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)

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.
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PMID:Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process. 328 38

The human TOP3alpha gene encoding DNA topoisomerase IIIalpha (hTop3alpha) has two potential start codons for the synthesis of proteins 1,001 and 976 aa residues in length. The sequence of the N-terminal region of the 1,001-residue form resembles signal peptide sequences for mitochondrial import, and fluorescence microscopy shows that the addition of as few as the first 34 aa of the 1,001-residue form of hTop3alpha to a green fluorescent protein can direct the chimeric protein to mitochondria. Biochemical analyses of subcellular fractions of HeLa cells further demonstrate that a distinctive fraction of hTop3alpha is present inside mitochondria, as evidenced by its resistance to proteinase K. This fraction constitutes several percent of the enzyme in the nuclear fraction, suggesting that the distribution of the mitochondrial and nuclear forms of hTop3alpha is roughly in proportion to the DNA contents of these cellular compartments. The presence of a type IA DNA topoisomerase in the mitochondria of other eukaryotes is supported by an examination of the amino acid sequences of mouse and Drosophila DNA topoisomerase IIIalpha and Schizosaccharomyces pombe DNA topoisomerase III. Given the presence of at least one type IA DNA topoisomerase in all forms of life examined to date, the finding of a type IA enzyme in mitochondria further supports the notion of a key role of such enzymes in DNA transactions.
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PMID:Dual localization of human DNA topoisomerase IIIalpha to mitochondria and nucleus. 1220 14

All microbial biofilms are initiated through direct physical contact between a bacterium and a solid surface, a step that is controlled by inter- and intramolecular forces. Atomic force microscopy and confocal laser scanning microscopy were used simultaneously to observe the formation of a bond between a fluorescent chimeric protein on the surface of a living Escherichia coli bacterium and a solid substrate in situ. The chimera was composed of a portion of outer membrane protein A (OmpA) fused to the cyan-fluorescent protein AmCyan. Sucrose gradient centrifugation and fluorescent confocal slices through bacteria demonstrated that the chimeric protein was targeted and anchored to the external cell surface. The wormlike chain theory predicted that this protein should exhibit a nonlinear force-extension "signature" consistent with the sequential unraveling of the AmCyan and OmpA domains. Experimentally measured force-extension curves revealed a unique pair of "sawtooth" features that were present when a bond formed between a silicon nitride surface (atomic force microscopy tip) and E. coli cells expressing the OmpA-AmCyan protein. The observed sawtooth pair closely matched the wormlike chain model prediction for the mechanical unfolding of the AmCyan and OmpA substructures in series. These sawteeth disappeared from the measured force-extension curves when cells were treated with proteinase K. Furthermore, these unique sawteeth were absent for a mutant stain of E. coli incapable of expressing the AmCyan protein on its outer surface. Together, these data show that specific proteins exhibit unique force signatures characteristic of the bond that is formed between a living bacterium and another surface.
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PMID:Simultaneous force and fluorescence measurements of a protein that forms a bond between a living bacterium and a solid surface. 1574 61