Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

The genotypic distribution among non-insulin-dependent diabetes mellitus (NIDDM) patients of a restriction fragment length polymorphism

The genotypic distribution among patients of a marker allele, or restriction fragment length polymorphism (RFLP), can be used to determine the mode of inheritance of a disease-predisposing gene, if the association of the marker with the disease is sufficiently high. In the case of noninsulin-dependent diabetes mellitus (NIDDM), the RFLP in the 5'-flanking region of the human insulin gene does not allow discrimination between dominant and recessive modes of inheritance, or between any intermediate model. Also, it is demonstrated, in general, that the observation of a higher odds ratio for individuals with two copies of a marker allele than for individuals with at least one copy does not in itself imply a gene-dosage model.     

Two individuals with elliptocytic red cells apparently lack three minor erythrocyte membrane sialoglycoproteins.

We have studied the erythrocytes of two individuals (P. L. and K. W.) who lack the Gerbich (Ge) blood-group antigen. The erythrocytes of P. L. and K. W. were not reactive with two monoclonal antibodies (NBTS/BRIC 4 and NBTS/BRIC 10) which reacted with normal erythrocytes. The membranes of P. L. and K. W. erythrocytes appeared to lack three minor sialoglycoproteins (beta, beta 1 and gamma). These three minor sialoglycoproteins were found to be associated with the cytoskeletons of normal erythrocytes. Approx. 10% of the erythrocytes of P. L. and K. W. were frankly elliptocytic. We suggest that one or more of the minor sialoglycoproteins may play a part in maintaining the discoid shape of the human erythrocyte.     

Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii.

Glyoxylate dehydrogenase (glyoxylate:NAD+ oxidoreductase) was purified 600-fold in three steps from crude extracts of the fungus Sclerotium rolfsii (Corticium rolfsii Curzi). Two of the purification steps involved dye-affinity chromatography. The enzyme is a tetramer of Mr 250 000, with identical subunits of Mr 57 000. Inhibition studies suggest that there is one essential thiol group per active site.     

Interactions of sulphide and other ligands with cytochrome c oxidase. An electron-paramagnetic-resonance study.

The effect of sulphide on resting oxidized cytochrome c oxidase was studied by both e.p.r. and optical-absorption spectroscopy. Excess sulphide causes some reduction of cytochrome a, CuA and CuB, and the formation of the cytochrome a3-SH complex after about 1 min. After several hours in the presence of excess sulphide only the e.p.r. signals due to low-spin ferricytochrome a3-SH persist, giving a partially reduced species. Re-oxidation of this partially reduced sulphide-bound enzyme by ferricyanide makes all of the metal centres except CuB detectable by e.p.r. We conclude that sulphide has reduced and binds to CuB as well as to ferricytochrome a3. Sulphide binding to cuprous CuB may raise its mid-point potential and make re-oxidation difficult. Addition of reductant (ascorbate + NNN'N'-tetramethyl-p-phenylenediamine) and sulphide together to the oxidized resting enzyme produces a species in which cytochrome a and CuA are nearly completely reduced and cytochrome a3 is e.p.r.-detectable as approx. 80% of one haem in the low-spin sulphide-bound complex. The g = 12 signal of this partially reduced derivative is almost unchanged in magnitude relative to that of the resting enzyme; this suggests that the g = 12 signal may arise from less than 20% of the enzyme and that it may be relatively unreactive to both ligation and reduction. Such a reactivity pattern of the g = 12 form of the oxidase is also demonstrated with the ligands F- and NO, which are thought to bind to cytochrome a3 and CuB respectively.     

Magnetic-circular-dichroism studies of haem a and its derivatives.

1. The Thy-1 membrane glycoproteins from rat thymus and brain bound deoxycholate to 24% of their own weight as measured by equilibrium dialysis. The binding occurred co-operatively at the critical micelle concentration of deoxycholate, suggesting that the glycoproteins bind to a micelle, and not to the detergent monomer. 2. From sedimentation-equilibrium and deoxycholate-binding data the molecular weights of the glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoproteins respectively. The molecular weight of the polypeptide part of the glycoprotein is thus 12500. 3. In the absence of deoxycholate, brain or thymus Thy-1 glycoprotein formed large homogeneous complexes of mol. wt. 270000 or 300000 respectively. The sedimentation coefficient of these was 12.8 S. The complex was only partially dissociated by 4M-guanidinium chloride. 4. After cleavage of brain or thymus Thy-1 glycoprotein with CNBr, two peptides were clearly identified. They were linked by disulphide bonds and both contained carbohydrate. This cleavage suggests there is only one methionine residue per molecule, which is consistent with the above molecular weights and the known amino acid composition.     

Cortisol binding in human breast cancer: Correlation with antitumor immunity

Summary The intensity of cortisol binding was measured in the cytosol fraction of the primary tumor obtained from 50 patients with stage I and II breast cancer. The state of cellular antitumor immunity of the same patients was investigated by the tube leucocyte adherence inhibition (LAI) test, performed with peripheral blood leucocytes 1–2 days preoperatively. It was found that the intensity of tumor cortisol binding correlates negatively with LAI values. Patients with high cortisol binding in their tumors have low LAI values, while low tumor cortisol binding is associated with higher antitumor immunity. The results suggest that high cortisol binding in the tumor might inhibit the tumor recognition process and/or the cellular immune defense mechanism and thus facilitate cancer development.     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.