Zum Nachweis von Aminosäuren im 10−10-Mol-Maßstab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonyl-aminosäuren auf Dünnschichtchromatogrammen

Seiler, Nikolaus; Wiechmann, Jutta

doi:10.1007/bf02150289

Cited by 120 publications

(16 citation statements)

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“…NH, (45:35:20, v/v) was used in the first direction and modified for a better resolution of dansyl-lysine and dansyl-arginine by increasing the amount of ammonia from 20 to 30 parts. The second direction was developed with chloroformmethanol-glacial acetic acid (75 : 20: 5, v/v) [29].…”

Section: Determination Of N-terminal Amino Acidsmentioning

confidence: 99%

“…When the lysine is bound to the murein with its C-terminal end, the dansyl-method should yield an cc/s-dansyl-lysine whereas when it is bound with the N-terminal end only mono-, presumably the s-dansyl lysine should appear. The murein with the lysine, prepared by an overnight digestion of the murein lipoprotein complex with trypsin and subsequent washing with hot 401" dodecylsulfate, was treated with dansylchloride and the dansyl labeled compounds, after acid hydrolysis, were chromatographed in the solvent system of Seiler and Wiechmann [29]. Due to the possibility that the high molecular weight substrate may be spun down, the excess of dansylchloride and the by-products of the reaction (the respective amide and the free sulfonic acid) could be removed with pyridine and acetone before the dansyl labeled macromolecule was hydrolyzed.…”

Section: E N D Group Determinationsmentioning

confidence: 99%

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Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the E. coli Cell Wall. The Specific Effect of Trypsin on the Membrane Structure

1969

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A decrease of the absorbance a t 578 nm of a cell wall suspension of mid log phase E . coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55O/, of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = I : 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein.This protein could be shown to be a lipoprotein with a part of the lipid probably covalently bound to the protein. It is called murein-lipoprotein. The link between the lipoprotein and the murein is lysine. After trypsin digestion lysine is the only additional amino acid remaining a t the murein. The ratio of the amount of lysine to the known constituents of the murein demonsstrates that on the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein (N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acidmeso-diaminopimelic acid-D-alanine). After 3 min incubation with trypsin, the isolated lipoprotein molecules have a lysine to arginine ratio of 4:4 as compared with 5 : 4 in the native molecule. The lipoprotein has an unusual amino acid composition since it contains about 65O/, polar amino acids and no glycine, cysteine, proline, phenylalanine, and histidine could be found. On a weight basis the lipoprotein accounts for more than 40°/, of the rigid layer. Since the murein is held together exclusively by covalent bonds one can get a fairly accurate idea of the distribution of the lipoprotein molecules in the cell wall. About lo5 lipoprotein molecules per cell should be distributed 103 A apart along the glycosidic chains of the murein.The lipoprotein has an important function in stabilizing the total structure of the cell wall.It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic examination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.There are several lines of evidence suggesting that cell walls are more than mere cell envelopes protecting the essential cell functions which are supposed to take place in the cytoplasm. For example cell walls of E. coli not only contain the systems of active transport but interact with colicins (bacteriocins) in such a way that suggests that the whole complex memUnwnutl Abbreviation. Dansyl-, l-dimethylaminonaphthalene-5-sulfonyl-.Enzymes. Trypsin (EC 3.4.4.4); chymotrypsin (EC 3.4.4.5) ; papain (EC 3.4.4.10) ; pronase (EC 3.4.4) ; lysozyme (EC 3

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Section: Determination Of N-terminal Amino Acidsmentioning

confidence: 99%

Section: E N D Group Determinationsmentioning

confidence: 99%

Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the E. coli Cell Wall. The Specific Effect of Trypsin on the Membrane Structure

1969

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“…The mixture was evaporated to dryness and the residue hydrolyzed h r 18 h a t 100" to repeat the conditions used in the determination of free amino groups in peptides. Because an 8.5-fold molar excess of diaminopimelic acid was used over the amount of the reagent, only one spot, presumably that of the mono-dansylated diaminopimelic acid, was obtained on thin-layer chromatography [13]. Diaminopimelic acid labeled a t both amino groups with the dansyl group, was prepared from 1Omg diaminopimelic acid in 2 m l 0.5M NaHC03 with 21 mg dansyl chloride in 2 ml acetone containing 0.05 ml pyridine.…”

