Analytical subcellular fractionation of rat pituitary homogenates, with special reference to prolactin proteolysis by lysosomes.
Prolactin proteolysis by rat pituitary homogenates was assayed by measuring the release of trichloroacetic acid-soluble peptides from 125I-labelled rat prolactin. There was a distinct optimum at pH 4.3, with only trace amounts of activity at neutral and alkaline pH. Rat pituitary homogenates were subjected to analytical subcellular fractionation by sucrose density gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their respective marker enzymes, including: cytosol (lactate dehydrogenase); plasma membrane (5'-nucleotidase); lysosomes (N-acetyl-beta-glucosaminidase, beta-glucuronidase); mitochondria (particulate malate dehydrogenase); endoplasmic reticulum (neutral alpha-glucosidase); prolactin granules (radioimmunoassayable prolactin). Acid prolactin protease had a similar distribution to the lysosomal marker enzymes. A localisation of the activity to lysosomes was confirmed by subcellular fractionation experiments in which the lysosomes were selectively disrupted with low concentrations of the membrane perturbant, digitonin. Experiments with specific inhibitors of the lysosomal cathepsins indicate that both cathepsins B and D are implicated in pituitary prolactin proteolysis.
D000818:Animals; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002458:Cell Fractionation / Q000379:methods; D002500:Centrifugation, Isopycnic; D005260:Female; D008247:Lysosomes / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000201:enzymology / Q000648:ultrastructure*; D011247:Pregnancy; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000032:analysis; D013997:Time Factors
D011388:Prolactin; D002403:Cathepsins