Cleaved prolactin and its 16K fragment are generated by an acid protease.
A cleaved form of rat PRL (cldPRL), which resolves after reduction of the disulfide bonds into two fragments of 16K and 8K mol wt, has been reported in the pituitary gland. To further understand the genesis of this cleaved PRL and its related 16K fragment (cldPRL/16K), pituitary proteins were labeled with [35S]methionine and then dialyzed and lyophilized before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pulse-chase experiments did not show a precursor-product relationship between PRL and cldPRL. However, if proteins were concentrated by trichloroacetic acid instead of dialysis, no cldPRL/16K was observed. Other experiments demonstrated that during dialysis cldPRL/16K is generated in vitro at the expense of intact PRL in a time-dependent manner. Labeled proteins dialyzed against water previously adjusted to different pHs showed after electrophoresis that cldPRL/16K was generated only at acidic pH. In the presence of the protease inhibitors phenylmethylsulfonylfluoride, tosylamidelysyl chloromethylketone, or tosylphenylalanine chloromethylketone or when the incubation medium was heated at 65 C for 45 min, the acid pH was unable to generate cldPRL/16K. Trypsin inhibitors did not prevent cldPRL generation. In conclusion, an acid protease, possibly of lysosomal origin, cleaves PRL in vitro. cldPRL/16K form was not found in the pituitary or incubation medium under physiological conditions. This PRL variant is generated as a preparative artifact unless experimental conditions are controlled.
Casabiell
X
X
Department of Medicine, Faculty of Medicine, Santiago University, Spain.
Robertson
M C
MC
Friesen
H G
HG
Casanueva
F F
FF
D000818:Animals; D016282:Aspartic Acid Endopeptidases; D003470:Culture Media; D046508:Culture Techniques; D003956:Dialysis; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D009479:Neuropeptides / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism*; D051381:Rats
D003470:Culture Media; D009479:Neuropeptides; D010446:Peptide Fragments; D011388:Prolactin; C045659:small cardioactive peptide A; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases