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)

Due to Triton X-114 fractionation of synaptosomes isolated from the electric organ of the fish Torpedo, the existence of a hydrophilic and an amphiphilic form of the enzyme choline-O-acetyltransferase (ChAT) was revealed. Amphiphilic ChAT which represents about 10% of total enzyme activity in synaptosomes, reached 40% of ChAT activity measured in preparations of synaptosomal plasma membranes (SPM) which were washed with solutions of increasing ionic strength. ChAT activity bound to washed SPM could be partially solubilized using proteinase K but not phospholipase C. No ChAT solubilization occurred by treating intact synaptosomes with proteinase K. Water/Triton X-114 partition coefficients of hydrophilic and amphiphilic ChAT were found to be 6.5 and 0.17, respectively. Sedimentation coefficients determined by centrifugation in linear density gradients of sucrose containing Triton X-100, were 4.2S and 4.4S for amphiphilic and hydrophilic ChAT, respectively. On the other hand, removal of Triton X-114 from the detergent phase containing amphiphilic ChAT activity led to enzyme aggregation. Finally, amphiphilic ChAT was slightly more acidic (pH 6.6) than was hydrophilic enzyme (6.8-7.0). We conclude that in Torpedo synaptosomes two forms of ChAT activity, a soluble and a membrane-bound form, are indeed present which differ in their hydrophobicity. The soluble form is hydrophilic. The membrane-bound form is amphiphilic and it aggregates upon removal of detergent. These are two characteristics of integral membrane proteins. Membrane-bound ChAT is most probably intracellularly oriented and not bound to membrane through a 'receptor' protein.
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PMID:Membrane-bound choline acetyltransferase of the torpedo has characteristics of an integral membrane protein and can be solubilized by proteolysis. 150 66

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.
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PMID:Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol. 171 49

The fluorescence properties of proteinase K are described and related to the X-ray model refined at 1.48 A resolution. Upon excitation of proteinase K at 295 nm the fluorescence is determined by the two tryptophan residues, Trp-8 and Trp-212. The tryptophans are partly buried just below the surface of the molecule. Neither Trp is in a highly hydrophobic environment, suggesting that this cannot be the explanation for the fluorescence at 330 nm: formation of exiplexes with adjacent peptide bonds would seem to be the more likely cause. Trp-8 is located in a 'cavity', close to an internal cluster of water molecules. The contribution of Trp-8 to the total indole emission is 60% and that of Trp-212 is 40%. The tryptophan fluorescence quantum yield is constant in the pH range 3-9. The fluorescence spectrum resulting from the simultaneous excitation of the tyrosyl and tryptophyl residues at 280 nm is dominated by the indole fluorophores: 61% of the light absorbed by the tyrosyl side chains is transferred to the two indole rings. Iodide and caesium are not efficient quenchers of the proteinase K tryptophan fluorescence, which is explained by restricted access of the ions to the somewhat buried Trp side chains and by electrostatic repulsion of caesium ions. Acrylamide quenching proceeds via both a dynamic and a static process and the data show homogeneity of the indole fluorescence arising from fluorophores in similar environments. The activation energy for the thermal deactivation of the excited tryptophans is 54 kJ mol-1. This value is substantially higher than those found for other proteinases from microorganisms and arises from the thermostability of proteinase K. Photooxidation of proteinase K in the presence of proflavine follows the kinetics of a first order reaction. The two tryptophans differ in their photoreactivity, Trp-212 being considerably more reactive.
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PMID:Fluorescence properties of native and photooxidised proteinase K: the X-ray model in the region of the two tryptophans. 173 54

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].
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PMID:Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution. 173 56

