Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.
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PMID:Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma. 753 18

Mast cells arise in cultures of murine bone marrow in medium supplemented with interleukin-3 (IL-3). In the present study, we report the development of long-term mast cell lines from murine bone-marrow-derived cultured mast cells (BMCMC) following inoculation with adenovirus 12-simian virus 40 (Ad12-SV40) hybrid virus. One culture of Ad12-SV40 immortalized BMCMC (designated as MCP-5) was selected for further analysis. These transformed cells appear similar in morphology and histochemistry to the primary BMCMC from which they are derived and did not shed infectious virus into the culture supernatants. In addition, these cells synthesize predominantly chondroitin sulfate proteoglycans and contain histamine which is released following a physiologic stimulus. Limiting-dilution single-cell cloning produced five independent mast cell lines (MCP-5.1 to MCP-5.5). Southern blot analysis of genomic DNA isolated from these single-cell clones demonstrates different patterns of viral integration in all the five clones. All clones retain responsiveness to an exogenous source of IL-3 for growth and proliferation. Each single-cell clone also demonstrates a unique pattern of cytokine gene expression in response to calcium ionophore A23187 and phorbol-12-myristate-13-acetate. This suggests that within a culture of BMCMC there are differences in cytokine gene expression that vary from one cell to another. The availability of immortalized mast cell lines derived from murine bone marrow which retain their growth factor responsiveness and the ability to respond to degranulating stimuli should facilitate future studies of mast cell biology.
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PMID:Immortalization of mouse bone marrow-derived mast cells with Ad12-SV40 virus. 768 24

Thermostable alkaline protease from an alkaliphilic thermophile Bacillus sp. B18' was purified by using DEAE- and CM-Toyopearl 650M column chromatographies. Molecular weights of the enzyme determined by SDS-PAGE and gel filtration were 30,000 and 28,000, respectively. The optimum pH and temperature toward the hydrolysis of casein were pH 12-13 and 85 degrees C, both of which are higher than those of a mesophilic alkaline protease from an alkaliphile, Bacillus sp. B21-2. The enzyme was stable at pH 5.0-12.0 and about 60% of the initial enzymatic activity was retained after a 60 min incubation period at pH 10.0 and 70 degrees C. Thermostability of the enzyme was enhanced by Ca2+. The enzyme activity was inhibited by DFP, suggesting that the enzyme is a serine protease. The NH2-terminal amino acid is Gln, which is that of many subtilisin-type proteases. The 20 residues of the NH2-terminal amino acid sequence have a comparative high homology with those of other alkaline proteases from alkaliphiles (40-50%), especially thermostable alkaline protease from Bacillus sp. No. AH-101 (95%) and Thermoactinomyces sp. HS682 (95%).
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PMID:Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp. 776 36

Intracellular proteins ligated to ubiquitin are degraded by the 26 S proteasome which is composed of the 20 S proteasome and a regulatory subunit complex. We have reported that ATP-dependent activity of the proteasome is activated periodically during the ascidian mitotic division cycle. In the present study, we examined changes in the activities and in the amounts of proteasomes during progression of the ascidian meiotic division cycle. During the metaphase-anaphase transition triggered by treatment with calcium ionophore, the activity of 26 S proteasome was found to be enhanced transiently and then decreased. The change in proteasome activity was completely abolished by pretreatment with a cell-permeable calcium chelating agent, BAPTA-AM, which indicates that proteasome activity is regulated by intracellular calcium mobilization. By immunoblot analyses, it was demonstrated that the 26 S proteasome underwent a change in amount in a manner similar to the change in its activities. The immunoblot analyses also indicated an inverse relation between the level of the 26 S proteasome and that of the 20 S proteasome throughout the cycle. These results, together with the fact that total amounts of the 26 S and 20 S proteasomes remain constant throughout the cycle, suggest that 26 S proteasome activity is regulated through interconversion between the 26 S and 20 S proteasomes induced by intracellular calcium mobilization during the meiotic metaphase-anaphase transition.
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PMID:Intracellular calcium mobilization regulates the activity of 26 S proteasome during the metaphase-anaphase transition in the ascidian meiotic cell cycle. 781 81

