Prolactin (PRL) processing by kallikrein: production of the 21-23.5K PRL-like molecules and inferences about PRL storage in mature secretory granules.
We have previously described a series of C-terminally-clipped forms of PRL, the 21-23.5K PRL-like molecules (PLMs). Because we noted estrogen (E2) induction of PLMs and E2 also induces a pituitary glandular kallikrein, we have investigated the possibility that processing of PRL by kallikrein is responsible for the production of the PLMs. Subcellular fractionation of pituitaries from control or E2-treated female rats showed total kallikrein to be concentrated 1- to 4-fold and 6- to 20-fold in the granules (vs. original homogenate) from control and E2-treated animals, respectively. Cleavage of purified PRL by kallikrein resulted in the formation of large quantities of a number of the PLMs. Incubation of secretory granules derived from control or E2-treated animals under the same conditions showed no cleavage of PRL in the absence of a limiting granule membrane and a small production of the PLMs in the presence of a granule membrane. Production in the latter instance was slightly greater in granules derived from E2-treated animals. Addition of purified kallikrein to secretory granules from control or E2-treated animals in the presence or absence of granule membranes, resulted in the additional production of large amounts of only the smallest PLM (PLM 9), indicating a lack of cleavage of the two most C-terminal sites for the enzyme. Increasing the concentration of beta-mercaptoethanol to 0.64 M, to ensure monomerization of the granule PRL, had no effect on endogenous kallikrein activity of the number of products resulting from the addition of exogenous kallikrein to granules. In summary: 1) purified kallikrein can cleave purified PRL to form products which run with the same isoelectric point and mol wt values as the PLMs; 2) kallikrein is present in PRL secretory granules; 3) some PLM production occurs in a membrane-bound secretory granule fraction, but none occurs after removal of the membrane; this intragranular production, like cleavage of purified PRL with purified kallikrein, is dependent on the presence of a detergent and a reducing agent; 4) cleavage of granular PRL by exogenous kallikrein is limited to a single site and the more C-terminal sites are protected; and 5) protection of the C-terminus is not removed by intermolecular and intramolecular disulfide bond reduction. We conclude: 1) that pituitary glandular kallikrein is a strong candidate for the enzyme responsible for the production of the PLMs, and 2) that there is some element of PRL storage, other than intermolecular disulfide bonds, which involves the C-terminus of the molecule.
Ho
T W
TW
Division of Biomedical Sciences, University of California, Riverside 92521-0121.
Balden
E
E
Chao
J
J
Walker
A M
AM
D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D007526:Isoelectric Point; D007610:Kallikreins / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism / Q000648:ultrastructure*; D011388:Prolactin / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D004958:Estradiol; D008623:Mercaptoethanol; D011388:Prolactin; D007610:Kallikreins; D014357:Trypsin; D002401:Cathepsin B