scholarly journals Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma

1989 ◽  
Vol 261 (3) ◽  
pp. 811-818 ◽  
Author(s):  
N M Hooper ◽  
A J Turner
Keyword(s):  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Isolation And Characterization ◽  
C Terminus ◽  
Terminal Amino ◽  
Membrane Isolation ◽  
Solubilized Form ◽  
Triton X ◽  
Hydrolysis Of

Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.

scholarly journals Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme

1990 ◽  
Vol 265 (2) ◽  
pp. 429-433 ◽  
Author(s):  
N M Hooper ◽  
J N Keen ◽  
A J Turner
Keyword(s):  
Human Kidney ◽  
Amino Acid Sequences ◽  
Kidney Cortex ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
C Terminus ◽  
The Difference ◽  
Solubilized Form ◽  
Triton X ◽  
High Degree

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.

Preparative isolation and characterization of the B875 complex from Rhodobacter sphaeroides 2.4.1

1988 ◽  
Vol 66 (5) ◽  
pp. 442-448 ◽  
Author(s):  
Rafael Picorel ◽  
Gabriel Gingras
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Phosphatidyl Ethanolamine ◽  
Sodium Dodecyl ◽  
Pigment Analysis ◽  
Preparative Isolation ◽  
Ethanolamine Phosphatidyl ◽  
Isolation And Characterization ◽  
Detergent System ◽  
Triton X

We have developed a simple and efficient method, using a mixed detergent system of sodium dodecyl sulfate and Triton X-100, for the preparative isolation of theB875 complex from Rhodobacter sphaeroides 2.4.1. As a bonus, the method allows the preparation of both the B875 and B800-850 complexes from the same batch of chromatophores. The preparations are spectrally pure, as indicated by absorption and circular dichroism spectroscopy. The latter method suggests that the Qy band of the B875 complex is due to weakly interacting bacteriochlorophyll molecules. Protein and pigment analysis shows that the B875 complex contains 2 mol of bacteriochlorophyll and 2 mol of sphaeroidene per mol of apoprotein (12 266 g), whereas the B800-850 complex contains 3 mol of bacteriochlorophyll and 1 mol of sphaeroidene per mol of apoprotein (11 497 g). While these stoichiometries are in accord with currently accepted models, they disagree with their published experimental basis. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol were found to be present in the B875 complex.

Purification and partial amino acid sequence of thuricin S, a new anti-Listeriabacteriocin from Bacillus thuringiensis

2007 ◽  
Vol 53 (2) ◽  
pp. 284-290 ◽  
Author(s):  
Sonia Chehimi ◽  
François Delalande ◽  
Sophie Sablé ◽  
Mohamed-Rabeh Hajlaoui ◽  
Alain Van Dorsselaer ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Proteinase K ◽  
Terminal Sequence ◽  
Partial Amino Acid Sequence ◽  
Ph Values ◽  
Heat Stable ◽  
Isolation And Characterization ◽  
Terminal Amino ◽  
Edman Sequencing

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3–10.5). The monoisotopic mass of thuricin S purified by high perfomance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.

Isolation and characterization of anti-inflammatory peptides derived from trypsin hydrolysis of microalgae protein (Synechococcus sp. VDW)

2019 ◽  
Vol 33 (4) ◽  
pp. 303-324 ◽  
Author(s):  
Rutairat Suttisuwan ◽  
Saranya Phunpruch ◽  
Tanatorn Saisavoey ◽  
Papassara Sangtanoo ◽  
Nuttha Thongchul ◽  
...  
Keyword(s):  
Isolation And Characterization ◽  
Trypsin Hydrolysis ◽  
Synechococcus Sp ◽  
Anti Inflammatory ◽  
Hydrolysis Of

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

scholarly journals Isolation and characterization of a novel glycosyl-phosphatidylinositol-anchored glycoconjugate expressed by developing neurons

1992 ◽  
Vol 203 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Joel NEDELEC ◽  
Michel PIERRES ◽  
Herve MOREAU ◽  
Jacques BARBET ◽  
Philippe NAQUET ◽  
...  
Keyword(s):  
Glycosyl Phosphatidylinositol ◽  
Isolation And Characterization ◽  
Developing Neurons

scholarly journals Peptides as antigens. Importance of orientation.

1986 ◽  
Vol 164 (4) ◽  
pp. 1344-1349 ◽  
Author(s):  
T Dyrberg ◽  
M B Oldstone
Keyword(s):  
Peptide Sequence ◽  
Carrier Protein ◽  
Binding Pattern ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Terminal Amino ◽  
Peptide Characterization ◽  
A Site ◽  
Peptide Antibodies

Factors known to be important in producing protein-reactive peptide antibodies include the accessibility of the region from which the peptide sequence is derived, the hydrophilic-phobic character of the sequence, and the length of the peptide. The data presented here indicate that the orientation of the peptide coupled to a carrier protein also influences the binding pattern of peptide antibodies. An octapeptide, representing a sequence from the alpha chain of the human acetylcholine receptor, was coupled either through an N- or C-terminal cysteine-glycine-glycine linker to a carrier protein and used to immunize rabbits. The resulting antisera reacted at comparable titers to the uncoupled immunizing peptides, but did not crossreact with the identical but opposite-linked peptide. Characterization of the binding to other homologous peptides showed that immunization with the N-terminal-linked peptide induced antibodies reactive specifically with the C-terminal amino acid(s). Immunization with the C-linked peptide resulted in antibodies reactive with a site of the peptide near the C-terminus.

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