title: Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases.
abstract: Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10-1.15 g . cm-3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5'-nucleotidase, had an apparent Km for 4-methylumbelliferyl phosphate of approx. 59 microM, and was inhibited by levamisole. The high-density (1.20-1.25 g . cm-3) peak was resistant to levamisole-inhibition, had an apparent Km of approx. 30 microM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.
mesh_terms: D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000145:classification / Q000302:isolation & purification*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D005260:Female; D007978:Levamisole / Q000494:pharmacology; D010902:Pituitary Gland / Q000201:enzymology*; D011388:Prolactin / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D007978:Levamisole; D011388:Prolactin; D000469:Alkaline Phosphatase