Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We compared the abilities of young and senescent fibroblasts to take up and degrade [3H]ribonuclease A (native and oxidized), [3H]ribonuclease4-13, [3H]hemoglobin, [3H]glyceraldehyde-3-phosphate dehydrogenase, [3H]beta-galactosidase, [3H]glycogen phosphorylase, and [125I]serum albumin. The endocytic uptake of these proteins ranged from fluid-phase to predominantly absorptive. Intralysosomal degradation rates of the different endocytosed proteins varied by an order of magnitude, but in no case was there a difference between cultures of young and senescent fibroblasts.
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PMID:Degradation of endocytosed proteins is unaltered in senescent human fibroblasts. 314 44

A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with beta-galactosidase linked by the tetrapeptide Ile-Glu-Gly-Arg. RNase A was cleaved from the fusion using factor Xa, and the resulting product purified and reconstituted. The isolated RNase A was chromatographically, catalytically, and immunologically identical with authentic RNase A. This work argues that the method suggested by Nagai and Thogersen [Nagai, K. & Thogersen, H. C. (1984) Nature (Lond.) 309, 810-812] for releasing fusion proteins is quite general, even when applied to particularly complicated expression problem. The procedure here makes RNase A available for the first time as a model for studying structure-function relationships in proteins using site-directed mutagenesis.
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PMID:Expression of bovine pancreatic ribonuclease A in Escherichia coli. 354 26

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
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PMID:Ribonuclease S-peptide as a carrier in fusion proteins. 845 73

The study of enzyme kinetics under steady-state conditions represents a common and very useful method for investigating the mechanisms of enzymatic reactions. We report the use of mass spectrometry (MS) coupled with HPLC for the kinetic analysis of enzymatic reactions in real time. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A (RNase A) and the substrate-specific hydrolysis of lactose with beta-galactosidase can be monitored using ion-spray (pneumatically assisted electrospray) mass spectrometry as a sensitive and specific detector for the native substrates. The resulting data can be used to calculate both KM and Vmax for each system. Kinetic parameters obtained for RNase A and beta-galactosidase paralleled those obtained by conventional techniques. These findings suggest the possibility of developing alternative techniques, based on mass spectrometric detection, for performing kinetic analyses of enzymatic processes where no simple spectrophotometric assay is feasible. In addition to enabling the determination of kinetic parameters for authentic substrates, and not chromogenic analogs, such assays would also be useful in situations where very high sensitivity and specificity are desired.
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PMID:Kinetic monitoring of enzymatic reactions in real time by quantitative high-performance liquid chromatography-mass spectrometry. 853 90

Tissue-specific delivery of variety of molecules has been a valuable technique for biological and medical research and for the diagnosis and therapy of cancer. We have therefore examined the ability of streptavidin-protein A (ST-PA) fusion protein complexed with monoclonal antibodies (mAbs) to transfer biotinylated proteins into specific type of cells. ST-PA/mAbs complexes could efficiently deliver biotinylated beta-galactosidase into a variety of cancer cell lines through molecules expressed on their surface. In addition, ST-PA/mAb complexed with either biotinylated glucose oxidase or biotinylated ribonuclease A could be transferred to specific cell types and made to display cytotoxic activity against the transduced cell. The flexibility of the system was enhanced by the fact that the cell-targeting specificity could be altered by just changing the mAb used and the "payload" molecule could be replaced by substituting one biotinylated protein or enzyme with another. This flexibility was achieved without the need to generate a covalent chemical link or engineering new recombinant molecules. Results obtained to date suggest that the ST-PA fusion protein may be used as a nearly "universal carrier" to transfer a variety of effector molecules into target cells with a high degree of specificity. Essentially, the ST-PA fusion protein effectively serves as a high-efficiency, modular "molecular bridge" for the transfer into cells of a wide variety of effector molecules.
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PMID:Cell-specific, multidrug delivery system using streptavidin-protein A fusion protein. 881 44

The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate and yield of hen egg white lysozyme was investigated. Separate experiments were conducted in which known amounts of individual contaminants were added to test their effect on renaturation kinetics. On the basis of a simplified model for the kinetic competition between folding and aggregation, it was found that none of the above contaminants had an effect on the rate of the folding reaction, but some of them significantly affected the rate of the aggregation reaction and, thus, the overall renaturation yield. While ribosomal RNA did not seem to affect the aggregation reaction, plasmid DNA and lipopolysaccharides increased the aggregation rate, resulting in a decrease of about 10% in the overall renaturation yield. Phospholipids were found to improve refolding yields by about 15% by decreasing the overall rate of the aggregation reaction without affecting the rate of the folding reaction. Proteinaceous contaminants which aggregate upon folding, such as beta-galactosidase and bovine serum albumin, were found to significantly decrease renaturation yields by promoting aggregation. This effect was strongly dependent on the concentration of the proteinaceous impurity. On the other hand, the presence of refolding ribonuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation kinetics of lysozyme, even at concentrations as high as 0.7 mg/mL.
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PMID:Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. 910 38