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Biomedical subjects

M Schleyer

Publications and source records attributed to M Schleyer.

At least 37 records · Page 2Linked to original sources
PubMed1993

Nucleotide sequence and 3'-end deletion studies indicate that the K(+)-uptake protein kup from Escherichia coli is composed of a hydrophobic core linked to a large and partially essential hydrophilic C terminus.

The kup (formerly trkD) gene from Escherichia coli encodes a minor K(+)-uptake system. The gene is located just upstream of the rbsDACBK operon at 84.5 min on the chromosome and is transcribed clockwise. kup codes for a 69-kDa protein, which may be composed of two domains. The first 440 amino acid residues appear to form an integral membrane protein that might traverse the cell membrane 12 times. The C-terminal 182 amino acid residues are predicted to form a hydrophilic domain located at the cytoplasmic side of the membrane. Deletion studies from the 3' end of kup showed that removal of almost the complete hydrophilic domain of the protein reduced, but did not abolish, K(+)-uptake activity.

Amino Acid Sequence↗
PubMed1991

Protein folding causes an arrest of preprotein translocation into mitochondria in vivo.

With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment.

Aminopterin↗
PubMed1990

Comparative analytical studies on recombinant and native human somatotropin.

In polyacrylamide gel electrophoresis and isoelectric focusing, somatotropin produced by recombinant DNA technology is as heterogeneous as highly purified native pituitary somatotropin. This heterogeneity is not attributable to different degrees of deamination of a single molecular species. In addition to the main protein of 22 kDa, the cloned products contain traces of interchain disulphide dimers of somatotropin. The quantitative amino acid analyses of the three cloned somatotropins investigated are neither identical nor do they correspond to the analysis of the native pituitary hormone. Moreover, there are discrepancies between the amino acid compositions of the hormones studied and the generally recognized amino acid analysis for human somatotropin.

Amino Acids↗
PubMed1988

Reconstitution of SEC gene product-dependent intercompartmental protein transport.

Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.

Adenosine Triphosphate↗
PubMed1985

Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes.

Translocational intermediates of precursor proteins of ATPase F1 beta subunit and cytochrome c1 across mitochondrial membranes were analyzed using two different approaches, transport at low temperature and transport after binding of precursor proteins to antibodies. Under both conditions precursors were partially transported into mitochondria in an energy-dependent manner. They were processed by the metalloprotease in the matrix but a major proportion of the polypeptide chains was still present at the outer face of the outer mitochondrial membrane. We conclude that transfer of precursors into the inner membrane or matrix space occurs through "translocation contact sites"; precursor polypeptides to F1 beta and cytochrome c1 enter the matrix space with the amino terminus first; and a membrane potential is required for the transmembrane movement on an amino-terminal "domain-like" structure but not for completing translocation of the major part of the polypeptides.

Antibodies↗
PubMed1985

Development of ischemia-induced damage in defined mitochondrial subpopulations.

Two mitochondrial subpopulations were isolated from guinea-pig heart by density gradient centrifugation. Under control conditions, both contain functionally intact mitochondria in which ischemic damage develops similarly. However, in one subpopulation adenine nucleotide content, adenine nucleotide translocase activity, oxidative phosphorylation and Ca2+ uptake are a quarter lower than in the other one when related to mitochondrial protein mass. Cytochrome contents and uncoupled electron flux are the same. Changes develop most evidently at the very beginning of ischemia for NAD-linked respiration. When ischemia progresses, cytochromes and the translocator protein are gradually lost or inactivated. Thereupon only partial recovery of mitochondrial function can be obtained after 20 min of reperfusion.

Adenine Nucleotides↗
PubMed1984

Proteinaceous receptors for the import of mitochondrial precursor proteins.

Mild trypsin treatment of isolated Neurospora mitochondria strongly inhibits their ability to bind and import the precursors of several mitochondrial proteins. Evidence is presented for two proteins, the ADP/ATP carrier and the mitochondrial porin, that specific binding of the precursors to the outer surface of the mitochondria is affected by the protease treatment. We suggest that the receptors that mediate the import of these two precursors are proteinaceous. Treatment of mitochondria with elastase also inhibits the binding and import of the ADP/ATP carrier and the porin. In contrast the import of the precursors of subunits 2 and 9 of the mitochondrial proton-translocating ATPase was unaffected by elastase treatment at the concentrations used. We suggest that the import pathways of the latter two proteins are distinct from those of the ADP/ATP carrier and the porin.

