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B6db references: 93037488

type Journal Article
authors Roper, M. D.; Kraus, J. P.
title Rat cystathionine beta-synthase: expression of four alternatively spliced isoforms in transfected cultured cells
journal Arch Biochem Biophys
ui 93037488
year (1992)
volume 298
number 2
pages 514-21.
keywords *Alternative Splicing
abstract The gene for rat cystathionine beta-synthase consists of 17 exons. Its transcripts are alternatively spliced, forming four distinct mRNA species. Type III consists of exons 1 through 12, 14, 15, and 17; type I also contains exon 16. The open reading frame of type IV spans exons 1-->13; type II, 3-->13. We cloned the corresponding cDNAs into appropriate expression vectors and inserted the constructs into Escherichia coli (I, III, and IV) and Chinese hamster (CH) cells (I through IV); all sequences were transcribed and translated. Catalytic activity was observed only for types I and III in lysates of transfected CH cells and transformed E. coli. The catalytic and kinetic properties of I and III were identical despite their structural difference (exon 16). Both isoforms exhibited 6 mM Km constants for homocysteine which were reduced approximately eightfold by AdoMet; this elucidates the mechanism by which AdoMet regulates synthase activity. The four isoforms were differentially degraded by transfected cultured cells. Type III (t1/2 = 18 h) was degraded at 1/3 the rate of type I (t1/2 = 6 h); thus the 14 amino acid residues encoded by exon 16 appear to enhance degradation of CBS. The half-lives of both types II and IV were markedly shorter (ca. 1 h). Western blots comparing rat liver to lysates from transfected CH cells revealed that hepatocytes express both isoforms. Type III was predominant, as predicted by its longer half-life and more abundant mRNA. PCR analysis of cDNA from various tissues revealed that type III mRNA was preferred in liver, kidney, and heart; equal amounts of I and III were found in brain.
last changed 2002/11/12 16:17

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