STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

已经研究了溶酶体酶-己糖氨酸酶(a)

The lysosomal enzyme beta-hexosaminidase has been studied (A.) at the level of it's genetic organization and (B.) by in vivo and in vitro expression of it's cDNAs. A. Genetic Organization. The gene encoding the beta-chain of beta-hexosaminidase has been isolated and it's genomic organization precisely characterized by restriction endonuclease mapping and sequencing of the intron-exon junctions. Camparison with the previously characterized alpha-chain gene revealed extensive similarities with some interesting differences. Both genes are about 40 kilobases in length, contain 14 exons and 13 introns, and have compact 3' ends and expanded 5' ends. The respective coding sequences are interrupted at homologous positions by 12 of the 13 introns. The most 5' intron of the two genes, which is the only intron to occur at a non-homologous position, interrupts both sequences at possible proteolytic processing sites. The extensive conservation of intron positions between the two genes demonstrates that they were derived from a common ancestor. B. In Vivo and In Vitro Expression. The beta-chain cDNA has been expressed in transfected COS cells under the control of the SV-40 late promotor. The expressed protein is enzymatically active and undergoes proteolytic processing consistent with transport to lysosomes. An in vitro expression system has been developed in which beta-chain mRNA, generated by transcription with SP6 polymerase, is translated in a rabbit reticulocyte lysate supplemented with microsomes. Within the microsomes the translated product assumes a native structure as indicated by reactivity with a conformation-sensitive antibody, assembly of the subunits into dimers and acquisition of catalytic activity. In addition, by expressing genetically engineered forms of the beta- cDNA, we have shown that the beta-chain contains two functional initiator AUG codons.