Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 998 bp fragment of plasmid pBR322, comprising part of the TEM-1 beta-lactamase gene, was labelled with biotin-11-dUTP for use as a DNA probe in a rapid non-isotopic spot hybridisation test. Diluted broth cultures of bacteria producing different beta-lactamases were filtered onto nitrocellulose and lysed in situ. Following pre-hybridisation treatment with proteinase K, hybridisation with the labelled probe was demonstrated using a commercially available streptavidine/polyalkaline phosphatase-based detection system. The probe was highly specific, reacting only with strains producing either the TEM-1 or structurally similar TEM-2 enzyme. An inoculum of 3-4 X 10(6) cells gave optimum positive discrimination. When 90 recent ampicillin-resistant strains of Escherichia coli isolated from patients with urinary tract infections were screened using the system, 72% gave a positive hybridisation signal.
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PMID:Detection of TEM beta-lactamase genes by non-isotopic spot hybridisation. 331 35

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.
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PMID:Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli. 333 Jul 53

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.
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PMID:Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export. 786 84

TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of TolR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.
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PMID:Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity. 837 53

The ExbB protein together with the ExbD and TonB proteins is involved in energy-coupled transport across the outer membrane of Escherichia coli. To understand this unusual process it is required to determine the subcellular location of ExbB and its transmembrane arrangement. Using ExbB-beta-lactamase fusion proteins as reporters for a periplasmic versus a cytoplasmic location of the fusion sites, and accessibility of ExbB in spheroplasts and cell lysates to aminopeptidase K, trypsin, and proteinase K, we arrived at a model of ExbB topology in the cytoplasmic membrane. Starting with the N terminus in the periplasm ExbB contains three transmembrane segments (residues 16-39, 128-155, 162-194) a small periplasmic loop and two large portions in the cytoplasm. Two of the 18 fusion proteins studied, ExbB34-beta-lactamase and ExbB41-beta-lactamase, conferred a high ampicillin resistance. Protease experiments revealed a high respectively low percentage of the molecules in a reverse transmembrane orientation. Both proteins were lacking positive charges at the inner side of the cytoplasmic membrane which determine the orientation of transmembrane segments.
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PMID:Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli. 844 62