Active and inactive ecto-5'-nucleotidase variants in liver of control and dystrophic Lama2dy mice.
The ecto-5'-nucleotidase (eNT) activity and the eNT protein content in liver of normal and merosin-deficient dystrophic Lama2dy mice were studied. After the solubilization procedure, the eNT activity in the final extract was 9.2+/-2.5U/mg (nmol of phosphate released from AMP per min and per mg protein) in normal liver, and it rose to 16.1+/-3.9U/mg (P=0.005) in dystrophic liver. The increase of activity was less pronounced in Lama2dy liver (1.7-fold) than the one reported in muscle (four-fold), which probably reflects the lower content of merosin in liver. Similarly to muscle, liver contained active and inactive eNT, as demonstrated by the higher level of immunoreactive protein in normal than in dystrophic liver in Western blots performed with samples containing the same units of eNT activity. PNGase F digestion decreased the size of liver and muscle eNT from 72 and 69kDa, to 63 and 60kDa. Oligoglycan cleavage did not alter eNT activity or the sedimentation coefficient, revealing that oligosaccharides are not required for catalysis or for maintaining the dimeric structure. The eNT protein content in samples of normal liver decreased by 55 or 80% after the trypsinolysis of native or deglycosylated enzyme, but the activity did not change. Such a high proportion of inactive eNT is unlikely to come from aged enzyme, which suggests the involvement of inactive enzyme in non-catalytic actions.
Morote-García
Julio C
JC
Departamento de Bioqui;mica y Biología Molecular-A, Edificio de Veterinaria, Universidad de Murcia, Apdo. 4021, E-30071 Espinardo, Murcia, Spain
García-Ayllón
M Salud
MS
Campoy
Francisco J
FJ
Vidal
Cecilio J
CJ
Muñoz-Delgado
Encarnación
E
D015720:5'-Nucleotidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D007797:Laminin / Q000172:deficiency* / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000201:enzymology; D009708:Nucleotidases / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012685:Sepharose / Q000031:analogs & derivatives*; D014357:Trypsin
D007797:Laminin; D009844:Oligosaccharides; C029427:adenosine monophosphate-sepharose; D012685:Sepharose; D009708:Nucleotidases; C018775:adenosine monophosphatase; D015720:5'-Nucleotidase; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase