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)

The turnover rate of an individual protein is a function of the rates of synthesis and loss of that protein. For most intracellular proteins, loss occurs through digestion by lysosomal or cytosolic proteases. Although a significant proportion of hepatic lysosomal enzymes is released from the hepatocyte by excretion into bile, the contribution of biliary excretion to the turnover of hepatic lysosomal enzymes has never been measured. Thus, we used in vivo pulse-labeling to determine the half-lives of two hepatic hydrolases, beta-galactosidase (beta-gal) and beta-glucuronidase (beta-glu). Each enzyme was purified by immunoisolation from hepatic lysosomes that were isolated at various times after injection of rats with 3H-labeled leucine. The decay curves for the specific radioactivities of beta-gal and beta-glu were used to calculate the half-lives of the proteins, which were 3.8 and 5.1 days, respectively. To determine the percent of total hepatic contents of each enzyme that was lost per day by biliary excretion, we collected bile from bile fistula rats for 24 hours and then used radioimmunoassays to quantitate the amounts of beta-gal and beta-glu in bile and liver samples of the same rats. We found that approximately 4% of the total hepatic contents of both beta-gal and beta-glu was excreted into bile per day. Finally, we used these data to calculate that 31% and 41% of hepatic losses of beta-gal and beta-glu, respectively, were due to biliary excretion. These results suggest that extracellular release through biliary excretion is a major mechanism contributing to the turnover of lysosomal hydrolases.
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PMID:Quantitative importance of biliary excretion to the turnover of hepatic lysosomal enzymes. 760 20

Short-lived proteins are targeted for turnover by sequence elements known as degradation signals. Because of the large size and heterogeneity of these signals, the structural features important for their function are not well defined. In this study, we have isolated three classes of degradation signals by screening short artificial sequences for the ability to destabilize a reporter protein. Class I and class II signals were derived by inserting random nonapeptide sequences after the second residue of beta-galactosidase. Class III signals contained five-residue homopolymers at the same position. Class I beta-galactosidase turnover was inhibited in mutants lacking either the ubiquitin-conjugating enzyme Ubc2 or the ubiquitin protein ligase Ubr1. Class I random inserts functioned to promote N-terminal proteolytic processing and define a novel pathway for exposure of residues that are destabilizing according to the N-end rule. Efficient degradation of proteins containing class II signals required at least three Ubc enzymes: Ubc6, Ubc7, and either one of the related enzymes Ubc4 and Ubc5. Analysis of 56 amino acid substitutions in the class II signal suggested that it is recognized in the form of an amphipathic alpha helix. Class III signals consisted of short tracts of hydrophobic residues such as Leu and Ile. Degradation of class III proteins involved the Ubc4 and Ubc5 enzymes but not Ubc2, Ubc6, or Ubc7. Clusters of hydrophobic residues appear to be critical for the recognition of both class II and class III signals.
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PMID:Synthetic signals for ubiquitin-dependent proteolysis. 762 4

The interactions between CD4 or CD8 and p56lck were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8 alpha to the DNA-binding protein LexA, and the unique amino-terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased beta-galactosidase activity and growth on leucine-deficient medium, indicating interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56lck with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8 alpha interacting with itself, since homotypic or heterotypic interactions between CD8 alpha and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full-length src family tyrosine kinases in yeast.
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PMID:Interactions between the amino-terminal domain of p56lck and cytoplasmic domains of CD4 and CD8 alpha in yeast. 766 3

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.
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PMID:In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs. 768 Mar 41

We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase-catalyzed processing. An artificial substance P-precursor is produced as a beta-galactosidase (1-459) fusion protein containing nine copies of the decapeptide sequence Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by alpha-chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted converted to substance P by papain-catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase-catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo- and polypeptides.
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PMID:Peptide production by a combination of gene expression, chemical synthesis, and protease-catalyzed conversion. 768 60

Comparative analysis of the enzymatic profiles of 58 spirochaetal isolates clearly differentiated borrelias from leptospires, serpulinas and a treponeme. Strains of both Borrelia burgdorferi and Borrelia hermsii characteristically produced significant amounts of leucine arylamidase. This enzyme activity was not unique to borrelias but was also detected amongst pathogenic and non-pathogenic leptospira serovars. This fact, however, did not hamper a correct differentiation of borrelias from these spirochaetes, because leptospires possessed unique enzyme profiles. The API ZYM system could not differentiate the human strains of B. burgdorferi from those isolated from ticks, or from B. hermsii. Treponema phagedenis could be differentiated from all the other spirochaetes by the production of alpha-fucosidase. Our results confirm and extend previous studies indicating that human and animal intestinal spirochaetes have many common enzyme activities. All strains produced reactions of maximum intensity when tested for the presence of beta-galactosidase activity. However the avian strains lacked esterase (C4) which was present in human and swine intestinal spirochaetes. All strains of Serpulina hyodysenteriae, and Serpulina innocens as well as the human intestinal spirochaete strain HRM-14 showed alpha and beta glucosidase activity. Both enzyme activities were absent or insignificant in most other intestinal spirochaetes examined: 25 different human strains, non-pathogenic swine strain M1 and the avian strain 4742. However, swine strain LL3 and avian strain 1380 showed some beta-glucosidase activity.
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PMID:Comparative study of the enzyme activities of Borrelia burgdorferi and other non-intestinal and intestinal spirochaetes. 776 Jul 53

