PubMed Health⌕ Search

Biomedical subjects

E Wachter

Publications and source records attributed to E Wachter.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.

Amino Acid Sequence↗
PubMed1991

Biogenesis of the mitochondrial phosphate carrier.

The mitochondrial phosphate carrier (PiC) is a member of the family of inner-membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat PiC suggested the existence of an amino-terminal extension sequence in the precursor of PiC. By expressing PiC in vitro, we found that PiC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino-terminus of the mature protein was generated. Import of PiC showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of PiC was reconstituted when starting with the presequence-carrying precursor. These results are discussed in connection with the recently postulated role of PiC as an import receptor located in the outer membrane.

Amino Acid Sequence↗
PubMed1991

cDNA sequence and deduced amino acid sequence of the precursor of the 37-kDa inner envelope membrane polypeptide from spinach chloroplasts. Its transit peptide contains an amphiphilic alpha-helix as the only detectable structural element.

We present the nucleotide sequence and the deduced amino acid sequence of a cDNA clone that encodes the entire precursor of the 37-kDa inner envelope membrane protein from spinach chloroplasts. The precursor protein consists of 344 amino acids (Mr 38,976). In vitro processing followed by radiosequence analysis of the in vitro transcribed and translated precursor protein revealed that its transit peptide consists of only 21 amino acid residues. The transit peptide has the potential to form an amphiphilic alpha-helix with a strong hydrophobic moment. It is speculated that this structural element represents an ancestral envelope-targeting domain. The in vitro synthesized precursor protein is directed to the chloroplasts and it is inserted into the envelope membrane in an ATP-dependent manner. The mature protein (323 amino acid residues, Mr 36,830) has a moderate hydrophobicity and contains only one membrane-spanning segment which is located at the C-terminus and possibly anchors the protein within the envelope membrane.

Amino Acid Sequence↗
PubMed1991

Evasion of Yersinia enterocolitica serotype 03 from complement-mediated killing.

Using isogenic strains of Y. enterocolitica serotype 03 (isolate 75) differing in LPS side chains (S and R), plasmid content (p+ and p-) or selective failure of YOP1 expression (YOP1-) we observed comparable C9 consumption via the alternative pathway by all strains. Differences became apparent in the bactericidal assays in which the 75S p+ strain was resistant whereas the plasmid-negative S and R strains were killed. Increased but submaximal resistance was observed with the 75R p+ and 75S YOP1- strains indicating that LPS side chains and plasmid-encoded factors other than YOP1 also contribute to serum resistance. Deposition of C9 and terminal complement complex (TCC) formation were greatly reduced on the resistant 75S p+ strain compared to the sensitive 75S p- strain. The 75S YOP1- variant behaved like the 75S p- strain with respect to C9 deposition and TCC formation suggesting that YOP1 prevents terminal C activation. A comparison of TCC deposited on resistant and sensitive Y. enterocolitica revealed no differences with respect to their salt- and protease-resistance and their size. We conclude from our experiments that serum resistance of 75S p+ depends on several surface components YOP1 being the most important. The YOP1 protein clearly interferes with C9 deposition and TCC formation and thereby contributes to serum resistance of plasmid-positive, YOP1 expressing Y. enterocolitica.

Centrifugation, Density Gradient↗
PubMed1990

Isolation and sequence of an FK506-binding protein from N. crassa which catalyses protein folding.

Slow protein-folding reactions are accelerated by a prolyl cis/trans isomerase isolated from porcine kidney which is identical to cyclophilin, a protein that is probably the cellular receptor for the immunosuppressant cyclosporin A. Catalysis probably involves the isomerization of prolyl peptide bonds in the folding protein chains. Cyclosporin A inhibits folding catalysis by cyclophilin. Here we report the isolation, cloning, sequencing and expression of another protein with prolyl isomerase activity from Neurospora crassa which is unrelated to cyclophilin and which also catalyses slow steps in protein folding. This protein does, however, show sequence similarity to a human protein that binds to another, recently discovered immunosuppressive drug, FK506. Moreover, it shares 39% identity with the carboxy-terminal 114 residues of a cell-surface protein from the bacterium Legionella pneumophila, the causative agent of Legionnaires' disease. Catalysis of folding by the FK506-binding protein from N. crassa is inhibited by FK506, but not by cyclosporin A. Thus, at least two different classes of conformationally active enzymes (conformases) exist that catalyse slow steps in protein folding. Both occur in a wide variety of cells and are inhibited by immunosuppressive drugs.

