Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The plasma enzyme, human lecithin-cholesterol acyltransferase (LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation.
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PMID:Lecithin-cholesterol acyltransferase (LCAT) deficiency with a missense mutation in exon 6 of the LCAT gene. 185 5

We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.
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PMID:A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency. 760 41

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.
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PMID:Optimized bacterial expression of human apolipoprotein A-I. 1250 90