Section: Reaction Of Diaminopimelic Acid With Dansyl Chloridementioning

confidence: 99%

The Covalent Murein‐Lipoprotin Structure of the Escherichia coli Cell Wall

Braun

Sieglin

1970

European Journal of Biochemistry

208

110

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After pronase treatment of the murein-lipoprotein complex (rigid layer) of the cell wall of E . coli B or E. coli K12 (W 945), lysine and arginine remain as the sole amino acids covalently bound to the murein (peptidoglycan, glycopeptide). These amino acids occur in equimolar amounts, each equal to the amount of lysine remaining with the murein after trypsin digestion of the murein-lipoprotein complex.From partial acid hydrolysates of such a murein, prepared by pronase digestion of the mureinlipoprotein complex, the following peptides have been isolated : ( I ) diaminopimelyl-lysyl-arginine : ( 2 ) alanyl-glutamyl-diaminopimelyl-lysyl-arginine ; (3) glucosaminyl-muramyl-alanyl-glutamyldiaminopimelyl-lysyl-arginine. Peptide 1 shows that the lipoprotein is bound by the a-amino group of the presumably N-terminal lysine to the carboxyl group of diaminopimelic acid. Peptide 2 consists of a peptide side chain of the murein to which the two amino acids of the N-terminal end of the lipoprotein, lysine and arginine, are attached. Peptide 3 constitutes a repeating unit of the murein to which the peptide lysyl-arginine of the lipoprotein is bound.The lipoprotein cleaved from the murein by a short trypsin digestion had an amino acid composition similar to the lipoprotein in the untreated murein-lipoprotein complex and arginine as N-terminal amino acid. The following structure is proposed : murein-lysyl-arginyl-lipoprotein.In the rapid reaction of trypsin with the cell wall the enzyme apparently cleaves at the C-terminal end of the lysine of the murein-lipoprotein linkage which results in murein-lysine and arginyl-lipoprotein. Some cleavage a t the C-terminal end of arginine gives rise to free arginine These results support the supramolecular structure of the murein-lipoprotein complex previously proposed. On the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein from which an average distance of 103 A between two lipoprotein molecules along the polysaccharide chains of the murein is deduced. (14 o/o).It was found that trypsin alone specifically causes a rapid decrease of the absorbance of cell wall preparations of E. coli 1112 and R [1,2]. The narrow specificity of trypsin and the high reaction rate of the digestion strongly suggested that one type of molecule in the cell wall was responsible for the trypsin effect. It was found that a lipoprotein was the target of the rapid trypsin reaction. Our results indicated that this lipoprotein is covalently linked t o the murein. The murein is an enormous macromolecule, the size of the cell, consisting of the polysaccharide strands -(N-acetylmuramyl-N-acetylglucosaminyl),-crosslinked by the peptide side chains (L-a~any~-D-g~utamy~-diaminopime~y~-o-a~anine), between the amino group of diaminopimelic acid of one chain and the carboxyl group of D-alanine of the other chain [3--51:where MurNAc = N-acetyl-muramic acid and Dpm = meso-2,B-cliaminopimelic acid. The lipoprotein molecules together with the murein form the rigid layer of the E . c...

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“…The reaction mixture was lyophilized, washed with acetone and with water, and hytirolyzed in 6 N HC1 for 24 hr at 110 °. After drying, the hydrolysate was dissolved in 50% (v/v) pyridine, and chromatographer on both polyamide sheets [19] and silica gel-G plates [20]. The DNS-amino acids were identified by comparison with DNS-amino acid standards.…”

Section: North-holland Publishing Company -Amsterdammentioning

confidence: 99%

C‐phycocyanin from the thermophilic blue‐green algaMastigocladus laminosus, isolation, characterization and subunit composition

1972

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Zum Nachweis von Aminosäuren im 10−10-Mol-Maßstab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonyl-aminosäuren auf Dünnschichtchromatogrammen

Cited by 120 publications

References 1 publication

Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the E. coli Cell Wall. The Specific Effect of Trypsin on the Membrane Structure

Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the E. coli Cell Wall. The Specific Effect of Trypsin on the Membrane Structure

The Covalent Murein‐Lipoprotin Structure of the Escherichia coli Cell Wall

C‐phycocyanin from the thermophilic blue‐green algaMastigocladus laminosus, isolation, characterization and subunit composition

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