Enzyme & protein最新文献

Proteasomes are intricate cellular proteases that are able to degrade many protein and peptide substrates in vitro. These particles are structurally complex; they are assembled from at least 14 small molecular mass polypeptide subunits to form mature 20S proteasomes. Recently, we demonstrated that proteasome subsets may be discriminated by serological criteria, and have found that subtle differences in the subunit composition of proteasomes can alter their cleavage specificity. Proteasome structural complexity is further enhanced when some proteasomes associate with additional proteins to form a 26S ATP- and ubiquitin-dependent protease. Herein we confirm the existence of distinct cellular forms of proteasomes, and show that they differ in their hydrophobic characteristics. We have reproducibly purified, using solely biochemical techniques, distinct proteasome subsets similar to the serologically defined LMP2+ and LMP2- proteasomes. These proteasome subsets differ in their expression of at least three polypeptides, and both lack several additional polypeptides as compared to the serologically defined LMP2+ and LMP2- proteasomes. Finally, we demonstrate that these structurally unique proteasomes differ in their capacity to cleave a defined panel of fluorogenic peptide substrates. It appears that mammalian cells might recruit and modify proteasomes to perform distinct cellular tasks.

Proteasomes are cylindrical particles which have a pseudohelical arrangement of subunits. On 2D-PAGE gels, rat liver proteasome preparations give rise to up to 25 proteins which are encoded by at least 16 different genes that are all members of the same family. Proteasomes are able to degrade protein substrates to acid soluble peptides. They have at least five different catalytic components which can be distinguished by the use of synthetic peptide substrates and inhibitors which have very different reactivity at the different sites. Proteasomes can undergo conformational changes when treated with various effectors of their multiple peptidase activities. They are found in the nucleus and in the cytoplasm and, in cultured cells, show changes in localization during the course of the cell cycle.

The activity of phosphofructokinase purified from rat kidney cortex has been assayed at two different pH values. At pH 7 the enzyme showed cooperativity for the binding of fructose 6-phosphate (Fru-6-P) and a strong allosteric inhibition by ATP. When the assays were done at pH 8 hyperbolic kinetics were observed for both substrates, a smaller inhibition by ATP was observed and the Vmax for ATP and for Fru-6-P was higher than at pH 7. A sequential reaction mechanism was inferred. Results are discussed in terms of the importance of a reduced hexose-phosphate cycling rate during metabolic acidosis induced by exercise.

The lobster proteasome is primarily a cytosolic enzyme in crustacean striated muscles, although a small amount (< 1% of total) occurs in aggregates associated with invaginations of the cell membrane. The complex exists in vitro in three distinct catalytic states (basal, SDS-activated, and heat-activated forms) which have identical subunit compositions. This review summarizes recent results showing that the branched-chain amino acid-preferring (BrAAP) activity mediates the hydrolysis of myofibrillar proteins by the heat-activated proteasome: (a) only the BrAAP activity is stimulated by heat treatment; (b) the BrAAP activity is strongly inhibited by protein substrates, and (c) both the BrAAP and proteolytic activities show similar sensitivities to cations and protease inhibitors.

The effect of chemical compounds like sodium dodecyl sulfate (SDS), fatty acid esters of glycerol, carnitine and coenzyme A, phospholipids, histones, polylysines as well as homobifunctional chemical cross-linkers on the various proteolytic activities of mammalian proteasomes have been tested. Most of the reagents enhance these activities, and some, e.g. fatty acid CoA esters, histones and the chemical cross-linkers, exert dual effects, i.e. activation and inhibition at the same time, depending on the activity measured. With optimally activating concentrations of SDS, no structural changes in proteasomes can be detected by electron microscopy. Formation of micelles at supra-optimal detergent concentrations may be a reason for irreversible denaturation of the proteasome.