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)

We have purified two chymotrypsin-like proteases from chum salmon sperm which have no apparent acrosome structure. Both of them were high molecular mass proteases (650 kDa and 950 kDa by gel filtration) and showed not only chymotrypsin-like activity but also trypsin-like activity. The 650 kDa protease was composed of at least eight or nine kinds of polypeptide with molecular masses ranging from 20 kDa to 30 kDa and was highly activated by low concentrations of SDS. Electron microscopy revealed that the 650 kDa protease was a ring-shaped particle. The 950 kDa protease was shown to contain at least one component that cross-reacts with an antibody against the 650 kDa protease. Finally, we revealed that the 650 kDa protease is located along the sperm flagella, by using immunofluorescence microscopy. The subunit composition, SDS-activation and molecular shape of 650 kDa salmonid protease were quite similar to those of the eukaryotic multicatalytic proteinase (proteasome), which is well known to participate in ATP-dependent degradation of ubiquitinated proteins; and, furthermore, the motility of demembranated sperm of salmonid fish is inhibited by chymotrypsin inhibitors in an ATP-dependent manner. Thus, the protease located in salmonid fish sperm flagella is a proteasome and is a strong candidate for the factor which regulates flagellar motility in an ATP-dependent manner.
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PMID:Purification of proteasomes from salmonid fish sperm and their localization along sperm flagella. 831 81

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.
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PMID:The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity. 831 14

The 26S protease complex was purified from chick skeletal muscle and shown to consist of unusually heterogeneous 21-140 kDa polypeptides, including the 21-32 kDa subunits of the 20S proteasome. Electron microscopic analysis revealed that the 26S complex may have a symmetric morphology with two large rectangular terminal domains attached to a thinner central 20S proteasome domain. The 26S complex was capable of degrading the peptide substrates of the 20S proteasome, including Suc-LLVY-AMC, N-Cbz-LLE-NA and N-Cbz-ARR-MNA. The two enzyme complexes showed similar sensitivities to various site-specific protease inhibitors, although their sensitivities to SDS were differed from each other. Immunoprecipitation with anti-26S complex antibody reduced peptide hydrolysis by the 20S proteasome. Similarly, anti-20S proteasome antibody inhibited peptide hydrolysis by the 26S complex. These results demonstrate that the 26S protease complex contains the 20S proteasome as a functional and structural component.
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PMID:Structure and properties of the 26S protease complex from chick skeletal muscle. 835 24

A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46). These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologous of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and "sterile" subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from "sterile" subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunoblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP. The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.
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PMID:Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and "sterile" subjects. 850 May 28

The cytosol fraction prepared from Xenopus laevis ovaries by one-step ultracentrifugation catalyzed the hydrolysis of succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide with or without SDS, an activator of 20S latent proteasomes. Both activities were lost on immunodepletion with antibodies against 20S proteasome. Storage of the cytosol at 4 degrees C led to abolition of the SDS-independent activity, but not the SDS-dependent activity. Upon DEAE-cellulose chromatography, the enzyme catalyzing the SDS-independent activity could be separated from that responsible for the SDS-dependent activity. These results indicate that the ovary cytosol contains a large proportion of a novel, "active" form of proteasomes which does not require SDS but is unstable and is readily converted to a latent form which requires SDS. ATP is known to stabilize 26S protease complex, but ATP reduced SDS-independent activity. It seems that the "active" form of proteasomes (molecular weight: about 1000-kDa) is different from 26S protease complex.
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PMID:A novel "active" form of proteasomes from Xenopus laevis ovary cytosol. 850 84

A ubiquitin (Ub)/ATP-dependent proteolytic complex (26S proteasome) purified from rabbit skeletal muscle was dissociated into two subcomplexes, a 20S proteasome and a regulatory subunit complex, by preparative non-denaturing polyacrylamide gel electrophoresis (PAGE). The isolated regulatory subunit complex preparation gave a single broad band on analytical non-denaturing PAGE, and several bands ranging between 33 and 110 kDa on SDS-PAGE. This complex was found to consist of about 20 subunits on the basis of two-dimensional PAGE, the pattern of which appeared identical or very similar to that of the 33-110 kDa 26S proteasome subunits. The apparent molecular mass of the complex was estimated to be 1100 kDa by Ferguson plot analysis and also by Superose 6 gel filtration. Unlike the 26S proteasome, neither ATPase activity nor protease activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Phe-Ser-Arg-MCA, Z-Leu-Leu-Glu-beta NA, [14C]-casein, [125I]-lysozyme and Ub-[125I]-lysozyme were significantly detectable in the regulatory subunit complex. This complex was found to be capable of associating with itself in MgATP-dependent manner. These results suggest that a regulatory subunit complex dissociated from the 26S proteasome comprises all the higher molecular mass subunits of the 26S proteasome, and has no detectable ATPase and protease activities, although the homo-oligomerization occurs in an ATP-dependent fashion.
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PMID:Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization. 854 9

A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.
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PMID:Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus. 862 29

In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pI 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt as a substrate for ubiquitin-dependent proteolysis (Obin, M. S., Nowell, T., and Taylor, A. (1994) Biochem. Biophys. Res. Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to Gt, included (i) the presence in ROS membranes "stripped" of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of 125I-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a "ladder" of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these 125I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of 125I-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (E1); (ii) four ubiquitin carrier proteins (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, an enzyme required for "N-end rule" proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass 125I-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties. Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.
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PMID:Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt and rhodopsin. 866 97

A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+. The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide- of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.
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PMID:Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. 870 14

Membrane cofactor protein (MCP, CD46) of the complement system is a measles virus (MV) receptor. Human lymphocytes express a heavily glycosylated (H) and a lightly glycosylated (L) form of MCP, which confers a two-band profile on SDS-PAGE the ratio of which is controlled genetically and organ-specifically. In contrast, granulocytes express a single heavily glycosylated form regardless of lymphocyte MCP phenotype. We investigated susceptibility to MV of granulocytes and lymphocytes from individuals with different lymphocyte MCP phenotypes. In any individual, granulocytes were > 10-fold less susceptible to MV than lymphocytes, and the lymphocytes with predominant H form were generally less susceptible to those with an increasing amount of L form. Thus, lymphocytes always exhibit high susceptibility to MV compared to granulocytes in all individuals. This finding may explain the lymphopenia and immunosuppression observed secondary to MV infection.
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PMID:Human lymphocytes are more susceptible to measles virus than granulocytes, which is attributable to the phenotypic differences of their membrane cofactor protein (CD46). 871 5


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