Linked Life Data

2016311

Source:http://linkedlifedata.com/resource/pubmed/id/2016311

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
1991-5-17
pubmed:abstractText
Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
266
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6999-7007
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:2016311-Animals, pubmed-meshheading:2016311-Base Sequence, pubmed-meshheading:2016311-Cell Line, pubmed-meshheading:2016311-Exons, pubmed-meshheading:2016311-Glucokinase, pubmed-meshheading:2016311-Hexokinase, pubmed-meshheading:2016311-Islets of Langerhans, pubmed-meshheading:2016311-Isoenzymes, pubmed-meshheading:2016311-Liver, pubmed-meshheading:2016311-Mice, pubmed-meshheading:2016311-Molecular Sequence Data, pubmed-meshheading:2016311-Mutagenesis, Insertional, pubmed-meshheading:2016311-Mutagenesis, Site-Directed, pubmed-meshheading:2016311-Oligonucleotide Probes, pubmed-meshheading:2016311-Pituitary Gland, pubmed-meshheading:2016311-Polymerase Chain Reaction, pubmed-meshheading:2016311-RNA, Messenger, pubmed-meshheading:2016311-RNA Splicing, pubmed-meshheading:2016311-Rats, pubmed-meshheading:2016311-Restriction Mapping, pubmed-meshheading:2016311-Sequence Homology, Nucleic Acid, pubmed-meshheading:2016311-Transfection
pubmed:year
1991
pubmed:articleTitle
Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.
pubmed:affiliation
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't