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The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the red blood cells of an individual, homozygous for the Mi-VII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic peptide comprising the residues 40-61 of glycophorin A was deduced from manual sequence analyses. The Mi-VII-specific glycophorin A was shown to exhibit an arginine → threonine and a tyrosine → serine exchange at the positions 49 and 52 respectively. The threonine-49 residue was found to be glycosylated. Inhibition assays demonstrated that one of the Mi-VII-specific antigen determinants (Anek) is located within the residues 40-61 of glycophorin A and comprises sialic acid residue(s) attached to O-glycosidically linked oligosaccharide(s). Our data contribute to an understanding of the Miltenberger system and provide an explanation at the molecular level for the previous finding that the erythrocytes from the Mi-VII homozygote lack a high-frequency antigen (EnaKT), located within the residues 46-56 of normal glycophorin A. [ABSTRACT FROM AUTHOR]

The hybrid glycophorin in Dantu-positive human erythrocytes of the N. E. variety was not cleaved by treatment of intact cells with various proteases, in contrast to normal glycophorins. Therefore, it could be purified by phenol/saline extraction of membranes from trypsin-treated and chymotrypsin-treated red cells and subsequent gel filtration in the presence of Ammonyx-LO. The complete structure of the hybrid molecule, comprising 99 amino acid residues, was elucidated by sequence analyses of peptides prepared by chymotrypsin, trypsin, cyanogen bromide or V8 proteinase treatment. The N-terminal 39 residues and the glycosylation of the molecule were found to be indistinguishable from those of blood-group-s-specific glycophorin B. Conversely, the residues 39- 99 were shown to be identical with the residues 71 - 131 of the major blood-group M-active or N-active sialoglycoprotein (glycophorin A). Hemagglutination inhibition assays revealed that the Dantu antigen represents a labile structure. The receptor might be located within the residues approximately 28-40 of the hybrid glycophorin, as judged from the effects of modifications of membranes. Our data provide an explanation for the previous findings that Dantu-positive cells (N. E. type) exhibit a protease-resistant N antigen and a qualitatively altered s antigen. [ABSTRACT FROM AUTHOR]

The N-termini of four mitochondrial translation products, the var 1 protein, cytochrome b, and subunits l and III of cytochrome c oxidase have been characterized in Saccharomyces cerevisiae and compared with the known DNA sequences of the respective structural genes. The four mature proteins correspond to the predicted primary translation products and retain the formylated methionine residue. Thus. subunit II of cytochrome oxidase studied previously [Pratje et al. (1983) EMBO J. 2, 1049 - 1054] is so far the only mitochondrial translation product carrying a N-terminal-extended transient presequence in S. cerevisiae. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the N-terminal tryptic glycopeptide from a minor human erythrocyte membrane sialoglycoprotein (component D or glycophorin C) was determined by manual sequencing. The glycosylation sites were identified by a new procedure for the detection of the glycosylaled derivatives released by Edman degradation. The fragment, comprising 47 residues, was found to contain an average of about 12 O-glycosidically linked oligosaccharides and one asparagine-linked carbohydrate chain. An identical hexapeptide sequence occurring in two regions of the glycopeptide provides evidence that it has developed by an internal gene duplication during evolution. In addition, a part of its structure shows a striking similarity to the sequence of a certain region of the MN and Ss erythroeyte membrane sialoglycoproteins (glycophorins A and B), suggesting that the molecules might be related. [ABSTRACT FROM AUTHOR]

DNA-binding protein I from Escherichia coli was purified from cells carrying the ssh gene on a multicopy plasmid. In comparison to the strain without the recombinant plasmid the DNA-binding protein was overproduced more than 20-fold. The amount of the protein was measured after the purification steps by gel electrophoresis and radial immunodiffusion. The protein was purified to homogeneity and was active in replication assays like the wild-type DNA-binding protein. The assays were enzymatic replication of single-stranded and double-stranded fd DNA. E. coli DNA-binding protein I was further subjected to amino acid sequence analysis. A monomer of the protein consists of 187 residues which correspond to a molecular weight of 19715 with 5% error in analysis. The sequence of the amino-terminal 40 residues was determined and includes several basic residues of the protein. Sequence comparison between the DNA-binding protein I from E. coli and that coded by bacteriophage fd reveals similarities suggesting that DNA-binding protein I may use amino-terminal residues for binding to DNA like the phage protein. [ABSTRACT FROM AUTHOR]

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind nonspecifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the aminoterminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases. [ABSTRACT FROM AUTHOR]

