STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

I. β-氨基己糖苷酶α-亚基中的Gly 269-> Ser突变 在3例非犹太人成人GM 2患者中发现了纯合子形式 神经节苷脂沉积症这与德系犹太人的情况形成鲜明对比 存在Gly 269-> Ser突变但总是在化合物中的患者 与Tay-Sachs等位基因无效的杂合性。 二. - 我们分离了编码α和β的近全长cDNA， 鼠β-bexosaminidase的β-亚基。的核苷酸序列 α-亚基已被确定。这些cDNA已经被用来分离 包含鼠α和β亚基部分的基因组克隆 基因. 三. 人类α和β亚基已经被整合到 已经用于过表达β-氨基己糖苷酶的杆状病毒载体 B（α-亚基二聚体）和β-氨基己糖苷酶S（α-亚基二聚体） .从重组大肠杆菌的培养基中纯化了这些酶， 通过伴刀豆球蛋白A亲和层析， 通过Mono S柱色谱法，用于B同工酶或苯基-Superose -S同工酶的柱层析。 四.已克隆了大鼠内质网羧基酯酶， 测序来自该基因家族的其他部分cDNA也已被克隆。 测序全长cDNA和突变的版本已经在大肠杆菌中表达。 COS 1细胞，以确定蛋白质决定簇， 滞留在内质网中。

I. The Gly269 -> Ser mutation in the beta-hexosaminidase alpha-subunit has been found in homozygous form in 3 non-Jewish patients with adult GM2 gangliosidosis. This is in contrast to the situation in Ashkenazi Jewish patients where the Gly269 -> Ser mutation is present but always in compound heterozygosity with a null Tay-Sachs allele. II. - We have isolated near full length cDNAs encoding alpha and beta-subunits of murine P-bexosaminidase. The nucleotide sequence of the alpha-subunit has been determined. These cDNAs have been used to isolate genomic clones encompassing portions of the murine alpha and beta-subunit genes. III. The human alpha and beta-subunits have been incorporated into baculovirus vectors which have been used to overexpress beta-hexosaminidase B (an alpha-subunit dimer) and P-hexosaminidase S (an alpha-subunit dimer) . The enzymes have been purified from culture medium of recombinant virus-infected SF9 cells by Concanavalin A affinity chromatography followed by Mono S column chromatography, for the B isozyme, or phenyl-Superose column chromatography for the -S isozyme. IV. A rat endoplasmic reticulum carboxyl esterase has been cloned and sequenced. Other partial cDNAs from this gene family have also been sequenced. The full-length cDNA and mutated versions have been expressed in COS 1 cells in order to identify the protein determinant that signifies retention in the endoplasmic reticulum.