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 gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (alpha and beta) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigorous determination of the nature of the initiation triplet for IF2 beta, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2 alpha and IF2 beta. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2 alpha synthesis. Under these conditions, IF2 beta is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with beta-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2 beta initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2 alpha start sequences but containing genetic information for this 18-base region. A hybrid protein with beta-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2 beta, demonstrating that GUG, located 471 bases downstream from the IF2 alpha external start codon, is the internal start codon for the shorter form of IF2.
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PMID:Characterization of the translational start site for IF2 beta, a short form of Escherichia coli initiation factor IF2. 211 58

The immunity protein to colicin A (Cai), which is constitutively expressed at a very low level in Escherichia coli strains, has been studied in recombinant plasmid constructs allowing expression of various immunity fusion proteins under the control of inducible promoters. The 13-amino acid NH2-terminal region of Cai was substituted by polypeptides from beta-galactosidase or from colicin A. Upon induction of the chimeric proteins, the rate of expression of the immunity protein could be correlated to the level of resistance to colicin A. The immunity protein has been "tagged" with an epitope from the colicin A protein for which a monoclonal antibody is available. Using this technique, we have directly demonstrated that the immunity protein is located in the cytoplasmic membrane. The results indicate that the NH2-terminal region of Cai is directed toward the cytoplasm and is probably not required for Cai insertion into the membrane or for its function.
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PMID:Use of a foreign epitope as a "tag" for the localization of minor proteins within a cell: the case of the immunity protein to colicin A. 244 80

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.
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PMID:Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. 246 43

vWF is a multimeric glycoprotein that serves as the major carrier in plasma of Factor VIII (FVIII). We have used an anti-human vWF MAb W5-6A to investigate the FVIII binding site on vWF. W5-6A inhibited FVIII binding to vWF-coated polystyrene tubes in a concentration-dependent manner with 90% inhibition of FVIII binding at a concentration of 10 micrograms/ml. The W5-6A epitope was identified by screening a vWF fragment library using the bacteriophage expression vector lambda gt11. DNA sequence analysis of 29 immunoreactive phage clones localized the W5-6A epitope to a nonadecapeptide spanning amino acid residues threonine 78 to threonine 96 at the amino-terminus of the mature vWF polypeptide. Purified beta-galactosidase/vWF fusion protein from one of these clones, vWF9, was incubated with radiolabeled W5-6A and caused near complete inhibition of W5-6A binding to vWF. Inhibitory activity was lost after vWF9 trypsinization or reduction and alkylation. These data indicate that (a) the antigenic determinant recognized by W5-6A localizes to a nonadecapeptide at the NH2 terminus of the mature vWF polypeptide, (b) disulfide bonds within vWF9 may be necessary to maintain the structure required for immunoreactivity with W5-6A, and (c) W5-6A recognizes an immunogenic region on vWF that may be at (or near) the major FVIII binding domain.
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PMID:A monoclonal antibody to von Willebrand factor (vWF) inhibits factor VIII binding. Localization of its antigenic determinant to a nonadecapeptide at the amino terminus of the mature vWF polypeptide. 247 30

The distribution and localization of acid stable trypsin inhibitor (ASTI) in normal and malignant human tissues from various organs were examined using immunohistochemical techniques that used goat antibody raised against highly purified ASTI from human urine. Tissues were assessed as positive only when they were stained by both the biotin-avidin-peroxidase complex system and biotin-streptavidin-beta-galactosidase complex system, and the staining was abolished by absorption with purified ASTI. Under normal conditions, ASTI immunoreactivity was observed in only a few organs. Positive tissues for ASTI immunoreactivity included the kidney proximal tubules, glial cells of the cerebrum, fibrillar structures of the lamina propria of the stomach and colon, and bronchial epithelial cells. No ASTI immunoreactivity was observed in the cardiovascular system, reproductive system, or other tissues examined. As is not the case for normal tissues, ASTI immunoreactivity was found to be widely distributed in malignant tumors. Staining was observed in the extracellular space, i.e., in the stroma of the tumor and in connective tissues around the tumor invasion, whereas no ASTI immunoreactivity was detected in the malignant cells. Considering the identity of the first 36 NH2-terminal residues of ASTI purified from plasma or urine with a recently reported endothelial cell growth factor, the present findings suggest that ASTI could play an important role, not limited to its function as a protease inhibitor, in the invasive growth of malignant neoplasms.
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PMID:Distribution of acid stable trypsin inhibitor immunoreactivity in normal and malignant human tissues. 247 69

