[33] Immobilized anhydrotrypsin as a specific affinity adsorbent for tryptic peptides

Ishii, Shin‐ichi; Yokosawa, Hideyoshi; Kumazaki, Takashi; Nakamura, Izumi

doi:10.1016/s0076-6879(83)91035-2

Cited by 47 publications

(23 citation statements)

References 17 publications

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“…1 reports only the Glu-C endoproteinase peptides that were useful for the reconstruction of the complete sequence. The C-terminal extremity of the protein was identified following the application of a procedure for C-terminal peptide isolation by anhydrotrypsin affinity chromatography of peptides originated from a tryptic cleavage of a succinylated protein sample [16]. In Fig.…”

Section: Resultsmentioning

confidence: 99%

The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes

Mignogna¹,

Allocati

Aceto

et al. 1993

European Journal of Biochemistry

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The complete amino acid sequence of glutathione transferase from Proteus mirabilis was determined. The sequence was reconstructed by analysis of peptides obtained after cleavage by trypsin, Glu-C and Asp-N endoproteinases. The enzyme subunit is composed of 203 amino acid residues corresponding to a molecular mass of 22856 Da. Comparison of this sequence with other known primary structures of the corresponding enzyme from different sources shows a low level of identity (17-26%) with only seven conserved residues in all the sequences considered. This novel glutathione transferase could represent the prototype of a new class, possibly including other bacterial enzymes.The cytosolic glutathione S-transferases (GST) constitute a family of multifunctional dimeric proteins that catalyse the conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. GST are also involved in intracellular binding and transport of hydrophobic molecules, including heme, bile acids, bilirubin and polycyclic hydrocarbons, participate in the synthesis of prostaglandins and leukotrienes, and may play a key role in the elimination of toxic organic hydroperoxides [l -41. The structure and function of mammalian cytosolic GST have been the subject of numerous investigations and the various forms so far characterized from different tissues of the same organism or from different organisms can be grouped into at least four distinct classes, Alpha, Mu, Pi and Theta [5]. The complete primary structures for many members of these classes are available, mostly deduced by analysis of cDNA. GST isoenzymes belonging to the same class show 70-80% identity in their primary structure, whereas GST belonging to different classes have less than 30 % sequence identity [2]. The amino acid sequences deduced from the cDNA of yeast [6], plant [7] and insect [8, 91 GST have also been determined. Recently, the three-dimensional structure of the Piclass GST from pig lung has been determined [lo] and the geometry of glutathione site (G site) has also been elucidated.

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Section: Resultsmentioning

confidence: 99%

The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes

Mignogna¹,

Allocati

Aceto

et al. 1993

European Journal of Biochemistry

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“…The sample was carefully adjusted to pH 5.0 with 5% (v/v) acetic acid, and an aliquot (equivalent to 1.0 nmol) was freeze-dried and redissolved in 50 mM sodium acetate-HCl, 20 mM CaCl 2 , pH 5.0 (equilibration buffer, 0.1 ml final volume). This was applied to an anhydrotrypsin (AHT)-Sepharose column prepared as described in Ishii et al (1983) (10 mg AHT, 3.5-ml bed volume, 1-ml void volume and pre-equilibrated in equilibration buffer (100 ml)). The column was sealed and mixed overnight, the AHT-Sepharose was allowed to settle in the column, and the first 15 ml of the run-through fraction was collected while equilibration buffer was applied at 10 ml/h.…”

Section: Analysis Of the Phosphatidylinositol Moieties Of The Membranmentioning

confidence: 99%

“…The AHT-Sepharose should therefore bind all the peptides except for the C-terminal peptide (Ishii et al, 1983). Following AHTSepharose chromatography, the run-through fraction was subjected to reverse-phase HPLC to assess the previous step, and this revealed that the AHT-Sepharose chromatography appeared to have bound all but three of the peptides in the trypsin-digested sample as three significant peaks were pres- FIG.…”

Section: Identification Of the Pi Moieties Of The Membrane Form Of Pomentioning

confidence: 99%

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase

Brewis

Ferguson

Mehlert

et al. 1995

Journal of Biological Chemistry

123

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The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man␣1-2Man␣1-6Man␣1-4GlcN (29% of the total population), Man␣1-2Man␣1-6(GalNAc␤1-4)Man␣1-4GlcN (33%), and Man␣1-2Man␣1-6(Gal␤1-3GalNAc␤1-4)Man␣1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose ␣(1-2)-linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as ThrAsn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser 368. This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein. Glycosyl-phosphatidylinositol (GPI)1 membrane anchors are present in organisms at most stages of eukaryotic evolution, including protozoa, yeast, slime molds, invertebrates, and vertebrates, and are found on a diverse range of proteins. They are primarily responsible for the anchoring of cell-surface proteins in the plasma membrane and may be considered as an alternative to the hydrophobic transmembrane polypeptide anchor of integral membrane proteins. Many other functions have been proposed for GPI anchors, including roles in intracellular sorting, transmembrane signaling, and the novel endocytic process of potocytosis. A GPI anchor might also allow the protein to associate in membrane microdomains or be selectively released from the cell-surface by phospholipases. These functions, as well as the structure, biosynthesis, and distribution of GPI anchors have been extensively reviewed (Ferguson and

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“…Our present investigation, in conjunction with a previous report (30), presents an independent study on the cloning of the Psel(doinonas elastase gene and determination of its DNA sequence, from which the primary structure of elastase was deduced. Among the tryptic digests, the C-terminal peptide fragment was obtained by using affinity chromatography on anhydrotrypsin agarose (Takara Shuzo Co., Ltd., Kyoto, Japan) according to the manufacturer's instructions (8). The lysyl or tryptic peptides were separated and purified by reversedphase high-pressure liquid chromatography (HPLC).…”

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confidence: 99%

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase

et al. 1989

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The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences.

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[33] Immobilized anhydrotrypsin as a specific affinity adsorbent for tryptic peptides

Cited by 47 publications

References 17 publications

The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes

The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes

Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase

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