45C53. time in = 300). B, Impurity C of Calcitriol Pollen treated for 1 h with 80 m MG-132, 45 min after transfer to inhibitor-free medium. C, DMSO controls, treated as explained above, 45 min after transfer to new medium made up of DMSO. D, Pollen after 105 min of incubation in the presence of 80 m MG-132. Because MG-132 can inhibit calpains as well as the proteasome (Rock et al., 1994), the effect of trans-epoxy succinyl-l-leucylamido-(4-guanidino) butane (E-64) ester, a cell permeable inhibitor of Cys proteases, was also investigated. As reported in Impurity C of Calcitriol Physique ?Physique7A,7A, 40 m E-64 did not affect pollen tube growth (no significant difference between the slopes at 0.5). At the higher concentration (80 m), the elongation rate was reduced to 85% of that of controls. The difference between the slopes of the linear regressions was significant ( 0.05); however, the production of abnormal pollen tubes and a decrease in percent tube emergence did not occur after treatment with E-64 (data not shown). Open in a separate window Physique 7 Effect of non-proteasomal protease inhibitors on kiwifruit pollen tube growth over time. Growth is expressed as 0.0001; Fig. ?Fig.4B).4B). At this time, the growth rate was reduced to about 16% of that of controls. Epoxomicin caused an appreciable inhibition at both the concentrations tested, causing a reduction of pollen tube growth rate of 25% (1 m) and 36% (5 m) compared with the control ( 0.01; Fig. ?Fig.44C). Non-proteasomal protease inhibitors phenylmethylsulphonyl fluoride (PMSF), pepstatin, and leupeptin, which inhibit Ser-proteases, aspartic-proteases, and Ser/Cys-proteases, respectively, did not affect tube emergence and growth rate at the concentrations tested (Fig. ?(Fig.7,7, BCD). In fact, no significant differences between the slopes of control and treated tube linear regressions were found ( 0.1). Proteasome Inhibitors Increase Impurity C of Calcitriol the Level of High-Molecular Mass Ubiquitin Conjugates Because inhibition of proteasome function should result in the accumulation of ubiquitinated proteins, the effect of MG-132 around the levels of ubiquitin-protein conjugates was analyzed by immunoblot. The addition of the inhibitor (40 m) to the culture medium resulted in the accumulation of multiple, high-molecular mass bands recognized by an anti-ubiquitin antibody (Fig. ?(Fig.8A).8A). The conjugates already were detectable after 30 min of incubation and their level increased with time. In parallel, a more pronounced decrease in the levels of free ubiquitin monomer compared with the control was observed (Fig. ?(Fig.8B).8B). Comparable results were obtained when -lactone was added to the culture, although Impurity C of Calcitriol the effects produced by this inhibitor were evident only later, starting from 60 min of incubation (Fig. ?(Fig.8A).8A). Open in a separate window Physique 8 Effect of proteasome inhibitors on accumulation of high-molecular mass ubiquitin-conjugated proteins in germinating kiwifruit pollen. A and C, Immunoblotting of total protein (20 g per lane) extracted from pollen incubated with 40 m MG-132, 80 m E-64, or 10 m -lactone for different Impurity C of Calcitriol times and from pollen incubated in the medium without the respective inhibitor. Total protein was electrophoresed on 10% (w/v) polyacrylamide Rabbit polyclonal to Caspase 8.This gene encodes a protein that is a member of the cysteine-aspartic acid protease (caspase) family.Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. gels and was immunoblotted using polyclonal anti-ubiquitin antibody (A) or an anti-actin antibody (C). B, Immunoblot detection of free ubiquitin (each lane was loaded with 5 g of protein). Molecular mass of standard proteins are indicated around the left (in kilodaltons). Accumulation of high-molecular mass ubiquitin conjugates and a decrease in free ubiquitin level were not detectable in pollen germinated for 180 min in the presence of 80 m E-64 (Fig. ?(Fig.8,8, A and B). Quantitative evaluation of ubiquitin conjugates performed with a solid-phase dot-blot immunoassay showed a 44% increase in ubiquitin conjugate levels after 180 min of incubation in MG-132-treated pollen, compared with the amount found in the control (Table ?(TableI).I). A 29% increase was induced by -lactone treatment after 270 min of incubation. No differences from controls were observed at 180 min in the E-64-treated tubes. Table I Content of ubiquitin-protein conjugates in extracts from kiwifruit pollen incubated in the presence of different inhibitors 0.001 compared with controls.? b?Not significantly different from controls ( 0.1).? c? 0.005 compared with controls.? Conversation Tube emergence and growth in kiwifruit pollen is usually strongly dependent upon de novo protein synthesis, as exhibited by total inhibition of germination in the presence of cycloheximide. Plants show wide differences in.