Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells.

Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Research Abstract Details 

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Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Abstract Text:

K Suzuki,Y Furukawa,H Tamura,N Ejiri,H Suematsu,R Taguchi,S Nakamura,Y Suzuki,H Ikezawa,

A glycosylphosphatidylinositol (GPI)-anchored protein, 5'-nucleotidase [EC 3.1.3.5], was released from the membrane of bovine liver by use of phosphatidylinositol-specific phospholipase C (PI-PLC) of Bacillus thuringiensis and purified by several column chromatographies to a homogeneous state. The purified protein has an apparent molecular mass of 61 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. From the partial amino acid sequence of a tryptic peptide, mixed oligonucleotides were synthesized and used to screen a lambda gt11 liver cDNA library, and one positive clone, pE1, was isolated. Since the insert of the clone lacked the NH2-terminal coding region, another lambda gt11 liver cDNA library was screened by using a synthetic probe corresponding to the 5' region of the insert of pE1. Three additional cDNA clones were obtained. Sequencing of these cDNAs revealed an open reading frame that encodes a 574-residue polypeptide with a calculated mass of 63,084 Da. The predicted structure showed two highly hydrophobic stretches at both ends of the protein, like those of rat and human 5'-nucleotidases. The NH2-terminal 26 residues comprise a signal peptide and the COOH-terminal hydrophobic stretch may serve as a signal for the posttranslational GPI modification. An expression vector of the cDNA, pSVNT, was constructed in a mammalian expression vector pSVL and the 5'-nucleotidase activity was transiently expressed in COS-1 cells. The expressed activity was about 8 times higher than the pSVL-transfected control activity. PI-PLC released 45% of the transiently expressed 5'-nucleotidase activity, indicating that the cDNA isolated here encodes this enzyme expressed as a GPI-anchored protein.

Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Publishing Authors By Initials

K Suzuki,Y Furukawa,H Tamura,N Ejiri,H Suematsu,R Taguchi,S Nakamura,Y Suzuki,H Ikezawa,

For similar biochemical phenomena, metabolism, and nutrition: biochemical phenomena: sequence homology: sequence homology, amino acid research abstracts see: biochemical phenomena, metabolism, and nutrition: biochemical phenomena: sequence homology: sequence homology, amino acid research

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Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 113

Page Numbers: 607-13

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: May

YEAR: 1993

Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 376600

Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Keywords Mesh Terms:

KEYWORDS: Sequence Homology, Amino Acid

MESH TERMS: enzymology

Chemical & Substance for Abstract: Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. Information

Substance Name: 5'-Nucleotidase

Registry Number: EC 3.1.3.5

Grant and Affiliation Information for Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells.

AFFILIATION: Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University.

Country: JAPAN

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MEDLINETA: J Biochem

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