Isolation of two glycosylated forms of membrane-bound alkaline phosphatase from avian growth plate cartilage matrix vesicle-enriched microsomes.
Isolation of two membrane-bound alkaline phosphatase (AP) species from avian growth plate cartilage matrix vesicle (MV) fractions is described. AP was first released from the membranes by phosphatidylinositol-specific phospholipase C (PIase C), followed by chromatography on DEAE-Bio-Gel A and Reactive-Red agarose. Two AP species having apparent Mr of 81.5 and 77 kDa by SDS-PAGE were purified in high yield and specific activity by this simple method. Treatment with neuraminidase to remove sialic acid residues reduced their size slightly, but did not diminish the difference in Mr between the two species. Digestion with N-glycanase, however, decreased both AP species to a common size of 59 kDa. This reveals that both enzymes are highly glycosylated and suggests that the two forms may result from differences in degree of glycation. The amino acid compositions of the two avian enzyme forms are very similar, but are markedly enriched in serine, glycine and glutamate when compared to those reported for mammalian liver-kidney-bone AP. Possible differences in amino acid sequence between the two avian forms have not been excluded. The cross-reactivity of polyclonal antibodies to these enzymes with bovine kidney, but not intestinal AP, indicate that the avian cartilage APs are of the liver-kidney-bone isozyme type.
Wu
L N
LN
Department of Chemistry, University of South Carolina, Columbia 29208.
Valhmu
W B
WB
Lloyd
G C
GC
Genge
B R
BR
Wuthier
R E
RE
D000469:Alkaline Phosphatase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D006031:Glycosylation; D006132:Growth Plate / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*; D009439:Neuraminidase
D000596:Amino Acids; D007527:Isoenzymes; D000469:Alkaline Phosphatase; D009439:Neuraminidase