PubMed Health⌕ Search

Biomedical subjects

M Wiedmann

Publications and source records attributed to M Wiedmann.

At least 109 records · Page 6Linked to original sources
PubMed1990

Synthesis of bovine leukemia virus antigens in Escherichia coli.

Plasmids were constructed by the use of pEX vectors that encode and express different parts of the bovine leukemia virus (BLV): main core protein p24, nucleic acid-binding protein p12, transmembrane protein gp30, and different segments of envelope protein gp51. Expression of fusion proteins with molecular weights higher than 117 kD for all recombinant plasmids was shown in Coomassie-blue stained gels and by Western blot analysis with rabbit anti-BLV sera. Coupling of a gp51-encoding with a p24-encoding DNA fragment in pEX vectors led to synthesis of a fusion protein that was recognized by monoclonal antibodies directed against gp51 and p24 epitopes. Using another vector, a gp51-encoding DNA fragment of BLV was expressed as a fusion protein with 100 amino acids of the MS2 polymerase. The fusion protein was recognized by monoclonal antibodies directed against gp51.

Animals↗
PubMed1989

Photocrosslinking demonstrates proximity of a 34 kDa membrane protein to different portions of preprolactin during translocation through the endoplasmic reticulum.

Photocrosslinking has been used to identify integral proteins of the endoplasmic reticulum membrane that are in proximity to nascent preprolactin during in vitro translocation. A photoreactive lysyl derivative was introduced into truncated preprolactin chains comprising 86 or 115 amino acids. Both with the 86mer, containing the reactive group in the signal sequence, and with the 115mer, containing the probe exclusively in the mature portion of the chain, photocrosslinking occurred to an approximately 35 kDa transmembrane glycoprotein, the signal sequence receptor (SSR). SSR is identical with a previously isolated abundant and ubiquitous 34 kDa membrane protein that appears to be essential for protein translocation.

Animals↗
PubMed1989

A membrane component of the endoplasmic reticulum that may be essential for protein translocation.

We have purified a glycosylated, membrane-spanning protein of relative molecular mass approximately 34,000 (Mr approximately 34 K) from canine microsomes that appears to be essential for protein translocation across the endoplasmic reticulum (ER) as shown by the inhibitory action of antibodies directed against it and of monovalent Fab-fragments produced from them. The ER membrane contains at least as many molecules of the 34 K membrane protein as bound ribosomes. The protein can be detected immunologically in tissues of various organisms, indicating an universal function.

Animals↗
PubMed1989

Substitution of a pentalenolactone-sensitive glyceraldehyde-3-phosphate dehydrogenase by a genetically distinct resistant isoform accompanies pentalenolactone production in Streptomyces arenae.

Pentalenolactone (PL), an antibiotic produced by Streptomyces arenae, is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The producer strain contains different isoforms of GAPDH: a PL-sensitive enzyme on nonproduction media and a PL-insensitive enzyme on production media. After induction of PL synthesis, the sensitive GAPDH disappears parallel to the disappearance of its activity, as shown by Western (immunoblot) hybridization. The two isoenzymes exhibit little immunological cross-reactivity and differ in size, amino acid composition, and several amino acid residues of their amino termini. Two different types of plasmids from a S. arenae genomic library, named pBRPLR1 and pBRPLR2, were cloned in Escherichia coli by selection for enhanced PL resistance. Both contain a GAPDH structural gene. Plasmid pBRPLR1 increases E. coli PL tolerance 7-fold, and plasmid pBRPLR2 increases it 30-fold. GAPDH from pBRPLR1 transformants shows biphasic PL inactivation kinetics. These cells contain PL-sensitive GAPDH from both E. coli and S. arenae. GAPDH from pBRPLR2 transformants tolerates higher PL concentrations than either E. coli or S. arenae PL-sensitive GAPDH but is less resistant than S. arenae PL-insensitive GAPDH. Nondenaturing polyacrylamide electrophoresis showed this GAPDH to be a hybrid of E. coli and S. arenae PL-insensitive GAPDH. The hybrid enzyme could be purified to homogeneity. Induction of the lacZ promoter of pUC subclones of both GAPDH genes had only a small effect on raising the level of intracellular GAPDH.

Amino Acid Sequence↗
PubMed1988

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

Affinity Labels↗
PubMed1988

Oxygen limitation induces indirectly the synthesis of cytochrome P-450 mRNA in alkane-growing Candida maltosa.

Candida maltosa cells grown on hexadecane under oxygen limitation have an up to 6-fold higher cytochrome P-450 content in comparison to cells cultivated at oxygen saturation. We show by mRNA quantification using an in vitro translation system and subsequent specific immunoprecipitation that the cytochrome P-450 induction occurs mainly on the transcriptional level. The signal for induction may be the enhanced intracellular hexadecane concentration owing to a reduced hydroxylation capacity of the cytochrome P-450 at oxygen limitation.

