High molecular weight glycosylated protease in calf brain. Purification and partial characterization.
A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.
Malik
M N
MN
Laboratory of Neuronal Proteases and Cytoskeletal Proteins, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
Fenko
M D
MD
Sheikh
A M
AM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D006031:Glycosylation; D006358:Hot Temperature; D006868:Hydrolysis; D008854:Microscopy, Electron; D008970:Molecular Weight
D000596:Amino Acids; D003902:Detergents; D010450:Endopeptidases