Circular pancreatic trypsin inhibitor. A novel substrate for studies on intracellular proteolysis.
Several recent studies indicate that substrates for ubiquitin-dependent proteolysis must possess unblocked alpha-amino termini. To examine further the importance of free amino groups for proteolytic susceptibility we selected pancreatic trypsin inhibitor (PTI) as a test substrate. PTI can be circularized to form cPTI, a molecule that lacks alpha-amino groups in the absence of an endoproteolytic cleavage. We compared the breakdown of radioiodinated PTI and cPTI in rabbit reticulocyte lysate and found that cPTI was not stabilized relative to PTI. In addition, proteolysis of PTI or cPTI was not inhibited upon conversion of their lysine residues to homoarginine. However, neither degradation of PTI nor cPTI required ATP, and ubiquitin conjugation to either molecule was minor relative to known substrates of the ubiquitin pathway. Thus, PTI and cPTI are cleaved by an ATP-independent endoprotease(s) that does not require the substrate to be ubiquitinated. Such an activity was identified in low salt fractions obtained upon DEAE chromatography of reticulocyte lysate. The ubiquitin/ATP-dependent protease and another large multisubunit protease, both of which elute from DEAE-Fractogel at higher salt concentrations, do not degrade PTI or cPTI. Although monomeric PTI was rapidly degraded in reticulocyte lysate, cross-linked PTI molecules were stable both in reticulocyte lysate and following introduction into cultured cells using red blood cell-mediated microinjection. Thus, increased rates of turnover do not necessarily correlate with greater molecular mass of the substrate.
Rote
K V
KV
Department of Biochemistry, University of Utah, Salt Lake City 84132.
Hough
R
R
Goldenberg
D
D
Rechsteiner
M C
MC
D000818:Animals; D007611:Aprotinin / Q000031:analogs & derivatives* / Q000378:metabolism; D004120:Dimethyl Suberimidate / Q000378:metabolism; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity
D010456:Peptides, Cyclic; D011480:Protease Inhibitors; C046916:circular bovine pancreatic trypsin inhibitor; D004120:Dimethyl Suberimidate; D007611:Aprotinin; D010447:Peptide Hydrolases