A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity.
A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.
Gee
N S
NS
Merck Sharp and Dohme Research Laboratories, Terlings Park, Harlow, Essex, U.K.
Howell
S
S
Ryan
G
G
Ragan
C I
CI
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D000939:Epitopes / Q000032:analysis; D005260:Female; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D010449:Peptide Mapping; D010744:Phosphoric Monoester Hydrolases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase