A study of the heterogenous structure of guinea pig lysosomal beta-mannosidase using a polyclonal antibody.
Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.
McCabe
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NR
Department of Pediatrics, University of Chicago, IL 60637.
Dawson
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G
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D044902:beta-Mannosidase
D008361:Mannosidases; D044902:beta-Mannosidase