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E Wachter

Publications and source records attributed to E Wachter.

At least 55 records · Page 3Linked to original sources
PubMed1982

Folding of the mitochondrial proton adenosinetriphosphatase proteolipid channel in phospholipid vesicles.

The mitochondrial H+-ATPase proteolipid from Neurospora crassa was incorporated into small unilamellar dimyristoylphosphatidylcholine vesicles and its conformation determined by circular dichroism spectroscopy (CD). While the largely alpha-helical conformation is relatively independent of the method of incorporation into vesicles, i.e., rehydration, detergent dialysis, or detergent dilution, the proteolipid conformation was significantly different in detergent micelles and in organic solvents. Only very slight changes in the CD spectrum were observed upon binding of the H+-ATPase inhibitor dicyclohexylcarbodiimide to the proteolipid in vesicles, thus suggesting that the inhibitor acts either by blocking the channel or by masking an essential charge group, rather by than causing an overall conformational change in the channel. Additionally, very similar CD spectra were obtained for vesicles with different lipid/protein mole ratios, indicating either that no substantial conformational differences exist between monomer and multimers or that monomers self-associate to form stable complexes during incorporation into vesicles. This study has provided a physical basis for model-building studies of the proteolipid channel structure.

Adenosine Triphosphatases↗
PubMed1981

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment.

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.

Amino Acid Sequence↗
PubMed1981

Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.

Actins↗
PubMed1981

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IV. The amino acid sequence of the human urinary trypsin inhibitor isolated by affinity chromatography.

An acid-resistant trypsin inhibitor from human urine and serum is released in vivo by limited proteolysis from the high molecular acid-labile inter-alpha-trypsin inhibitor. The inhibitor shows an apparent molecular mass of 30 000 Da and is composed of two Kunitz-type domains. The domains are released in vitro by prolonged tryptic hydrolysis. The C-terminal domain is responsible for antitryptic activity. For the other domain no inhibitory activity towards proteinases, i.e. chymotrypsin, trypsin, pancreatic and leucocytic elastase has been demonstrated so far. The polypeptide chain comprising both domains consists of 122 residues and has a molecular mass of only 13 400 Da. In this work we have found that both, the N-terminal extension peptide with 21 residues and the "inactive" domain are linked O-glycosidically and N-glycosidically, respectively, with large carbohydrate moieties. The N-terminal amino acid sequence of the human urinary trypsin inhibitor was determined by solid-phase Edman degradation of a single peptide. The molecular mass calculated for the total polypeptide chain of 143 residues should be 15 340 Da; from the difference to the measured value (30 000 Da) it is concluded that the glycopeptide contains a considerable carbohydrate moiety.

Alpha-Globulins↗
PubMed1981

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, V. Attachments of carbohydrates in the human urinary trypsin inhibitor isolated by affinity chromatography.

The inhibitory active part of the inter-alpha-trypsin inhibitor with a known amino acid sequence is present as an acid-resistant inhibitor in human serum, in urine, in bronchial and in nasal mucus. The inhibitor molecule has a 50% carbohydrate content. Carbohydrate side chains are attached in two positions. One chain is linked to the polypeptide O-glycosidically via the serine residue in position 10 in the N-terminal extension peptide. The second side chain is attached N-glycosidically via the asparagine residue in position 24, located in the inactive "inhibitory" Kunitz-type domain of the inhibitor. The composition of the carbohydrate side chains were determined.

Alpha-Globulins↗
PubMed1980

N,N'-dicyclohexylcarbodiimide binds specifically to a single glutamyl residue of the proteolipid subunit of the mitochondrial adenosinetriphosphatases from Neurospora crassa and Saccharomyces cerevisiae.

The N,N'-dicyclohexylcarbodiimide-binding proteolipid subunit of the mitochondrial adenosinetriphosphatases (ATP phosphohydrolase, EC 3.6.1.3) of Neurospora crassa and Saccharomyces cerevisiae were purified from mitochondria incubated with the radioactively labeled inhibitor. The specifically labeled subunit was cleaved with cyanogen bromide and N-bromosuccinimide, and the resultant fragments were separated by gel chromatography in the presence of 80% (vol/vol) formic acid. The N,N'-dicyclohexylcarbodiimide label was recovered in each organism exclusively in a 17-residue fragment. Further analysis by automated solid-phase Edman degradation revealed that the bound label was present at only one position, corresponding to a glutamyl residue. The N,N'-dicyclohexylcarbodiimide-modified glutamyl residue is the only identical acidic position in both proteins and occurs in the middle of a hydrophobic sequence of about 25 residues.

