Purification, preliminary characterization, and immunological comparison of hog lens leucine aminopeptidase (EC 3.4.11.1) with hog kidney and beef lens aminopeptidases.
Leucine aminopeptidase (LAP) was purified from hog lenses by application of the Himmelhoch procedure for isolation of hog kidney LAP [S. R. Himmelhoch (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 508-513, Academic Press, New York.] This involved treating crude hog lens homogenates with hexadecyltrimethylammonium bromide, DEAE-cellulose adsorption and elution, ammonium sulfate fractionation (53-84% of saturation), and gel filtration on a Bio-Gel A-1.5m column. Purifications ranging from 2080- to 4700-fold with activity yields from 28 to 100% were achieved. The hog lens LAP appeared homogeneous by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Bio-Gel chromatography of the native enzyme and SDS-PAGE of dimethylsuberimidate-crosslinked LAP indicated a molecular weight of 326,000. SDS-PAGE of untreated LAP showed a subunit weight of 54,000, consistent with a hexameric enzyme structure. By immunodiffusion, LAP from hog lens and kidney were identical while hog lens and beef lens enzymes demonstrated only partial identity. Electrophoresis of the native enzymes showed a slightly lower mobility for the hog lens LAP than for beef LAP at pH 8.7.
D000818:Animals; D002417:Cattle; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007668:Kidney / Q000201:enzymology*; D007908:Lens, Crystalline / Q000201:enzymology*; D007931:Leucyl Aminopeptidase / Q000276:immunology / Q000302:isolation & purification*; D011489:Protein Denaturation; D013045:Species Specificity; D013552:Swine
D007931:Leucyl Aminopeptidase