The multicatalytic proteinase. Multiple proteolytic activities

The multicatalytic proteinase is a high molecular weight nonlysosomal proteinase which has been isolated from a variety of mammalian tissues and has been suggested to contain several distinct catalytic sites. The enzyme degrades protein and peptide substrates and can cleave bonds on the carboxyl side of basic, hydrophobic, and acidic amino acid residues. The three types of activity have been referred to as trypsin-like, chymotrypsin-like, and peptidyl-glutamyl peptide bond hydrolyzing activities, respectively. All of these proteolytic activities are associated with a single band on native polyacrylamide gels. The pH optimum of the proteinase (pH 7.5-9.5) depends on the substrate. Using synthetic peptide substrates it was possible to demonstrate two distinct activities. Trypsin-like activity is inhibited at concentrations of the peptide aldehyde inhibitors leupeptin and antipain or of N-ethylmaleimide which have little or no effect on chymotrypsin-like activity. Results of mixed-substrate experiments also suggest that there are at least two distinct types of catalytic sites. All proteolytic activity is lost following dissociation by urea or by acid treatment. Polyclonal antibodies raised against the intact multicatalytic proteinase precipitate the complex but have little effect on its proteolytic activities.     

Gibberellin A13 7-aldehyde: A proposed intermediate in the fungal biosynthesis of gibberellin A3

Gibberellin A₁₃ 7-aldehyde, previously proposed as an intermediate in the fungal biosynthesis of gibberellin A₃, has been prepared from gibbere     

Phosphorylation of ATPase subunits of the 26S proteasome

The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [³²P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.     

Selection and assembly of indigenous bacteria and methanogens from spent metalworking fluids and their potential as a starting culture in a fluidized bed reactor

Waste metalworking fluids (MWFs) are highly biocidal resulting in real difficulties in the, otherwise favoured, bioremediation of these high chemical oxygen deman (COD) wastes anaerobically in bioreactors. We have shown, as a proof of concept, that it is possible to establish an anaerobic starter culture using strains isolated from spent MWFs which are capable of reducing COD or, most significantly, methanogenesis in this biocidal waste stream. Bacterial strains (n = 99) and archaeal methanogens (n = 28) were isolated from spent MWFs. The most common bacterial strains were Clostridium species (n = 45). All methanogens were identified as Methanosarcina mazei. Using a random partitions design (RPD) mesocosm experiment, we found that bacterial diversity and species–species interactions had significant effects on COD reduction but that bacterial composition did not. The RPD study showed similar effects on methanogenesis, except that composition was also significant. We identified bacterial species with positive and negative effects on methane production. A consortium of 16 bacterial species and three methanogens was used to initiate a fluidized bed bioreactor (FBR), in batch mode. COD reduction and methane production were variable, and the reactor was oscillated between continuous and batch feeds. In both microcosm and FBR experiments, periodic inconsistencies in bacterial reduction in fermentative products to formic and acetic acids were identified as a key issue.     

The photodecomposition of tryptophan peptides

Aqueous solutions of the four trytophan peptides, Ala-Trp-Val, Val-Trp-Ala, Ala-Ala-Trp-Val and Ala-Gly-Trp-Leu, have been irradiated with light of wavelengths >295 n. The major changes were destruction of the tryptophan residue, liberation of ammonia and the formation of photoproducts of increased molecular weight. Up to 40% of Ala-Ala-Trp-Val and Ala-Gly-Trp-Leu were converted to products with molecular weights ranging from two to four times those of the original tetrapeptides. Most of the yellow material formed during irradiation in air was present in the high molecular weight fractions. Irradiation of Ala-Gly-[¹⁴C]Trp-Leu gave the following identifiable photoproducts: Ala-Gly-Asp-Leu, Ala-Gly-(N′-formyl)Kyn-Leu, Ala-Gly-Oia-Leu, and ammonia, where Kyn means kynurenine and Oia, β-(3-oxidolyl)alanine.     

Peptide inhibitors of melittin action

The sequence of peptides necessary to inhibit melittin-induced lysis was studied using 13 peptide analogues of the inhibitor Ac-IVIFDC-NH₂. Although this inhibitor is a disulfide-linked dimer, inhibition was equally effective if the thiol SH was blocked or replaced by methionine or lysine. The substitution of phenylalanine with other aromatic residues preserved activity, as did the replacement of aspartic acid by asparagine. The results suggest that the cytolytic activity of melittin can be inhibited by a short peptide of four hydrophobic residues followed by two other nonspecific residues. Fluorescence studies showed that the inhibitor caused a blue shift in the Trp emission spectrum. A spin label attached to the N-terminus of the inhibitor significantly quenched the fluorescence. These data confirmed the involvement of Trp 19 with the inhibitor, also predicted by molecular modeling of the probable binding site. Density gradient studies with large unilamellar vesicles indicated that the inhibitor prevented melittin from reacting with the lipid bilayer.     

Proteasome response to interferon-gamma is altered in senescent human fibroblasts

We have investigated immunoproteasomes in human fibroblasts during replicative senescence. Unlike levels of constitutive proteasome catalytic subunits and 26S proteasome regulatory subunits, levels of immunosubunits did not decrease dramatically in senescent cells. However, the induction of immunosubunits by interferon-gamma (IFN-gamma) was lost in senescent cells. In contrast, levels of the 11S proteasome regulator, PA28, were increased by IFN-gamma even in senescent cells, and both immunosubunits and PA28 increased with the reversible growth arrest in confluent cell cultures. The results highlight differences in the mechanisms of regulation of immunoproteasomes compared to constitutive proteasomes and in the irreversible growth arrest of senescent cells compared to reversible contact-induced growth arrest.     

Sequence requirements for the activity of membrane-active peptides.

Synthetic peptides were constructed with the sequence of the first 20 residues of melittin and terminating with a range of different amino acid amides. These were found to have haemolytic and cytolytic activity similar to that of melittin, provided that certain charge constraints were observed. The nature of the 21st residue was not critical except when the residue introduced a negative charge. The presence of at least two positive charges in the molecule was found to be essential for activity. One of these charges could be the amino-terminal amine. Peptides could be inactivated by the addition of a non-acidic presequence which was acetylated at the N-terminus. Introducing a protease cleavable sequence into an N-terminal extension of the peptides produced analogues with low haemolytic activity that could be activated by proteolytic action. A peptide with extra positive charges introduced on the hydrophilic face of the helix possessed a haemolytic activity that was greater than that of melittin.