Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.
N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.
Slusher
B S
BS
Department of Neuroscience and Pharmacology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Robinson
M B
MB
Tsai
G
G
Simmons
M L
ML
Richards
S S
SS
Coyle
J T
JT
D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004150:Dipeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D043425:Glutamate Carboxypeptidase II; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009928:Organ Specificity; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000201:enzymology
D000936:Antigen-Antibody Complex; D004150:Dipeptidases; D043425:Glutamate Carboxypeptidase II