Recombinant fused protein containing human erythrocyte NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2.) was produced in Escherichia coli, which was linked to the NH2 terminus of beta-galactosidase of the vector pUC13 via a recognition sequence of alpha-thrombin. Cleavage of purified fused protein with alpha-thrombin yielded the enzyme whose apparent molecular weight (32,000) was the same as the native enzyme. The amino-acid sequence from Phe-1 to Leu-10 was determined to be identical to that of the authentic enzyme. The purified enzyme showed an identical absorption spectrum and similar catalytic properties to the native enzyme. Establishment of the expression system would make it possible to determine the reaction mechanism of the enzyme.
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PMID:Expression of human erythrocyte NADH-cytochrome b5 reductase as an alpha-thrombin-cleavable fused protein in Escherichia coli. 250 Jan 49

We describe the identification of the gene encoding an immunodominant 32-kilodalton (kDa) protein of Mycobacterium tuberculosis. The 32-kDa antigen is abundantly secreted into the culture supernatant of a variety of mycobacteria and appears to be a major stimulant of cellular and humoral immunity against mycobacteria. Recombinant clones expressing a 140- or 125-kDa beta-galactosidase fusion protein reactive with rabbit polyclonal anti-32 kDa protein serum were detected. The corresponding DNA sequence contains a 1,008-base-pair coding region. The deduced amino acid sequence corresponds to a 336-residue protein including the previously determined NH2-terminal sequence of the 32-kDa protein (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, M. Weckx, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Upstream of this NH2-terminal region, the gene codes for a signal peptide required for the secretion of a 294-amino-acid-long mature protein. A putative promoter sequence could be located upstream of the open reading frame. Comparison of the M. tuberculosis 32-kDa antigen with the Mycobacterium bovis BCG alpha-antigen (K. Matsuo, R. Yamaguchi, A. Yamazaki, H. Tasaka, and T. Yamada, J. Bacteriol. 170:3847-3854, 1988) revealed 73.8% homology between DNA sequences and 72.8% homology between amino acid sequences (signal and mature protein). Finally, the 140-kDa fusion protein could selectively be recognized by human tuberculous sera. This result confirms our previous finding that the 32-kDa antigen could be a valuable tool for the serological diagnosis of tuberculosis. Moreover, the availability of recombinant proteins opens perspectives for the localization of relevant B- and T-cell epitope regions on the 32-kDa antigen.
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PMID:Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. 250 31

The construction of two fused genes is described. One involves the in-frame fusion of the yeast prepro-alpha-factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH2-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the alpha-factor promoter, expressed active beta-galactosidase in alpha haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF alpha 1-lacZ fusion junction provided significantly higher levels of beta-galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.
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PMID:Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae. 250 25

Extracellular beta-galactosidase from P. canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration. The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5. In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium. The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined.
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PMID:[Molecular characteristics of beta-galactosidase secreted by Penicillium canescens]. 251 Aug 32

A total of six amino acid sequences encoded in conserved regions of the HIV-env (three from gp120 and three from gp41) were selected as potential antigenic domains. These sequences (11-20 amino acids) were fused to the NH2 terminus of beta-galactosidase by recombinant DNA techniques, and the purified chimeric proteins were used to titer (by immunodots) 75 sera from HIV-infected individuals of various stages. All the HIV antigens were recognized by some or all the HIV-seropositive sera but by none of the control sera. Of the three conserved domains in gp41, two are highly immunodominant. All (100%) HIV-seropositive sera reacted with one of these immunodominant domains in titers (approximately 1:100,000) almost two orders of magnitude higher than any other tested domain. This emphasizes the diagnostic value of the epitopes (ERYLKDQLLGIWGCSGKLIC) previously (see Refs. 11 and 12) identified in this domain. A decrease in average antibody titers is observed in late stages of infection for all the antigens tested, yet distribution of antibody reactivity was independent of stage for only three of the six domains. A significantly higher proportion of reactivity of seropositive sera in early stage (62%) compared with late stage (11%) of infection was found for a domain (NVTENFNMWKN) mapped at the NH2 terminus of gp120; serum antibody reactivity with this domain also correlated with a lack of culturable HIV in blood mononuclear cells.
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PMID:Patterns of antibody recognition of selected conserved amino acid sequences from the HIV envelope in sera from different stages of HIV infection. 254 48


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