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)

DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5-100 micrograms/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5-6 micrograms/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6-100 micrograms/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 micrograms/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by "intermediate-type" protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.
 ...
PMID:Changes in DNA topology during spermatogenesis. 349 Mar 60

The sedimentation of L1210 nucleoids has been used to demonstrate a hyperthermia-associated increased protein to DNA ratio and an apparent inhibition of processes involved in the restoration of the protein to DNA ratio. The distance of nucleoid sedimentation increased as a function of exposure temperature and exposure time, and was proportional to an increased protein to DNA ratio in the nucleoids. Studies in which control and hyperthermia-treated cells were mixed prior to nucleoid preparation indicated that the association of protein with the nucleoid occurred during hyperthermia treatment and not during nucleoid preparation. However, double-labelling studies suggested an interaction between control and hyperthermia-treated cells since the presence of control cells during lysis resulted in near normalization of nucleoid sedimentation. Treatment with proteinase K also restored the nucleoid sedimentation. Incubation at 37 degrees C following hyperthermia revealed a rapid restoration of nucleoid sedimentation and a slower restoration of the protein to DNA ratio. Ethidium bromide-induced changes in nucleoid sedimentation were altered by the hyperthermia-associated increased protein content of nucleoids and the alterations were overcome by enzymatic digestion of the protein prior to the ethidium bromide exposure. Thus, hyperthermia caused, and inhibited the repair of, an increased protein content of nucleoids. The restoration of the increased protein possibly occurs by a heat-sensitive proteolytic enzyme. The temporal use of an appropriate chemotherapy agent to inhibit the restoration of hyperthermia-associated changes may be a useful treatment option.
 ...
PMID:Restoration of hyperthermia-associated increased protein to DNA ratio of nucleoids. 355 98

The molecular mass of proteinase K was determined by gel electrophoresis in the presence of sodium dodecyl sulfate and by active site labelling with diisopropyl fluorophosphate. Both methods indicate molecular masses in the range of 27 000-29 000 Da. These values differ significantly from that of 18 500 formerly determined by gel filtration (Ebeling et al. (1974) Eur. J. Biochem. 47, 91-97). Proteinase K was inactivated with [3H]diisopropyl fluorophosphate. Afterwards the labelled protein was reduced, S-carboxymethylated and digested with cyanogen bromide. The chain lengths of the isolated CNBr-fragments are indicative of a molecular mass of proteinase K of at least 28 000 Da. Two CNBr-fragments were sequenced. The radioactively labelled fragment contains 69 residues and the sequence around the labelled residues was found to be -Ile-Ser-Gly-Thr-SER-Met-Ala-Thr-Pro-. This sequence is typical for that around the active site residue of the subtilisins. From the determined sequences it is concluded that the fungal proteinase K is phylogenetically related to the bacterial subtilisins.
 ...
PMID:Proteinase K from Tritirachium album limber. I. Molecular mass and sequence around the active site serine residue. 392 77

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.
 ...
PMID:Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis. 618 37

A sensitive, modified 3,5-diaminobenzoic acid (DABA), fluorometric DNA assay was developed and compared to mithramycin and ethidium bromide assays in determining the DNA content of dense connective tissues including: Swarm rat chondrosarcoma, rabbit, dog, monkey, and most importantly, adult human articular cartilage. In the more cellular cartilages, the three methods gave equivalent results. However, in the relatively acellular human cartilage, the DABA method was shown to be superior. Both the mithramycin and ethidium bromide gave falsely high values compared to the DABA method, which by subtraction after DNase digestion approached the DABA value. The latter was completely DNase sensitive. With the DABA method, the DNA content of human cartilage can be obtained on less than 5 mg wet weight of fresh, alcohol-fixed, or lyophilized material. While the DNA can also be released by digestion with papain or protease from Streptomyces griseus, proteinase K was preferable. The comparison of literature values for other fluorometric and spectrophotometric assays of human cartilage suggest these methods overestimate human articular cartilage DNA concentrations, whereas the DABA values were in line with those predicted from previous morphometric analysis. Thus, the modified method represents an improvement in DNA analysis of dense connective tissues.
 ...
PMID:Fluorometric determination of DNA in cartilage of various species. 623 38

Polymerase chain reaction (PCR) primered with primers 3,4 and 5,6 was performed using DNA extracted from dried blood spot of a falciparum malaria patient from Mengpeng Township, Yunnan using four different methods, including STE-proteinase K-SDS, methanol-proteinase K-SDS, Chelex-100 and Chelex-100-proteinase K to amplify DNA of apical membrane antigen 1 (AMA-1). A 900 bp DNA band could be seen on the ethidium bromide-stained agarose gel electrophoresis of the PCR products when DNA was extracted using all the above-mentioned methods except STE-proteinase K-SDS method. DNA extracted with Chelax-100 was recommended.
 ...
PMID:[Application of Plasmodium falciparum DNA extract from dried blood spot of falciparum malaria patient in polymerase chain reaction]. 755 60

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.
 ...
PMID:Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region. 764 33

DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.
 ...
PMID:PCR amplification of DNA from stained cytological smears. 768 6

A polymerase chain reaction (PCR) test was developed to detect Mycobacterium bovis in tissues. The test was based on amplification of a 248 bp segment of the insertion sequence, IS1081, present in six copies in strains of M. bovis and other members of the tuberculosis complex. The procedure involved digestion with proteinase K, lysis with sodium dodecyl sulphate, and extraction with hexadecyl tetramethyl ammonium bromide and phenol:chloroform:iso-amyl alcohol. When agarose gel electrophoresis was used for detection, the method was able to detect 1 fg of pure DNA, or 0.2 genome equivalents. It could also detect as few as 10 organisms from pure cultures and between 200-500 organisms from tissues spiked with cultured organisms. Detection by hybridization was only marginally more sensitive. The method was tested on 110 selected tissues recovered post mortem from a variety of animals. Fifty three of 58 samples diagnosed as M. bovis culture positive, including all samples containing microscopically visible acid-fast bacilli, were positive on duplicate testing by PCR. Five of 52 culture negative samples were also positive by PCR including three which contained large numbers of acid-fast organisms. Ten of the culture negative samples came from animals in a herd known to be free of bovine tuberculosis and all these were negative by PCR.
 ...
PMID:Detection of Mycobacterium bovis in tissues by polymerase chain reaction. 774 Jul 61

Amplification inhibitors can lead to false-negative results for PCR. In order to evaluate the reliability of PCR for the detection of Chlamydia pneumoniae, the presence of PCR inhibitors in 75 bronchoalveolar lavage specimens was assessed after treatment by various sample preparation methods. Specimens were collected from patients with acute respiratory infections, including four cases of proven C. pneumoniae infection. Substances inhibitory to the amplification of chlamydial DNA continued to be present in 12% of the samples treated according to the commonly used single-step proteinase K digestion and in 31% of the samples processed by heat treatment. However, the complexing of DNA-contaminating proteins and polysaccharides from digested specimens to cetyltrimethylammonium bromide (CTAB) followed by DNA extraction efficiently removed inhibitors from all experimental samples and provided subsequent identification of all positive clinical samples by PCR. The CTAB method and proteinase K treatment had comparable detection limits of approximately 0.01 inclusion-forming units. CTAB-based DNA purification of respiratory specimens is recommended to increase the diagnostic sensitivity of PCR and confidence in negative results.
 ...
PMID:Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR. 781 12


<< Previous 1 2 3 4 5 6 7 Next >>