mixed infections with several protozoa. Acute and recurrent diarrhoea have been found to be the most common symptoms, whereas abdominal pain was most common in children with chronic infections. Peripheral blood eosinophilia was seen in a third of the children with dientamoebiasis. Metronidazole, oxytetracycline, doxycycline, and erythromycin were effective drugs in the treatment of D. f. infections. The therapy coincidentally led to the elimination of protozoal infections as well as the abdominal complaints. These results underline the pathogenic role of D. f. in children with gastrointestinal symptoms.

Abdominal Pain↗
PubMed1990

The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents.

Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane.

Amino Acid Sequence↗
PubMed1990

New ELISA test for detection of bovine leukemia virus infections in cattle, using bacterially synthesized p24.

A new ELISA test is described for the detection of antibodies to bovine leukemia virus protein p24. This test employs a bacterially synthesized p24 antigen which represents a hybrid protein consisting of beta-galactosidase and about 70% of the mature viral p24. The antigen preparation was enriched from Escherichia coli cells to 95% purity and was used for the detection of antibodies in cattle. In a selected set of 100 positive field sera, 97 could be verified by the new test.

Animals↗
PubMed1989

Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins.

The growth-related 25-kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25. Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA-derived RNA probes demonstrated that the abundance of p25 mRNA is also growth-related. High-level expression of p25 in Escherichia coli has been established by oligonucleotide-directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7-promoter expression vector. Recombinant p25 from the expressed cDNA sequence has been shown to comigrate with EAT p25 in electrophoresis and to react with antibodies against the EAT p25. On the amino acid level, p25 shows about 80% sequence homology to the human stress protein hsp27. Furthermore, p25 has similar isoforms of phosphorylation as demonstrated for small mammalian stress proteins from rat and human. From the results obtained, it is concluded that p25 is a mammalian stress protein, the abundance of which is related to growth characteristics of the Ehrlich ascites tumor.

Amino Acid Sequence↗
PubMed1989

The growth-related protein P23 of the Ehrlich ascites tumor: translational control, cloning and primary structure.

p23 is a protein of Ehrlich ascites tumor cells, preferentially synthesized in the exponentially growing tumor. In vitro, serum and actinomycin D rapidly induce p23 synthesis. Using transcription inhibitors and a wheat germ cell-free translation system, evidence is provided that the synthesis of p23 is under translational control. Actinomycin D even results in superinduction of p23. Polymerase chain reaction, cloning and sequencing of p23 cDNA suggest p23 to be identical with a 21 kDa protein of mouse erythroleukemia cells, the synthesis of which was shown to be controlled also at the translational level (Chitpatima, S. T., Makrides, S., Bandyopadhyay, R., and Brawerman, G. (1988) Nucleic Acids Res. 16, 2350).

Amino Acid Sequence↗