In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.
- MeSH
- akrosin metabolismus MeSH
- akrozom enzymologie MeSH
- analýza rozptylu MeSH
- histologické techniky veterinární MeSH
- imunoelektronová mikroskopie veterinární MeSH
- inhibitory proteas farmakologie MeSH
- motilita spermií účinky léků fyziologie MeSH
- neparametrická statistika MeSH
- proteasy farmakologie MeSH
- rosanilinová barviva MeSH
- ryby fyziologie MeSH
- sperma enzymologie MeSH
- spermie účinky léků enzymologie fyziologie MeSH
- tosylfenylalanylchlormethylketon farmakologie MeSH
- tosyllysinchlormethylketon farmakologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We recently demonstrated that TLCK and TPCK could act as potent but nonspecific inhibitors of mature caspases [Frydrych and Mlejnek [2008] J Cell Biochem 103:1646-1656]. The question whether TLCK and TPCK inhibit simultaneously caspase activation and/or processing remained, however, open. In this article, we demonstrated that TPCK even enhanced caspase-3 and caspase-7 processing although it substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity in HL-60 cells exposed to various cell death inducing stimuli. Under the same conditions, TLCK had no effect or affected caspase-3 and caspase-7 processing marginally depending on cell treatment used. Importantly, TLCK substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity irrespectively to the treatment used. Interestingly, treatment of cells with toxic concentrations of TPCK alone was accompanied by full caspase-3 and -7 processing even if it induced necrosis. In contrast, treatment of cells with concentrations of TLCK that caused necrosis was accompanied by only partial caspase-3 and caspase-7 processing. Our results clearly indicated that TPCK and TLCK did not inhibit caspase-3 and -7 enzymatic activity by prevention of their activation and/or processing.
- MeSH
- apoptóza MeSH
- HL-60 buňky MeSH
- inhibitory kaspas MeSH
- inhibitory serinových proteinas farmakologie MeSH
- kaspasa 3 metabolismus MeSH
- kaspasa 7 metabolismus MeSH
- lidé MeSH
- tosylfenylalanylchlormethylketon farmakologie MeSH
- tosyllysinchlormethylketon farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) exhibit multiple effects on cell death pathways in mammalian cells. Thus, they are able to induce apoptosis by itself or promote cell death induced by other cytotoxic stimuli [King et al., 2004; Murn et al., 2004]. On the other hand, TLCK and TPCK were reported to prevent apoptosis by inhibiting the processing of caspases in response to some cell death inducing stimuli [Stefanis et al., 1997; Jones et al., 1998]. We observed that the pretreatment of HL-60 cells with TLCK or TPCK diminished caspases 3 and -7 (DEVDase) and caspase-6 (VEIDase) activity in response to various cell death inducing stimuli such as staurosporine (STS), etoposide (ETP), or N6-(2-isopentenyl)adenosine. In addition, TLCK but not TPCK inhibited collapse of mitochondrial transmembrane potential Delta Psi m (delta psi) in dying HL-60 cells. Such effects used to be considered as protective, however, the protection was only presumable since neither TLCK nor TPCK actually prevented cells from death. Our results further indicated that serine protease inhibitors TLCK and particularly TPCK acted as efficient direct inhibitors of mature caspases. Indeed, experiments with human recombinant caspases provided unequivocal evidence that TLCK and TPCK are very potent but non-specific inhibitors of activated caspases, namely caspases 3, -6, and -7. Interestingly, TPCK exhibited similar efficiency towards human recombinant caspases to that found for panspecific caspase inhibitor Boc-D-CMK. Such properties of TLCK and TPCK, previously considered as specific inhibitors of serine proteases, might offer novel consistent explanation for several protective or protective-like effects on apoptotic cells. 2007 Wiley-Liss, Inc.
- MeSH
- apoptóza MeSH
- financování organizované MeSH
- HL-60 buňky MeSH
- inhibitory kaspas MeSH
- inhibitory serinových proteinas farmakologie MeSH
- kaspasy chemie metabolismus MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- rekombinantní proteiny antagonisté a inhibitory chemie metabolismus MeSH
- tosylfenylalanylchlormethylketon farmakologie MeSH
- tosyllysinchlormethylketon farmakologie MeSH
- Check Tag
- lidé MeSH