Upon in vitro processing of the recombinant HIV-1/gag p24 protein, expressed in Escherichia coli as a fusion protein, by HIV-1 protease, a cleavage site within the staphylococcal protein A fusion partner was found. N-terminal sequencing of the protein A fragments showed that HIV-1 protease cleavage occurred between phenylalanine-235 and tyrosine-236 within the sequence Gln-Asn-Ala-Phe/Tyr-Glu-Ile-Leu (QNAF/YEIL) in the IgG-binding domain C of the protein A encoded by the pRIT2T fusion gene vector (Pharmacia). Results presented here have proven that the protease-sensitive site is viable in vitro on the protein A alone and other chimeric protein, protein A/beta-galactosidase. A possible significance of this phenomenon in biotechnology work is discussed.
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PMID:Staphylococcal protein A is a novel heterologous substrate for the HIV-1 protease. 776 14

Thirteen Escherichia coli strains of different biotypes isolated from urine and faeces cultures were studied for metabolic and compositional changes during starvation in seawater at different timepoints. Additionally, the antibiotic susceptibility of the starved E. coli cells was evaluated over time on Mueller-Hinton agar (Bauer-Kirby method). All starved E. coli cells lost beta-galactosidase activity gradually with time and acquired the ability to degrade gelatine. Nine of the E. coli strains lost the ability to decarboxylate lysine and seven to acidify melibiose. C4 esterase, C8 esterase lipase, leucine arylamidase and C14 lipase activity increased during starvation, while alkaline and acid phosphatase and phosphoamidase activity decreased. Most of the E. coli strains underwent alterations in their electrophoretic protein pattern. The traditional Bauer & Kirby method was shown to be inadequate for testing antibiotic susceptibility of starved strains.
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PMID:Metabolic and compositional changes in Escherichia coli cells starved in seawater. 784 33

Cathepsin A (also named "protective protein" and carboxypeptidase L) stabilizes beta-galactosidase and activates neuraminidase by forming with them a high-molecular-weight lysosomal complex. We determined the main forms of the supramolecular organization of human placental cathepsin A and the quantitative relationship between them, using an affinity chromatography on agarose-Phe-Leu for direct purification of cathepsin A. We found that cathepsin A in human placenta exists as the following three forms: a 1270-kDa complex with beta-galactosidase and neuraminidase (about 1% of total cathepsin A), a 680-kDa complex with beta-galactosidase (30-40% of total), and a free 98-kDa cathepsin A dimer (60-70% of total). All forms are in dynamic equilibrium with each other, but almost all placental beta-galactosidase is associated with cathepsin A in the 680-kDa complex. The main properties of free cathepsin A (including the capacity to associate with beta-galactosidase) were found to be identical to those of cathepsin A obtained by dissociation of the 680-kDa complex. The presence of a free cathepsin A pool in the lysosome is connected with its sixfold overproduction in the cell compared to beta-galactosidase and may be necessary to ensure cathepsin A proteolytic function in addition to its protective role for beta-galactosidase and neuraminidase in the lysosomal multienzymatic complex. Such a dual function of cathepsin A is also confirmed by our finding that it is the only carboxypeptidase of placenta extract able to catalyze the hydrolysis of both carbobenzoxy (CBZ)-Glu-Tyr and CBZ-Phe-Leu dipeptide substrates.
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PMID:Direct affinity purification and supramolecular organization of human lysosomal cathepsin A. 805 88

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA binding domain is composed of three C2H2 zinc fingers. To identify its nuclear localization signal (NLS), wild type NGFI-A and various mutants were transfected into COS cells and their cellular location assayed by indirect immunofluorescence. Although wild type NGFI-A was located exclusively within the nucleus, deletions lacking the highly basic zinc finger region were not efficiently translocated to the nucleus. However, DNA binding per se is not required for nuclear localization, as an NGFI-A mutant (A Y339G), which does not bind DNA, is still faithfully directed to the nucleus. To determine the minimal region(s) of NGFI-A sufficient to direct nuclear localization, the cellular location of various NGFI-A/beta-galactosidase fusion proteins was examined. Fusion proteins containing all three zinc fingers were found in the nucleus, but those containing only two zinc fingers were predominantly cytoplasmic. When the zinc finger structure was altered by mutating a zinc-chelating cysteine residue in any one of the three zinc fingers, the resulting domain was no longer capable of directing beta-galactosidase to the nucleus. Furthermore, the mutation of an arginine residue in the third zinc finger of NGFI-A, a position which is occupied by a leucine residue in most C2H2 zinc fingers, abolished nuclear localization, but had no effect on DNA binding. These studies suggest that NGFI-A contains a novel NLS which is dependent on the overall structure of the DNA binding domain and not solely upon its highly basic nature.
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PMID:The nuclear localization signal of NGFI-A is located within the zinc finger DNA binding domain. 813 43


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