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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ubiquitin has been purified to homogeneity, through a dialysis membrane having a NMW cutoff of 12 kDa, by taking advantage of its non-dialysable nature under these conditions. The dialysate was continuously recycled through a CM-52 cation exchange column at pH 4.5. The adsorbed fraction was eluted selectively at pH 7.2. Ubiquitin (25 mg) was obtained from 500 ml of packed RBCs. On SDS PAGE, ubiquitin showed varying mobility depending on the time of boiling in SDS. With 2 min of boiling, the molecular weight seemed to be 10.5 kDa, whereas 10 min of boiling resulted in a molecular weight of 8.5 kDa. Ubiquitin showed a slow intrinsic proteolytic activity against SDS-denatured beta-galactosidase in the absence of ATP. For the first 4 hr, there was no detectable degradation, but degradation was nearly complete after 8 hr. These data are not in agreement with those of Freid et al. [Proc. Natl Acad. Sci, USA, 84 (1987), 3685] who have reported a proteolytic activity comparable to that of other proteolytic enzymes.
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PMID:Ubiquitin with a non-ATP-dependent slow intrinsic proteolytic activity: a mild and rapid purification procedure. 132 85

In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease. With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension. These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase. The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease. Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.
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PMID:Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae. 205 Jun 95

The baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV, which is representative of the MNPV subtype in which the virions may contain many nucleocapsids within a single viral envelope) encodes a protein, v-ubi, that has 76% identity with the eukaryotic protein ubiquitin. Transcriptional mapping indicated that the gene for v-ubi was transcribed during the late phase of viral infection. Two transcriptional start sites potentially encoding v-ubi were identified. Both sites were contained within a sequence motif common to baculovirus late genes. A recombinant virus, AcUbi-beta Gal, encoding a ubiquitin-beta-galactosidase fusion protein was constructed to monitor the temporal regulation of v-ubi gene during viral infection. The fusion protein was expressed maximally at 14-18 hr postinfection, consistent with its classification as a late protein. The amount of ubiquitin-beta-galactosidase fusion protein that accumulated in AcUbi-beta Gal-infected cells by 48 hr postinfection was approximately 14% of the level of beta-galactosidase that was synthesized under control of the polyhedrin promoter. Transcriptional analysis confirmed that synthesis of the fusion protein was directed by the v-ubi gene promoter. AcUbi-beta Gal also produced normal levels of authentic viral ubiquitin message. Southern blot analysis of AcUbi-beta Gal and 15 additional isolates revealed that the fusion sequences had not recombined at the ubiquitin locus. A polyubiquitin gene was isolated and sequenced from Spodoptera frugiperda, a lepidopteran host cell line for AcMNPV. The predicted amino acid sequence of the product of the host gene is identical to animal ubiquitin.
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PMID:Identification of a viral gene encoding a ubiquitin-like protein. 215

The lymphocyte cell surface receptor for the high endothelial venules (HEV's) of peripheral lymph nodes is specifically recognized by the monoclonal antibody MEL-14. Three independent complementary DNA (cDNA) clones, each of which encodes the protein ubiquitin, were detected by virtue of the expression of the MEL-14 antigenic determinant on cDNA-beta-galactosidase bacterial fusion proteins. The antigenic determinant defined by MEL-14 resides in the carboxyl terminal 13-amino-acid proteolytic peptide of ubiquitin, but is undetected in intact undenatured ubiquitin and other cellular ubiquitinated proteins. Antisera and monoclonal antibodies to ubiquitin determinants bind to the surface of both HEV-receptor positive and negative cell lines. The MEL-14-identified cDNA clones hydridize to RNA transcripts that encode tandemly repeated ubiquitins. Sequence analysis of these polyubiquitin cDNA's does not identify a leader sequence for export to the cell surface. The expression of the MEL-14 epitope of ubiquitin depends upon its local environment. The steady-state levels of expression of the ubiquitin messenger RNA's do not correlate with either the tissue derivation of the RNA or the expression of the lymphocyte HEV receptor. Regulation of the expression of the HEV receptor is not likely to reflect the transcriptional control of ubiquitin genes, but rather to reflect control of the expression of the HEV core polypeptide or its level or form of ubiquitination.
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PMID:Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species. 300 14

Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N epsilon-ubiquitinyl-cytochrome-c, N epsilon-K48polyubiquitinyl-lysozyme, or an N alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.
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PMID:Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. 952 56

We have recently identified a cDNA for a ubiquitin-specific protease (UBP), UBP41, that encodes the smallest functional UBP identified to date, using an Escherichia coli-based in vivo screening method. In the present study we isolated highly related cDNAs encoding a new family of UBP enzymes, named UBP46, UBP52 and UBP66. These UBPs have virtually identical catalytic domains spanning the sequence of UBP41 between the active-site Cys and the His box (95% identity). However, they possess distinct N- and/or C-terminal extensions. Moreover, they are more closely related to each other than to any other members of the UBP family. Thus these chick UBPs must define a novel family of de-ubiquitinating enzymes and should represent the first example among the UBP family enzymes, whose multiplicity is achieved by variation in their N- and C-terminal extensions. The chick UBPs were expressed in E. coli, and purified from the cells to apparent homogeneity using 125I-labelled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. Each of the purified UBP46, UBP52 and UBP66 enzymes behaved as proteins of similar sizes under both denaturing and non-denaturing conditions, suggesting that all of them consist of a single polypeptide chain. The UBP enzymes cleaved the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes and thus are active against ubiquitin-beta-galactosidase as well as a ubiquitin C-terminal extension protein of 80 amino acids. All UBPs except UBP66 released free ubiquitin from poly-His-tagged di-ubiquitin. However, the isopeptidase activity for hydrolysing polyubiquitinated lysozyme conjugates was not detected from these UBPs, which makes these UBPs distinct from UBP41. These results suggest that the chick UBPs may play an important role in production of free ubiquitin from linear polyubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.
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PMID:A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct N- and C-terminal extensions. 972 77

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.
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PMID:The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase. 1096 18

The ubiquitin-specific proteases (Ubps) are a family of largely dissimilar enzymes with two major conserved sequence regions, containing either a conserved cysteine residue or two conserved histidine residues, respectively. The murine Unp oncoprotein and its human homologue, Unph, both contain regions similar to the conserved Cys and His boxes common to all the Ubps. In this study we show that Unp and Unph are active deubiquitinating enzymes, being able to cleave ubiquitin from both natural and engineered linear ubiquitin-protein fusions, including the polyubiquitin precursor. Mutation of the conserved Unp Cys and His residues abolishes this activity, and identifies the likely His residue in the catalytic triad. Unp is tumorigenic when overexpressed in mice, leading to the suggestion that Unp may play a role in the regulation of ubiquitin-dependent protein degradation. We have demonstrated here that the high-level expression of Unp in yeast does not disrupt the degradation of the N-end rule substrate Tyr-beta-galactosidase (betagal), the non-N-end rule substrate ubiquitin-Pro-betagal, or the degradation of abnormal, canavanine-containing proteins. These data suggest that Unp is not a general modulator of ubiquitin-dependent proteolysis. However, Unp may have a role in the regulation of the degradation of a specific, as yet undescribed, substrate(s).
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PMID:Characterization of the ubiquitin-specific protease activity of the mouse/human Unp/Unph oncoprotein. 1101 21

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