Linked Life Data

10380109

Source:http://linkedlifedata.com/resource/pubmed/id/10380109

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1999-8-24
pubmed:databankReference
pubmed:abstractText
Although 5'-nucleotidases are ubiquitous in higher vertebrates, the arthropod enzymes have been little studied. The cDNA sequence of the mature 5'-nucleotidase from the tick Boophilus microplus was therefore determined (GENBANK accession number: U80634). The enzyme has 39-41% sequence identity with the vertebrate 5'-nucleotidases and contains binuclear metal ion binding sites. There are no significant introns within the coding region of the genomic sequence. Southern blot analysis indicates the presence of multiple related genes encoding 5'-nucleotidases. Recombinant tick 5'-nucleotidase was expressed in both Escherichia coli and in baculovirus-infected insect cells. The E. coli recombinant protein was truncated, inactive and produced in abundance. The enzyme was expressed in baculovirus-infected insect cells as a secreted, soluble, glycosylated and enzymatically active protein. This represents the first successful expression and characterization of enzymatically active recombinant 5'-nucleotidase from any organism. Supplementation of the culture medium with 25 microM zinc resulted in a twofold increase in the activity of the expressed protein. The enzyme was purified to homogeneity. It exists under non-denaturing conditions as a homodimer, with an apparent molecular mass of 135 kDa. The Km for the hydrolysis of AMP was 0.37 microM and the k(cat) = 11.5/s, in agreement with data for the native enzyme.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0962-1075
pubmed:author
pubmed:issnType
Print
pubmed:volume
8
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
257-66
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Cloning and expression of ecto 5-nucleotidase from the cattle tick Boophilus microplus.
pubmed:affiliation
CSIRO Tropical Agriculture, Long Pocket Laboratories, Indooroopilly, Queensland, Australia.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't