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The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.

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The adsorption of cytochrome c onto different mesoporous molecular sieves (C 12 -MCM-41, C 16 -MCM-41 and SBA-15) is studied at different solution pHs. Adsorption isotherms were recorded up to final solution concentrations of ca. 250 µmol/L and were found to be of the pseudo-Langmuir type. Cytochrome c (cyt c) adsorption was observed to be pH-dependent with maximum adsorption near the isoelectric point of the protein. SBA-15 showed a larger amount of cyt c adsorption as compared to MCM-41. The increased cyt c adsorption capacity may be due to the larger pore volume and pore diameter as compared to C 12 -and C 16 -MCM-41. It has been discovered that the amount of cyt c adsorption can be increased by the introduction of aluminum into the pure silica materials. The observed increase is most likely a consequence of the strong electrostatic interaction between the negative charges on the aluminum sites and the positively charged amino acid residues of cyt c. Furthermore, the rate of cyt c adsorption has been studied and no significant differences in adsorption rate were found for the mesoporous materials with different pore diameters between 3 and 9 nm. While the adsorption capacity is reduced upon bead formation (due to the reduction in specific pore volume), the rate of adsorption is mainly unchanged.

The adsorption of lysozyme on the mesoporous molecular sieves MCM-41 and SBA-15 from buffered solutions with different pH values has been studied as a model protein adsorption system. The amount adsorbed depends on the solution pH as well as on the pore volume and the composition of the adsorbent. The adsorption isotherms at pH 6.5 to 10.5 fitted the Langmuir model (type L isotherm), while the isotherms recorded at pH 12 are of the S type. The maximum amount adsorbed was observed for AlSBA-15 at pH 9.6 and amounted to 47.2 µmol/g (580 mg/g). The stability of SBA-15 toward to the buffer solution is higher for SBA-15 as compared to MCM-41, which is probably a consequence of the higher wall thickness of the former material. Diffuse reflectance Fourier transform infrared spectra of the adsorbed lysozyme confirm that the adsorption of the enzyme did not result in denaturation of Lz.

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Carbon mesoporous molecular sieves have been prepared from SBA-15 materials synthesized at different temperatures. This allows the synthesis of hexagonally arranged mesoporous carbons with pore diameters between 3.0 nm (CMK-3-100) and 6.5 nm (CMK-3-150). The novel materials have been studied in the adsorption of horse heart cytochrome c from solutions with different pH. A maximal adsorption capacity of 18.5 µmol/g was found for CMK-3-130 (d p ) 4.5 nm) at pH 9.6, which is near the isoelectric point of cytochrome c (pI ) 9.8).