Adenosine Triphosphatases↗
PubMed1979

Amino acid sequence of the aminoterminal segment of dermatosparactic calf-skin procollagen type I.

The N-terminal procollagen peptide of the pN alpha 1(I) chain from dermatosparactic calf skin contains 139 amino acid residues. For the determination of the amino acid sequence the procollagen peptide was treated with pyroglutamate aminopeptidase, protease from Staphylococcus aureus V8 and trypsin. The fragments obtained were separated by molecular sieve and ion-exchange chromatography and submitted to automated Edman degradation. The procollagen peptide consists of three segments, an N-terminal globular domain which contains all the cysteine residues and most of the hydrophobic residues present in the entire peptide, a triple helical part with a relatively high content of proline and hydroxyproline, and a short nonhelical region which forms the connection to the nonhelical region of the alpha 1(I) chain and which contains the proline-glutamine bond specifically split by the N-terminal procollagen peptidase during conversion of procollagen to collagen.

Amino Acid Sequence↗
PubMed1979

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.

Amino Acid Sequence↗
PubMed1979

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, I. Determination of the amino acid sequence of the antitryptic domain by solid-phase Edman degradation.

The acid-stable trypsin inhibitor of human serum and urine is released in vivo by limited proteolysis from the high molecular weight, acid-labile inter-alpha-trypsin inhibitor. When complexed with trypsin, both this acid-stable, active derivative and the inter-alpha-trypsin inhibitor can be degraded in vitro by prolonged digestion with trypsin to a low molecular weight "minimal" inhibitor. This minimal trypsin inhibitor was sequenced and found to be homologous to the known Kunitz-type inhibitors (e.g. the basic trypsin-kallikrein inhibitor from bovine organs). This indicates that the antitryptic activity of the big inter-alpha-trypsin inhibitor is due to a Kunitz-type domain.

Alpha-Globulins↗
PubMed1979

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, II. Characterization of a second inhibitory inactive domain by amino acid sequence determination.

A short digestion with excess of trypsin releases an inhibitor with an apparent molecular weight of 14,000 from both the inter-alpha-trypsin inhibitor and the ITI-related acid-stable inhibitor. The amino acid sequence of this inhibitor was determined. The inhibitor is composed of two covalently linked homologous Kunitz-type domains. One domain has antitryptic activity, as reported. This paper characterizes the second, inactive domain as also of the Kunitz type.

Alpha-Globulins↗
PubMed1979

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, III. Sequence of the two Kunitz-type domains inside the native inter-alpha-trypsin inhibitor, its biological aspects and also of its cleavage products.

The human inhibitor HI-14 consists of two Kunitz-type domains covalently connected. They are liberated from the human ITI by limited tryptic proteolysis. The inhibitor HI-14 is formed via a trypsin inhibitor complex. We have reported the amino acid sequences of the domain with antitryptic activity and the homologous domain without activity. Here we present the sequence of the domains as present in ITI. The domain lacking antitryptic activity is the N-terminal part of the inhibitor HI-14, whereas the domain with antitryptic activity represents the C-terminal part of HI-14 and probably the C-terminus of the ITI-molecule, too.

Alpha-Globulins↗
PubMed1979

The covalent structure of calf skin type III collagen. I. The amino acid sequence of the amino terminal region of the alpha 1(III) chain (position 1--222).

The amino terminal 227 amino acid residues of the alpha 1(III) chain contain four CNBr peptides: alpha 1(III)CB3A (79 residues), CB3B, CB3C (6 residues each), CB7 (37 residues) and CB6 (99 residues). Fragmentation of the CNBr peptides was carried out using trypsin, chymotrypsin and the protease from Staphylococcus aureus V8. The fragments obtained were isolated by a combination of molecular sieve and ion exchange chromatography. The sequence analysis was performed according to the automated Edman degradation procedure.

Amino Acid Sequence↗