Aerobiosis↗
PubMed1988

Post-translational transport of proteins into microsomal membranes of Candida maltosa.

We have isolated from the yeast Candida maltosa microsomal membranes that are active in the translocation of proteins synthesized in cell-free systems derived from C. maltosa, Saccharomyces cerevisiae or wheat germ. Translocation and core glycosylation of prepro-alpha-factor, a secretory protein, were observed with yeast microsomes added during or after translation. The signal peptide is cleaved off. Cytochrome P-450 from C. maltosa, the first integral membrane protein studied in a yeast system, is also inserted both co- and post-translationally into Candida microsomal membranes. Its insertion into canine microsomes occurs efficiently only in a co-translational manner and is dependent on the function of the signal recognition particle.

Candida↗
PubMed1987

Protein translocation across wheat germ microsomal membranes requires an SRP-like component.

Different wheat germ extracts were tested for the presence of membranes capable of translocating and processing nascent secretory proteins. One lysate was found in which nascent prehuman-placental lactogen (phPL) was translocated and processed to mature human placental lactogen (hPL). Processing was found to occur concomitant with translocation across membranes. Translocation across the wheat germ membrane required a component which is similar to the mammalian signal recognition particle (SRP). It bound to DEAE-Sepharose, had a sedimentation coefficient of 11S and contained a 7S RNA. In addition to hPL, the plant protein zein and the bacterial protein beta-lactamase were translocated across and processed by wheat germ membranes. Transport was found to occur only co-translationally. Our results show that the wheat germ protein translocation system is similar to the mammalian one. Unlike the mammalian SRP, the particle purified from wheat germ did not arrest elongation of nascent secretory proteins.

Journal Article↗
PubMed1987

Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

Animals↗
PubMed1986

N-alkanes induce the synthesis of cytochrome P-450 mRNA in Candida maltosa.

In the yeast Candida maltosa the level of cytochrome P-450 was 100-300-fold higher in alkane-grown cells than in glucose-grown ones (detected by a radioimmunoassay). It was shown immunochemically (1) that this was not the result of an assembly of preexisting apoenzyme with the prosthetic heme group. By cell-free translation of total poly(A)RNA in a wheat germ system and subsequent immunoprecipitation it was shown that the amount of mRNA coding for cytochrome P-450 paralleled its concentration in the cell.

Alkanes↗
PubMed1986

A signal sequence is required for the functions of the signal recognition particle.

It is shown that removal of the signal sequence of carp preproinsulin by gene technology abolishes the interaction between the polypeptide chain and the signal recognition particle with respect to both its functions: the inhibition of translation by the signal recognition particle in the absence of microsomal membranes, and the reconstitution of the protein translocation competence of high-salt inactivated microsomes. It is concluded that a signal sequence is absolutely required for the functions of the signal recognition particle.

Animals↗
PubMed1986

Internally transposed signal sequence of carp preproinsulin retains its functions with the signal recognition particle.

It is shown that the signal sequence of carp preproinsulin is functional with the dog pancreatic signal recognition particle (SRP) both when present at its normal location at the amino-terminus of the protein or when engineered to an internal location. Inhibition of translation by SRP in the absence of microsomal membranes, reconstitution by SRP of the translocation competence of high-salt inactivated microsomes and signal peptide cleavage all occur with the signal sequence being preceded by a highly charged peptide segment of 39 amino acid residues (the distance from the amino-terminus to the cleavage site of the signal peptidase is increased to 56 residues).

Amino Acid Sequence↗
PubMedDate not supplied

Xenopus oocytes can secrete bacterial beta-lactamase.

Most secretory proteins are synthesized as precursor polypeptides carrying N-terminal, hydrophobic sequences which, by means of a signal recognition particle (SRP), trigger the membrane transfer of the polypeptide and are subsequently cleaved off. The signal sequences appear to be interchangeable between prokaryotes and eukaryotes. In bacteria, secretion only involves the crossing of a membrane, whereas in eukaryotes the secretory process can be separated into two distinct phases: translocation across the membrane of the rough endoplasmic reticulum and subsequent intraluminal transport by processes involving vesicle budding and fusion. Since secretory proteins must be distinguished from other soluble proteins destined for various sites in the reticular system, it is conceivable that eukaryotic secretory proteins possess additional markers distinct from the signal peptide to guide the polypeptide after its transfer through the membrane. Proteins are secreted at different rates from a eukaryotic cell, suggesting a role in intracellular transport for receptors with differing affinities for some topogenic features in secretory proteins. We have tested this possibility by introducing into the lumen of eukaryotic rough endoplasmic reticulum a prokaryotic protein which, by virtue of its origin, had not been adapted to the eukaryotic secretory pathway. We reasoned that secretion of the bacterial protein would indicate that after membrane transfer no topogenic signal(s) and corresponding recognition system(s) are required. We report here that this is indeed the case.

Animals↗
PubMedDate not supplied

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle.

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.

Affinity Labels↗