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)

NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis after mineral or organic acid hydrolysis. The free mobility of cytochrome P-450 reductase in sodium dodecyl sulfate was identical to that of several soluble proteins used as standards (i.e. ovalbumin, bovin serum albumin, erythrocuprein, beta-galactosidase). Molecular weight estimates from sedimentation equilibrium studies in the presence of guanidine hydrochloride (76,500) are consistent with those determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at various per cent gel concentrations (79,000 to 80,000). Computer analysis of circular dichroism spectra of cytochrome P-450 reductase in the far ultraviolet region indicated the presence of 34 per cent alpha helical and 16 per cent beta structure. The amount of random structure was calculated to be 50 per cent.
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PMID:NADPH-cytochrome P-450 reductase. Circular dichroism and physical studies. 1 69

Eleven independent insertion mutations were isolated that prevented expression of major outer membrane protein 1b. Seven of the mutations were Mucts insertions located at ombP. These ompB::Mucts strains fell into two phenotypic classes with regard to expression of proteins 1a and 1b. The remaining four mutants were comprised of one Tn5 and three Mucts insertions mapping at par. The Mucts insertions at par were used to construct fusions of the lac operon to the par promoter. Expression of beta-galactosidase in these fusion strains reflected known regulatory properties of protein 1b. When an ompB allele was introduced into the par-lac fusion strains, beta-galactosidase activity was reduced 14- to 31-fold. Transcriptional regulation of the par gene and the existence of two functions at ompB are discussed. The results suggest that par is the structural gene for protein 1b and that an ompB gene product is a diffusible, positive regulatory element controlling expression of par.
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PMID:Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b. 11 44

Studies have been conducted to characterize further the interaction between 125I-labeled bovine thyrotropin (TSH) and bovine thyroid plasma membranes. Sequential subcellular fractionation of thyroid homogenates yielded preparations of progressively greater specific binding activity, highest activity being found in fractions previously shown to contain predominately plasma membranes (Amir, S. M., Carraway, T.F., Kohn, L.D., and Winand, R.J. (1973) J. Biol. Chem. 248, 4092-4100). Although binding of 125I-TSH by plasma membranes was greatest at pH 6.0, studies were conducted at pH 7.45 as well as pH 6.0, and results obtained differed quantitatively, but not qualitatively. Binding was maximal at 0 degrees, 15 degrees, and 22 degrees and steady state values remained unchanged for at least 22 hours. At 37 degrees, binding was decreased by 40% at 1 hour; the loss was even greater (65%) at 50 degrees. A similar loss of binding was evident when membranes were preincubated without TSH at 37 degrees or higher and were then incubated with 125I-TSH at 0 degrees. Lineweaver-Burk analysis indicated that preincubation resulted in loss of receptor sites without change in affinity of residual receptors. Addition of Ca2+ (1 to 10 mM) to the preincubation medium prevented the effect of preincubation at 37 degrees by preserving the number of receptor sites without altering their affinity. Under similar conditions, Na+ and K+ were without protective effect. Membranes bound 45Ca2+ in a specific and saturable manner. Scatchard plots indicated a dissociatiion constant (Kd) of 9 X 10(-5) M and a capacity (n) of 54 nmol/mg of membrane protein. 45Ca2+ was also displaced from membranes by Mg2+ and Mn2+. Ca2+ had a biphasic effect on binding; low concentrations (1 to 10 muM) added to the incubation mixture stimulated binding, while higher concentrations (0.1 mM) caused inhibition. Mg2+ and Mn2+, at comparable concentrations, were also inhibitory, Na+ and K+ less so. In the case of Ca2+, both the stimulatory and inhibitory concentrations were lower than those required to achieve saturation of Ca2+-binding sites. Proteolytic enzymes (trypsin, alpha-chymotrypsin, and pronase) sharply reduced binding of 125I-TSH, owing to a decrease in receptor sites. Phospholipases A and C enhanced binding of TSH, while neuraminidase and beta-galactosidase were without measurable effect.
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PMID:Properties of the interaction between bovine thyrotropin and bovine thyroid plasma membranes. 18 81

Escherichia coli strains have been isolated that produce hybrid proteins comprised of an NH2-terminal sequence from the lamB gene product (an outer membrane protein) and a major portion of the COOH-terminal sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23; a cytoplasmic protein). These proteins exhibit beta-galactosidase activity. One such strain, pop 3105, produces a hybrid protein containing very little of the lamB gene protein; the protein is found in the cytoplasm. The protein found in a second strain, pop 3186, contains much more of the lamB gene protein; a substantial fraction of the beta-galactosidase activity is found in the outer membrane, probably facing outward. These results indicate that information necessary to direct the lamB gene product to its outer membrane location is located within the lamB gene itself. The properties of such fusion strains open up the prospect of a precise genetic analysis of the genetic components involved in protein transport.
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PMID:Use of gene fusions to study outer membrane protein localization in Escherichia coli. 41 21

