عنوان

Molecular and biochemical characterization of a putative membrane skeletal anchor in Euglena

Number

TLpq304419715

.Language of Text, Soundtrack etc

انگلیسی

Title Proper

Molecular and biochemical characterization of a putative membrane skeletal anchor in Euglena

General Material Designation

[Thesis]

First Statement of Responsibility

S. Sodin-Semrl

Name of Publisher, Distributor, etc.

University of Illinois at Chicago

Date of Publication, Distribution, etc.

1997

Specific Material Designation and Extent of Item

110

Dissertation or thesis details and type of degree

Ph.D.

Body granting the degree

University of Illinois at Chicago

Text preceding or following the note

1997

Text of Note

Cytoskeletal anchor proteins represent an important link between the plasma membrane and the underlying cytoskeleton, serving both the extracellular and intracellular medium. In Euglena gracilis the unique framework of the membrane skeleton is anchored to the plasma membrane by the major integral membrane protein, IP-39. Known to be phosphorylated, IP-39 is also a casein kinase, instrumental in assembly of the cytoskeleton (Fazio et al., 1995). The majority of membrane anchors identified to date are glycosylated. PAS staining of the NaOH membrane insoluble fraction separated by SDS-PAGE yielded 2 positive bands. This suggested IP-39 has an external glycosylated domain. Following electroelution, acid hydrolysis and TLC putative carbohydrate moieties were linked to IP-39/68. IP-39 in SDS polyacrylamide gels also stains positive with Sudan black B, indicating that lipids may be covalently bound to the protein. However, after metabolic labeling of E. gracilis with (H)-palmitic acid, no signal was found associated with IP-39, which excluded palmitoylation as a possible lipid modification of IP-39. Since the IP-39 cDNA sequence had not been determined, it seemed pivotal to resolve the sequence and thus confirm its biochemical characteristics. Microsequencing of the protein identified 2 short internal peptide sequences; PYPAPY ("PYP") and VGQRT ("VGQ"). Degenerate primers were designed for both sequences, but the more specific primer from "PYP" produced useful PCR products, following PCR screening of the cDNA library. Optimization of the PCR reactions with inclusion of an initial denaturing step, hot start PCR and addition of acetamide resolved the 5 and 3 IP-39 PCR products, which were cloned and sequenced. We conclude that IP-39 is an undescribed protein with a unique cDNA sequence following sequence matching in various databanks (Blitz, Fasta, Genbank).

Biological sciences

cytoskeleton

Euglena gracilis

integral membrane proteins

Molecular biology

Pure sciences

S. Sodin-Semrl

Electronic name

p

[Thesis]

276903

a

Y