The complete amino-acid sequence of the acyl carrier protein of citrate lyase from Klebsiella aerogenes (ATCC 13882) was determined by automated sequential Edman degradations of the protein and of peptides in a liquid-phase sequencer of Edman and a solid-phase sequencer of Laursen. The sequence composed of 78 residues does not contain tyrosine or histidine residues. The primary structure has been established as Met- Glu- Met- Lys- Ile- Asp- Ala- Leu- Ala- Gly- Thr- Leu- Glu- Ser- Ser- Asp- Val- Met- Val- Arg- Ile- Gly- Pro- Ala- Ala- Gln- Pro- Gly- Ile- Gln- Leu- Glu- Ile- Asp- Set- Ile- Val- Lys- Gln- Glu- Phe- Gly- Ala- Ala- Ile- Gln- Gln- Val- Val- Arg- Glu- Thr- Leu- Ala- Gln- Leu- Gly- Val- Lys- Glu- Cys- Asp- Asn- Val- Gin- Leu- Ala- Arg- Val- Gln- Ala- Ala- Ala- Leu- Arg- Trp- Gln- Gln. The molecular weight of the acyl carrier protein calculated from the amino acid sequence and the phosphoribosyl dephospho-CoA prosthetic group is 9378. This prosthetic group is bound in phosphodiester linkage to serine-14 of the acyl carrier protein. Circular dichroism measurements indicate that citrate lyase acyl carrier protein has a high degree of α-helical structure. In this respect and in molecular size the acyl carrier protein from citrate lyase resembles the acyl carrier protein from Escherichia coli fatty acid synthetase. However, with exception of two very short sequences the amino acid sequences of these two proteins are completely different. [ABSTRACT FROM AUTHOR]

The primary structure of the histidine-containing phosphocarrier protein HPr of the phosphoenol-pyruvate-dependent phosphotransferase system from Staphylococcus aureus was determined by automated Edman degradation. The complete sequence was deduced from the direct analysis of the protein by automated Edman degradation in a liquid-phase sequencer of Edman and from the sequence of tryptic, thermolytic and cyanogens bromide peptides as obtained by automated Edman degradation in a solid-phase sequencer of Laursen. The amino-acid sequence was found to be Met-Glu-Gln-Asn-Ser-Tyr-Val-Ile-Ile-Asp-Glu-Thr-Gly-Ile-His-ala-Arg-Pro-Ala-Thr-Met-Leu-Val-Gln-Thr-Ala-Ser-Lys-Phe-Asp-Ser-Ile-Asp-Gln-Gly-Gly-Tyr-Asp-Ser-Met-Gln-Leu-Lys-Ser-Leu-Gly-Val-Gly-Lys-Asp-Glu-Glu-Ile-The-Ile-Tyr-Ser-Ala-Asp-Lys-Glu-Gly-Leu-Thr-Lys-Met-Ser-Ile-Val. The 70 residues correspond to a molecular weight of 7685. The one histidine involved in the phosphotransfer reaction of this protein was found at position 15 as part of a region of the sequence which has no predictable secondary structure. It is suggested that this protein belongs to the group of male proteins with the active center located on a protrusion rather than a cleft. [ABSTRACT FROM AUTHOR]

The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion-exchange chromatography, gel filtration and preparative thin-layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl-Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19. The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N-terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication. [ABSTRACT FROM AUTHOR]

The filamentous amyloid protein aggregates found in the brain of patients affected with Alzheimer's disease principally consist of a peptide termed βA4, according to its secondary structure of β-pleated sheets and its molecular mass of about 4 kDa. It has a length of up to 42 or 43 residues. By chemical means, we have synthesized peptide analogs corresponding to the human and rodent βA4 sequences. We describe structural and functional properties of peptides spanning residues 1–43, 10–23, 1–27 and 4–27 of βA4. The peptides have been tested for their ability to form filaments in vitro. Their solubilities and secondary structures in solution and in the solid state have been used to detect differences between the properties of human and rodent βA4 sequences. We show that mouse and rat βA4 homologs are as amyloidogenic as the human sequence. The absence of amyloid deposits in the brain of aged rats and mice is therefore not due to the three amino acid substitutions identified within the sequence which is homologous to βA4 of humans. Moreover, peptides corresponding to residues 1–27 of human and rodent βA4 are solubilized under physiological conditions; thus they are very unlikely to form stable filaments in vivo. [ABSTRACT FROM AUTHOR]