Adenosine Triphosphatases↗
PubMed1984

Transport of ADP/ATP carrier into mitochondria. Precursor imported in vitro acquires functional properties of the mature protein.

Precursor to ADP/ATP carrier synthesized in vitro is transferred into isolated mitochondria to a protease-resistant location. This process requires an electrical potential across the inner membrane. We show now that precursor imported in a cell-free system exhibits the same resistance to protease as the mature endogenous carrier. Furthermore, transferred protein, but not receptor-associated precursor, binds carboxy-atractyloside, a specific inhibitor of the mature carrier and can be isolated by the purification procedure for the mature carrier. At least 70% of the precursor associated with mitochondria in the presence of a membrane potential acquires this functional characteristic. Finally, the binding of carboxyatractyloside can be modulated by treatment of the imported protein with sulfhydryl reagents in a manner indistinguishable from the authentic carrier protein. We conclude that import in vitro leads to correct assembly and orientation of the ADP/ATP carrier in the mitochondria.

Animals↗
PubMed1983

Transfer of proteins into mitochondria. Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria.

The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.

Cell-Free System↗
PubMed1983

Preparative purification of human placental colony-stimulating factors.

For preparative purification of colony-stimulating factors (CSF) from human placental conditioned medium, cation exchange chromatography, gel filtration, and isoelectric focusing were combined. The individual fractions were tested using the growth in agar of cryopreserved human bone marrow (dose-response curves). Furthermore, the active preparations were characterized by analytical isoelectric focusing. Two fractions with highly enriched biological activity were isolated. They have the isoelectric points 5.4 and 5.6, respectively. Their specific activity exceeds that of the crude material by a factor of 60. Like crude material, they induce predominantly granulocytic colonies. The biochemical heterogeneity of biological activity in placental supernatants is discussed.

Carboxymethylcellulose Sodium↗
PubMed1983

The importance of the intramolecular disulfide bridges in porcine somatotropin for its biological activity.

The dependance of the porcine somatotropin activity on the entireness of the intrachain disulfide bridges is investigated in three biological systems: bioassay in vivo, radioimmuno assay and receptor assay with rat liver membranes. Cleavage of the disulfide bridges is accomplished by reduction, oxidation and sulfitolysis, respectively. In no case is the hormone activity completely abolished with regard to measurements in the bioassay and radioimmuno assay. In the radioligand assay with rat liver membranes neither native porcine somatotropin nor the treated hormone preparations are bound by the receptors.

Amino Acids↗
PubMed1982

Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria.

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation.

Adenosine Triphosphatases↗
PubMed1982

Heterogeneity of porcine somatotropin.

A chromatographical method is presented by which highly purified porcine somatotropin can be separated preparatively into five subfractions. The rechromatographed subfractions show comparable biological activities and molecular sizes, but they differ characteristically in the polyacrylamide gel electrophoresis and in the isoelectric focusing. Relative small differences are found in the amino acid composition of these fractions. The question is discussed, whether the term multiple forms or isohormones of porcine somatotropin is suitable for the growth promoting fractions.

Amino Acid Sequence↗
PubMed1982

Dependence of human somatotropin activity on interchain disulfide bridges.

The importance of the disulfide bridges for human somatotropin activity is investigated. The activity of somatotropin is tested by the tibia, radioimmuno-, and radioligand assays. The cleavage of disulfide bridges by sulfitolysis, reduction with dithiothreitol, or oxidation with performic acid does not completely abolish hormone activity. There is only one exception: in the radioligand assay, oxidized somatotropin is not able to displace native somatotropin from rat liver membranes. The diminution of hormone activity is independent of the charges of the groups introduced to the cysteine residues. The radioimmuno- and radioligand assays are more sensitive to conformational alterations in the somatotropin molecule than the biological test system.

Amino Acids↗