Synthesis and assembly of a catalytically active lysosomal enzyme, beta-hexosaminidase B, in a cell-free system.
The synthesis and dimerization of beta-chains during the formation of catalytically active beta-hexosaminidase B were studied in a cell-free system. beta-chain mRNA, transcribed from the cloned cDNA with SP6 polymerase, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of dog pancreas microsomal membranes and oxidized glutathione. Under these conditions, the primary beta-chain translation product was translocated into the microsomal vesicles and modified by the addition of N-linked oligosaccharide chains. After transfer into the microsomal vesicles, the beta-polypeptide assumed a conformation resembling the native state as determined by antibody reactivity. Like the authentic precursor enzyme, the microsomally located chains were assembled into dimers and were catalytically active. In intact human fibroblasts, dimerization of beta-chains occurred within 15 min after their synthesis, consistent with a site of assembly in the rough endoplasmic reticulum. The cell-free expression system was also useful in establishing the functionality of beta-chain initiator methionine codons. By expression of beta-chain mRNAs with altered methionine codons, we demonstrated that polypeptides initiating from any of the first three methionine codons in the beta-chain sequence contain a functional signal sequence and form catalytically active enzymes.
Sonderfeld-Fresko
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Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Proia
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003062:Codon; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011817:Rabbits; D001619:beta-N-Acetylhexosaminidases / Q000096:biosynthesis* / Q000378:metabolism
D003062:Codon; D001619:beta-N-Acetylhexosaminidases