Amino Acid Isomerases↗
PubMed1990

Molecular cloning of subunits of complex I from Neurospora crassa. Primary structure and in vitro expression of a 22-kDa polypeptide.

A lambda gt11 cDNA expression library was screened with antibodies directed against individual subunits of complex I from Neurospora crassa. Clones encoding cytoplasmically synthesized polypeptides with apparent molecular masses of 22, 29, 31, and 33 kDa were isolated. Northern blot analysis revealed that the corresponding genes are transcribed into mRNA species of about 0.85, 0.95, 1.3, and 1.4 kilobases, respectively. Further characterization of clones encoding the 22-kDa subunit was performed. A cDNA insert of 755 base pairs containing the complete coding sequence was used to express the polypeptide in vitro. A precursor of the protein is synthesized on cytoplasmic ribosomes without a cleavable signal sequence. Our data indicate that after import into the organelle and before assembly into complex I, the 22-kDa polypeptide forms intramolecular disulfide bridge(s). Nucleotide sequencing revealed an open reading frame coding for a protein of 183 amino acids. A molecular mass of 20,828 daltons was calculated. The polypeptide is hydrophilic and contains no obvious membrane-spanning domains. Eight cysteine residues arranged in a regular pattern are found in the primary structure of the protein. Therefore, this subunit is a good candidate to bind at least one of the iron-sulfur centers present in complex I of the respiratory chain.

Amino Acid Sequence↗
PubMed1990

Matrix processing peptidase of mitochondria. Structure-function relationships.

The mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP) cooperate in the proteolytic cleavage of matrix targeting sequences from nuclear-encoded mitochondrial precursor proteins. We have determined the cDNA sequence of Neurospora MPP after expression cloning. MPP appears to contain two domains of approximately equal size which are separated by a loop-like sequence. Considerable structural similarity exists to the recently sequenced yeast MPP as well as to Neurospora and yeast PEP. Four cysteine residues are conserved in Neurospora and yeast MPP. Inactivation of MPP can be achieved by using sulfhydryl reagents. MPP (but not PEP) depends on the presence of divalent metal ions for activity. Both MPP and PEP are synthesized as precursors containing matrix targeting signals which are processed during import into mitochondria by the mature forms of MPP and PEP.

Amino Acid Sequence↗
PubMed1989

Primary structure of a proteinase inhibitor released from goat serum inter-alpha-trypsin inhibitor.

An acid-resistant trypsin inhibitor was released from goat serum inter-alpha-trypsin inhibitor and isolated by affinity chromatography. The primary structure of the inhibitor was established and the inhibitory properties were estimated. The inhibitor designed gIK-14 was characterized as a serine proteinase inhibitor from the family of the double-headed Kunitz-type inhibitors as suggested.

Alpha-Globulins↗
PubMed1989

Sequence of a human keratin 13 specific cDNA encompassing coil 1B through the 3' end.

An expression library established in lambda gt11 with cDNA from squamous epithelium of the human upper digestive tract was screened with an antibody raised against keratin 13. A 1.2 kb fragment from the most strongly reacting plaque was sequenced and compared to known type I keratin sequences. The highest degree of homology was detected with the murine 47K type I keratin, which we consider to be the counterpart of human keratin 13. Tryptic peptides of keratin 13 were separated on a HPLC column and one peptide was sequenced. The amino acid sequence obtained supports the identity of the cDNA. An eight codon motif has been tandemly repeated in the C-domain of keratin 13. In spite of substantial divergence by point mutations and deletions, the remaining sequence homologies suggest that the C-domains of both the human keratin 13 and the orthologous murine protein have originated from a common ancestor.