The rational development of peptide vaccines requires the identification of both B- and T-cell epitopes. In this study, potential T-helper cell epitopes of Semliki Forest virus (SFV) were identified on the basis of their ability to induce delayed-type hypersensitivity (DTH) in mice using recombinant SFV fragments produced as hybrid proteins with beta-galactosidase in Escherichia coli and synthetic peptides coupled to beta-galactosidase. Although the tested fragments spanned almost the entire amino acid sequence of the structural proteins of SFV, only one DTH-inducing region (located between amino acid 137 and 151 of the SFV E2 membrane protein) was identified. Peptides containing this E2 region stimulated lymph node cells from SFV-primed mice in vitro. The ability of the identified T-cell epitope to induce a specific T-helper response in mice was evaluated using synthetic peptides that contained combinations of the DTH-inducing region and different previously identified linear B-cell epitopes of E2. These peptides proved able to induce an antipeptide IgG response in mice in an H-2d-restricted fashion. One of the peptides was also able to induce high titres of IgG reactive with SFV-infected cells and protected 70-100% of the peptide-immunized mice after challenge with virulent SFV. Our findings suggest that DTH and T-helper activity are mediated by different doses of the same T-cell epitope.
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PMID:A delayed-type hypersensitivity-inducing T-cell epitope of Semliki Forest virus mediates effective T-helper activity for antibody production. 128 93

The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.
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PMID:Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans. 131 49

Mice exposed to radiation-attenuated cercariae of Schistosoma mansoni are highly resistant to challenge infection, and sera from these mice can confer partial resistance when transferred to naive recipients. These sera recognize Ag present in schistosomular and adult worms, among them an Ag of 200 kDa. A cDNA encoding a 62-kDa portion of this Ag was cloned; the deduced amino acid sequence of this cDNA clone shares homology with myosins of other species. To assess the immunoprophylactic potential, we carried out vaccination trials in mice using the recombinant polypeptide expressed as a fusion protein with beta-galactosidase presented in the form of proteosome complexes with the outer membrane protein of meningococcus. The level of protection achieved was 32%, and this level could be increased to 75% by removal of those amino acids included in the fusion protein that were derived from the vector to yield a polypeptide, designated rIrV-5. A similar level of protection was achieved when mice were immunized with the same dose of rIrV-5 in the form of protein complexes but without outer membrane protein, suggesting that protection did not require the use of adjuvant. However, at least three immunizations were necessary to achieve protection. Using mAb and sera from mice vaccinated with rIrV-5, we demonstrated that the native protein recognized by antibodies against rIrV-5 is a 200-kDa protein that is expressed on the surface of newly transformed schistosomula. The protection achieved with rIrV-5 in mice encourages additional studies of its potential as a vaccine candidate for the prevention of schistosomiasis.
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PMID:Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen. 143 Nov 31

We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process. This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming. The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment. prlF1 and similar alleles are gain-of-function mutations. Null mutations in this gene confer no obvious phenotypes. Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process. The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested. Lon protease activity has been measured in prlF1 cells and shown to be increased. However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation. Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner. It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone. Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions.
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PMID:Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent. 151 98

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.
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PMID:Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein. 158 63

The RAG1 gene encodes a membrane protein involved in the low-affinity glucose/fructose transport system of the yeast Kluyveromyces lactis. Analysis of steady-state mRNA levels analysis and quantitation of expression by beta-galactosidase from RAG1-lacZ fusions assays revealed that the RAG1 gene was poorly expressed in cells grown under gluconeogenesis conditions, but was induced more than ten-fold when they were grown on various sugars. These sugars included glucose, fructose, mannose, sucrose, raffinose, as well as galactose. Nucleotide sequence and deletion analysis of the 5' flanking region of the RAG1 gene showed that an essential cis-acting element required for induced transcription of the RAG1 gene resided between -615 and -750 from the coding sequence. This region contained a 22 bp purine stretch, and a pair of 11 bp direct repeat sequences. The 11 bp repeats harbor a CCAAT motif, a consensus sequence for binding of the yeast and mammalian HAP2/3/4-type protein complex. The transcription of the RAG1 gene was dramatically affected by three unlinked mutations, rag4, rag5 and rag8. We discuss the possible roles of RAG4, RAG5 and RAG8 gene products in the expression of the RAG1 gene, as well as the importance of the inducible RAG1 gene in the fermentative growth of K. lactis.
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PMID:Glucose transport in the yeast Kluyveromyces lactis. II. Transcriptional regulation of the glucose transporter gene RAG1. 160 79


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