Chemical characterization and subunit structure of human N-acetylhexosaminidases A and B.
Human hexosaminidases A and B were purified from placentae, using two stages of affinity chromatography, to a high degree of purity. Each enzyme was purified 5000-6000-fold, and isolated in 25-40% yield. Enzyme preparations appeared homogeneous in the analytical ultracentrifuge and by acrylamide gel electrophoresis. Hexosaminidase A contained 1.65 residues of sialic acid per molecule, whereas no sialic acid was present in hexosaminidase B. The molecular weights of the A and B isozymes as determined by gel filtration and sedimentation equilibrium are 100 000 and 108 000, respectively. In 5 M guanidine-HCl each of the enzymes yielded a 50 000-dalton species, which can further be dissociated into 25 000-dalton polypeptide chains by reduction and alkylation. The hexosaminidase B yielded one type of polypeptide chain, denoted beta, whereas the product from hexosaminidase A could be separated by ion-exchange chromatography into two species of chains, denoted alpha and beta, in equal amounts. The amino acid compositions of the separated alpha and beta chains were determined, and were found to correlate well with those of the intact enzymes. These findings enable the construction of a plausible model for the molecular structure of both enzymes. According to this model hexosaminidase A is composed of two subunits alpha2 and beta2, in which the two polypeptide chains are linked by a disulfide bridge. The structure of hexosaminidase B is, in parallel, beta2beta2. The suggested model is discussed in view of the accumulated information about the interrelationships between hexosaminidase A and B and the genetic metabolic disorders with which they are involved.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005260:Female; D006146:Guanidines; D006596:Hexosaminidases / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding; D011487:Protein Conformation; D012794:Sialic Acids / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D004220:Disulfides; D006146:Guanidines; D007527:Isoenzymes; D046911:Macromolecular Substances; D012794:Sialic Acids; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D006596:Hexosaminidases