Glycophorin C (GPC) and D (GPD) are minor glycoproteins which are believed to be important for the structural integrity of the red cell membrane. We have investigated the structural relationship between these glycoproteins by both immunological and structural investigations: 1. A rabbit anti-serum produced against GPD reacts strongly with GPC and the abnormal glycoproteins of Gerbich: -2,-3 and Gerbich: -2,3 red cells, and recognizes most probably the homologous C-terminal portions of GPC and GPD. The two molecules however differ at their N-terminus. 2. One-dimensional mapping of the peptides obtained after tryptic, chymotryptic, V8 protease or acid cleavage of 125I-labelled GPC and GPD, indicated that GPC and GPD are structurally related but some differences were found indicating that additional peptides were generated from GPC. 3. The partial primary structure of GPD was determined. The sequencing data are consistent with the assumption that GPD represents an abridged version of GPC that comprises residues approximately 21/29-128 and exhibits a N-terminal residue that is blocked by an as yet undefined group. [ABSTRACT FROM AUTHOR]

Analyzes the relevance of conformation for the antigenic site A of the hemagglutinin of influenza virus. Synthesis of two peptides with variant sequences of the central part of site A; Formation of a loop structure on the sequence stretch; Assumption that the loop structure approximates to the structure of the cognate protein sequence; Production of anti-pedtide antibodies; Revelation of antibodies reactive against the influenza virus.

The N-terminal structures of the MN and Ss erythrocyte membrane sialoglycoproteins (glycophorins A, B) from two Henshaw (He) blood-group heterozygotes were determined by manual sequencing of tryptic glycopeptides and various secondary fragments. No structural alteration of the MN glycoprotein could be detected. The He-specific portion of the Ss glycoprotein was found to exhibit the N-terminal sequence Trp-...-...-...-Gly- (+ =glycosylation). Thus it differs at three positions from its normal counterpart which possesses 'N' activity and exhibits the N-terminal structure Leu-...-...-...-Glu-. Analysis of the Ss glycoprotein from 15 He-negative erythrocyte samples did not reveal any of the three He-specific structural alterations. The presence of a glycine residue at the fifth position of the blood-group-M-active MN glycoprotein as well as in the He-specific Ss glycoprotein provides an explanation for the occurrence of antisera (anti-Me) reacting with the M and He antigens. [ABSTRACT FROM AUTHOR]

The N-terminal structure of the Miltenberger (Mi-) blood group class I and II specific human MN erythrocyte membrane sialogycoproteins were determined by manual sequencing of tryptic glycopeptides and various secondary fragments. The Mi-I and Mi-II active glycoproteins were found to exhibit a threonine → methionine and theonine → lysine exchange, respectively, at position 28 which prevents N-glycosylation of asparagines 26. Due to the absence of the N-glycosidic oligosaccharide chain the monomeric form of the Mi-I and Mi-II specific glycoproteins possesses a slightly increased sodium dodecyl sulfate/polyacrymide gel electrophoretic mobility, in comparison to its normal counterpart. Serological studies suggest that antibodies, specific for Mi-I or Mi-II red cells, react with the structurally altered region of the MN glycoprotein. [ABSTRACT FROM AUTHOR]

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded amino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few amino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form. [ABSTRACT FROM AUTHOR]

A procedure for the reconstitution of the lactose carrier protein, a galactoside : proton symporter in Escherichia coli, is described. Starting from cytoplasmic membranes derived from carrier-overproducing strains, essentially all proteins including 89 % of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl O-β-D-maltoside. In the miceIlar state the carrier binds substrates with reduced affinity. Addition of E. coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50 - 60-nm diameter). In these vesicles, about 70 % of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding. After fusion of the small vesicles into larger vesicles (1 -5 μm), rapid countertransport of galactosides is demonstrated. Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons. [ABSTRACT FROM AUTHOR]

The ultimate step in Alzheimer's disease Abeta generation involves gamma-secretase, which releases Abeta from its membrane-bound precursor. A similar presenilin-dependent proteolytic activity is implicated in the release of the Notch intracellular domain. We have developed a novel assay for gamma-secretase activity based on green fluorescent protein detection. This involves cotransfection of a substrate-activator based on the amyloid precursor protein or the Notch sequence and a fluorescent reporter gene. Stable fluorescent cell populations were selected by fluorescent activated cell sorting and characterized. This assay enabled the identification and sorting of populations, which differ in their levels of gamma-secretase activity, with high fluorescent cells producing more Abeta than low fluorescent cells. Specific gamma-secretase inhibitors, L-685,458 and MW167, reduced cell fluorescence in a dose-dependent manner that paralleled inhibition of Abeta secretion. Overexpression of presenilin 1 increased the cell fluorescence. Cells expressing presenilin with different aspartate mutations (D257A, D385A and D257A/D385A) or exon 9 deletion mutation showed reduced fluorescence. The single aspartate mutations showed a concomitant reduction in Abeta secretion, whereas the D257A/D385A and DeltaE9 mutations had no effect on Abeta secretion.