The polymerase chain reaction (PCR) is an extremely sensitive technique that has been used for detection of DNA sequences in formalin-fixed, paraffin-embedded tissues. In order to verify that hepatitis B virus (HBV) DNA sequences are adequately preserved in routinely processed liver tissues, we performed PCR with five different primer pairs for HBV sequences on DNA extracted by two different methods from paraffin and frozen liver sections. The amount of PCR products obtained with DNA templates extracted by the proteinase K-SDS method from frozen sections was significantly larger than that from paraffin sections. However, boiling of deparaffinized sections in water containing Chelex-100 resulted in ample amounts of PCR products irrespective of the primers used. On Southern blots, the location of the bands of amplified DNA obtained by the different methods was consistent with the predicted size, suggesting that the viral sequences had not been altered by processing. Although freezing of fresh tissue yields quantitatively more HBV DNA, formalin fixation qualitatively preserves the viral DNA sequences adequately for detection by PCR. Therefore, formalin-fixed, paraffin-embedded tissues may be used for the detection of viral DNA sequences by PCR. Application of the described procedure to routinely processed tissues significantly broadens the applicability of this powerful diagnostic and investigative method.
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PMID:Comparative studies on the detection of hepatitis B virus DNA in frozen and paraffin sections by the polymerase chain reaction. 175 69

The reactions were studied of N-acyl-L-amino acid esters with various D-amino acid amides catalyzed by free alpha-chymotrypsin, trypsin and proteinase K in acetonitrile containing 80 or 5 vol. % of water. In the medium with low water content the incorporation of D-amino acid amides into peptides proceeded with satisfactory yield sometimes approaching that of analogous L-L dipeptides. In the media with high water content negligible or low yields of L-D dipeptides were achieved. Synthesis of Boc-L-Trp-D-Phe-NH2 catalyzed by alpha-chymotrypsin was performed at molar ratio L: D = 3 : 2 in acetonitrile with 5 vol.% of water and the dipeptide was isolated in larger quantity. However, synthesis of the peptide bond did not occur at all when diastereomeric dipeptides having D-residue in the N-terminal P1' position were used even in the media with low water content.
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PMID:Serine proteinase-catalyzed incorporation of D-amino into model peptides in acetonitrile with low water content. 182 59

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.
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PMID:Membrane proteins exposed on the external side of the intestinal brush-border membrane have fusogenic properties. 191 72

A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.
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PMID:An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication. 193 Oct 37

The three-dimensional crystal structure of thermitase complexed with eglin-c in the presence of 100 mM calcium has been determined and refined at 2.0-A resolution to a R-factor of 16.8%. This crystal structure is compared with previously determined structures of thermitase at 0 and 5 mM calcium concentration. In the presence of 100 mM calcium all three calcium binding sites in thermitase are fully occupied. At 100 mM CaCl2 the "weak" calcium binding is occupied by a calcium ion, which is chelated by three protein ligands and four water molecules in a pentagonal bipyramid geometry. Thermitase has, apparently, a monovalent and divalent cation binding position at 2.5-A distance from each other at this site. At low calcium concentrations the monovalent-ion position is occupied by a sodium or potassium ion. The "medium strength" binding site shows in the presence of 100 mM CaCl2 a square antiprism arrangement with eight ligands, of which seven are donated by the protein. At low calcium concentrations we observe a distorted pentagonal bipyramid coordination at this site. The largest difference between these two conformations is observed for ligand Asp-60, which has two conformations with 0.8-A difference in C alpha positions. The "strong" calcium binding site has a pentagonal bipyramid coordination and is fully occupied in all three structures. Structural changes on binding calcium to the weak and "medium strength" calcium binding sites of thermitase are limited to the direct surroundings of these sites. Thermitase resembles in this respect subtilisin BPN' and does not exhibit long-range shifts as have been reported for proteinase K.
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PMID:Calcium binding to thermitase. Crystallographic studies of thermitase at 0, 5, and 100 mM calcium. 199 69

Giardiasis is the most common human parasite infection in the United States, causing a lengthy course of diarrhea. Transmission of Giardia species is by the fecal-oral route, and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia species in drinking water through the Surface Water Treatment Rule. Current methods for detection of Giardia species in water rely primarily on microscopic observation of water concentrates with immunofluorescence techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 bp long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of the M13 vector with an insert was isolated from lysed host Escherichia coli XL1-Blue and used for production of the cDNA probe by nick translation with 32P-labeled nucleotides. Six different protocols were tested for extracting nucleic acids from the cysts. With the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates with dot blot hybridization assays.
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PMID:Detection of Giardia cysts with a cDNA probe and applications to water samples. 205 51


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