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)

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigen in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K. A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.
 ...
PMID:Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis. 280 Mar 7

Various immunological methods were used to localize a glycylprolyl protease previously isolated from Bacteroides gingivalis ATCC 33277. The results obtained by enzyme-linked immunosorbent assay, indirect immunofluorescence staining, and indirect immunogold labeling suggest that the glycylprolyl protease is present on the surface of the cell outer membrane and is specific to B. gingivalis strains. The enzyme was removed from the cell envelope by treatment of the whole cells with sodium dodecyl sulfate, Triton X-100, sodium deoxycholate, and proteinase K.
 ...
PMID:Surface location of a Bacteroides gingivalis glycylprolyl protease. 280 24

Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations.
 ...
PMID:Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose. 283 4

Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.
 ...
PMID:Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue. 283 96

A rapid and efficient method to purify lambda gt11 DNA is described. This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment. The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography. The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification. Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods.
 ...
PMID:Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts. 284 87

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.
 ...
PMID:Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles. 286 45

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing [3H]diaminopimelic acid and treated by a hot (96 degrees C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60%). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and less than 2% protein. Radiochemical analyses indicated that 3H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelic acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived peptidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of greater than 95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl++ +-alanine.
 ...
PMID:Structure of Bordetella pertussis peptidoglycan. 288 47

Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.
 ...
PMID:Isolation and structural studies of the intact scrapie agent protein. 289 Mar 30

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.
 ...
PMID:The preparation of poly(A)+mRNA from the hagfish slime gland. 289 86

A simple procedure is described for the preparation of photosystem I (PSI) particles from Triton X-100-solubilized thylakoid membranes of the unicellular cyanobacterium Synechococcus 6301. The purified PSI complex contained the full complement of antenna chlorophylls, 130 +/- 5/P700, displayed the electron paramagnetic resonance signals characteristic of iron-sulfur centers X, A, and B, and had a protein/chlorophyll ratio of 2.9. Determination of the polypeptide composition, utilizing a uniformly 14C-labeled complex, showed that it contained polypeptides of 70, 18, 17.7, 16, and 10 kDa, in a molar ratio of 4.0:0.7:1.0:0.5:1.6. The relative amount of the lower molecular weight polypeptides showed progressive decrease with increase in Triton X-100 concentration and time of exposure to detergent. Consequently, it is proposed that in vivo the composition of the complex is [70 kDa]4 [18 kDa]1 [17.7 kDa]1 [16 kDa]1 [10 kDa]2. Relative to 130 mol of chlorophyll a, the PSI complex contained 16 mol of carotenoids, 13.7 +/- 1.0 g atoms of Fe, and 12.2 +/- 1.1 g atoms of labile sulfide. The properties of complexes fully depleted of the low-molecular weight polypeptides by treatment with sodium dodecyl sulfate or with proteinase K are also described.
 ...
PMID:Characterization of a cyanobacterial photosystem I complex. 298 Dec 24


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>