Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides.
Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and beta-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004386:Duodenum / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005994:Glycerophosphates; D008055:Lipids / Q000032:analysis*; D051379:Mice; D008968:Molecular Conformation; D010455:Peptides / Q000032:analysis*; D010636:Phenols; D010710:Phosphates; D012967:Sodium Dodecyl Sulfate
D005994:Glycerophosphates; D008055:Lipids; D010455:Peptides; D010636:Phenols; D010710:Phosphates; D012967:Sodium Dodecyl Sulfate; D000469:Alkaline Phosphatase