Amino Acid Sequence↗
PubMed1989

Two out of the three kinds of subunits of inter-alpha-trypsin inhibitor are structurally related.

Inter-alpha-trypsin inhibitor is a 240-kDa plasma-protein complex of three different types of glycoproteins. Their stoichiometric relation in the complex is not yet known. One subunit results from proteolytic processing of a precursor protein composed of alpha 1-microglobulin and a double-headed Kunitz-type proteinase inhibitor protein. From this, only the inhibitor protein becomes part of the inter-alpha-trypsin inhibitor complex. Another subunit whose function is not yet understood is structurally unrelated to the first one as well as to other proteins of various data collections. Now we have obtained a cDNA clone coding for 837 amino acid residues of a precursor protein of the third subunit. Its primary structure is 40% identical to that of the completely known second-subunit precursor. Peptide sequences obtained from isolated inter-alpha-trypsin inhibitor represent a distinct part of only about two thirds of the predicted polypeptide precursor, suggesting that its maturation is very similar to that of the second subunit. Therefore, we conclude that the deduced primary structure covers about 98% of the mature third subunit.

Alpha-Globulins↗
PubMed1989

A family of mitochondrial proteins involved in bioenergetICS and biogenesis.

The respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. The functions of the subunits that do not contain prosthetic groups are largely unknown. The cytochrome reductase complex of Neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins I and II (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid of redox centres. Significantly, a cytochrome reductase fraction lacking these two subunits was inactive in electron transfer, and in yeast mutants with defective genes for either of the two subunits, assembly of the reductase is disrupted. Most mitochondrial proteins are imported into the mitochondrion as precursor proteins, and two proteins are necessary for cleaving their presequences, namely the matrix processing peptidase (MPP) and the processing enhancing protein (PEP), the latter strongly stimulating the activity of the former. Temperature-sensitive yeast mutants, which are affected in PEP or MPP, accumulate precursors at the nonpermissive temperature. We report here that subunit I of the cytochrome reductase can be grouped as members of the same protein family.

Amino Acid Sequence↗
PubMed1989

Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.

Animals↗
PubMed1989

The amino-acid sequence of the double-headed proteinase inhibitor from badger (Meles meles) submandibular glands.

Badger submandibular glands contain a double-headed secretory proteinase inhibitor. Its amino acid sequence was determined. Extensive homologies were found between this inhibitor and the corresponding inhibitors of fox, dog, lion and cat in both domains. As in fox and dog inhibitor, the trypsin-inhibiting domain of badger inhibitor contains an Arg residue in the reactive site in contrast to a Lys residue in the inhibitors of lion and cat. Domains I and II of badger inhibitor are structurally related both to the sequenced inhibitors of fox, dog, lion and cat and to the sequenced monovalent secretory pancreatic trypsin inhibitors. The sequence of the badger inhibitor is N-terminally extended by four amino acids in comparison to fox and dog inhibitors and extended by eight amino acids in comparison to lion and cat inhibitors. Furthermore, the badger inhibitor is C-terminally extended by two amino acids in comparison to the lion inhibitor and by three amino acids in comparison to all other sequenced inhibitors.

Amino Acid Sequence↗
PubMed1988

Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms.

Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins.

Amino Acid Sequence↗
PubMed1988

Complementary DNA and derived amino acid sequence of the precursor of one of the three protein components of the inter-alpha-trypsin inhibitor complex.

Inter-alpha-trypsin inhibitor is composed of three distinct protein components. These protein components stem from independently encoded and proteolytically processed precursor proteins. Only the structure of the protein component responsible for the inhibitory activity has been established so far. We now present the complete amino acid sequence of the precursor of the second protein component derived from cloned cDNA. The precursor molecule includes both a signal peptide and a propeptide sequence and seems to be further processed prior to the assembly of the inter-alpha-trypsin inhibitor complex.

Alpha-Globulins↗
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↗