alpha-haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca(2+)-binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca(2+)-binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca2+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg2+, Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of alpha-haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45Ca2+ binding reveals three equivalent, independent sites, with Kd approximately 0.11 mM. No 45Ca2+ binding is observed when the protein is incubated with Zn2+; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three-dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357-3364] it is suggested that alpha-haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca(2+)-binding experiments. In addition, structural similarities and conservation of ion-binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.
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PMID:The binding of divalent cations to Escherichia coli alpha-haemolysin. 788 8

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.
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PMID:Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. 802 Dec 48

The proteasome from the thermoacidophilic archaeon Thermoplasma acidophilum in its native state represents a 20S particle with significant secondary structure (approximately 35% alpha helix) of its subunits. Electron microscopy, ultracentrifugal and spectral analysis demonstrate that at pH of less than 3 dissociation to partially denatured subunits occurs. Upon dialysis against near neutral pH buffers, at low protein concentration, reconstitution occurs, leading to the restoration of up to 90% of the native fluorescence signal. The recovery of activity depends on several parameters, including the buffer system, the pH used to dissociate the complex, and the duration of exposure to low pH. High concentrations of Ca2+ and Mg2+ cause partial dissociation of the Thermoplasma proteasome, yielding distinct subcomplexes. Neither the completely nor the partially dissociated complexes have proteolytic activity, indicating that function is linked to fully assembled proteasomes.
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PMID:Dissociation and reconstitution of the Thermoplasma proteasome. 805 45

The crystal structure of the 50 kDa metalloprotease from the Gram-negative bacterium Serratia marcescens has been solved and refined to a crystallographic R-factor of 0.192 at 1.80 A resolution. The structure is very similar to that of alkaline protease from Pseudomonas aeruginosa, in particular the calcium binding "parallel beta roll" motif is completely conserved. The N-terminal proteolytic domain shows the typical "metzincin" fold. The active sites of the two enzymes are slightly different, Tyr216 is a Zn ligand in the Serratia metallo protease. The loops 70-77 and 122-132, which encompass the active site cleft, differ due to insertions and deletions so that the Serratia metallo protease seems to have a more open site than the alkaline protease.
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PMID:Crystal structure of the 50 kDa metallo protease from Serratia marcescens. 808 45

A previously undescribed high molecular mass protein (HMP) from human erythrocyte membranes was solubilized by Triton X-100 and purified on a calmodulin-agarose column in the presence of Ca2+. It was shown to have a native molecular mass of 522-560 kDa, comprised of a single subunit of a molecular mass of 28 kDa (p28). The protein is associated with the lipid bilayer rather than with the cytoskeletal component of the membrane. The purified HMP showed peptidase-hydrolyzing activity toward substrates containing hydrophobic amino acids at the P1 position of the P2-P1 cleavage site. The activity was inhibited by serine proteinase inhibitors (leupeptin, phenylmethansulfonyl fluoride) and chymotrypsin inhibitors in particular (chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone). The enzyme exhibited maximal activity at slightly alkaline pH (7.5-8.5) and at 37 degrees C and was stimulated over a narrow range of SDS concentrations (maximal at 0.05%). HMP was found to cross-react in Western blots with an antibody raised against the rabbit multicatalytic proteinase. The single subunit of HMP therefore contains both the catalytic activity and a sequence necessary for its association into a multimeric complex. The properties of the human erythrocyte membrane HMP described indicate that it is a novel peptidase related to the ubiquitous multicatalytic proteinase.
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PMID:Characterization of a novel high molecular mass protein with peptidase activity purified from the human erythrocyte membrane by calmodulin affinity chromatography. 814 98

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway.
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PMID:Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes. 818 44


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