title: [Isolation and characteristics of hexosaminidase A and activator protein from human kidney].
abstract: Hexosaminidase A (HA) was isolated from human kidney and purified to an electrophoretically homogeneous state. The purification procedure included ion-exchange chromatography on DEAE-cellulose, gel filtration on Toyopearl HW-55 and chromatofocusing on PBE 94 (enzyme yield 26.6%, 1133.6-fold purification). The physico-chemical and kinetic properties of HA are as follows: Mr of the purified enzyme is approximately 100,000; Km for 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside is 0.6 mM; pH optimum is at pH 4.4-4.6; pI is 5.0. The amino acid composition of the purified enzyme was determined. A specific anti-HA antiserum was raised, which did not immunoprecipitate with fibroblast extracts characterized by a mutational blockade of HA synthesis. GM2 was isolated and purified from murine liver as well as from the brain of a female patient who died of Tay-Sachs disease. The label was introduced by way of treatment of GM2 with tritiated acetic anhydride. The specific radioactivity of [3H]GM2 was 521 and 2065 Ci/M, respectively. The label was introduced into the N-acetylneuraminic acid and GalNAc residues of these GM2 preparations. An activator protein capable of solubilizing the natural substrate of HA was isolated from human kidney and partially purified (with a 19.9% yield and 480-fold purification). The Mr of the purified activator protein was approximately 21,000. Purified HA hydrolyzed [3H]GM2 only in the presence of the activator protein. An addition of the activator to the incubation medium containing normal fibroblast culture extracts and [3H]GM2 caused an increase in the rate of substrate hydrolysis, tenfold, on the average.
authors: E L
authors: EL
authors: Aronovich
authors: V S
authors: VS
authors: Akhunov
authors: K D
authors: KD
authors: Krasnopol'skaia
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000302:isolation & purification*; D005732:Gangliosides / Q000302:isolation & purification*; D006023:Glycoproteins / Q000302:isolation & purification*; D054818:Hexosaminidase A; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013661:Tay-Sachs Disease / Q000201:enzymology / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D049289:G(M2) Activator Protein; D005732:Gangliosides; D006023:Glycoproteins; C483345:PSAP protein, human; D011506:Proteins; C483344:Psap protein, mouse; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D005678:G(M2) Ganglioside; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases
vernacular_title: Vydelenie i kharakteristika geksozaminidazy A i aktiviruiushchego belka iz pochki cheloveka.