Inhibitor Report for: Spermine

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states-closed and open-in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

We have previously shown that the naturally occurring polyamines, spermidine and spermine, reverse effectively the in vitro inhibition of prolyl oligopeptidase (POPase) by its endogenous inhibitor by forming a kinetically significant complex (Soeda et al., J. Neurochem. (1986) 46, 1304-1307). In this study, we examined changes in the activities of POPase and its endogenous inhibitor and in the concentrations of polyamines during the regeneration of rat liver. POPase activity in the liver cytosol peaked 2 days after partial hepatectomy and then decreased near to control activity by 9 days, without its altered synthetic levels. Total polyamine concentrations also peaked at 2 days and remained elevated by 9 days, while cytosolic POPase inhibitor activity was minimal (56% of control) at 2 days. Treatment of the animals with a synthetic POPase inhibitor, Z-Gly-Pro-CHN2 (4 mg/kg), resulted in an obvious suppression of the liver regeneration. These results imply that the activity of POPase involved in nonlysosomal proteolytic pathway is exquisitely regulated by changes not only in its endogenous inhibitor levels but also in intracellular cationic potentials such as polyamines, and that POPase plays a crucial role for the growth and differentiation of liver cell.

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.