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

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.
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PMID:A method for the preparation of high molecular weight yeast DNA. 333 61

Macrophage migration enhancement factor (MEF), a lymphokine produced in the spleen by suppressor-like lymphoid cells, may be an important immunoregulatory molecule of macrophage function. MEF appears to be a potent positive chemokinetic factor and is unusual in that it lacks chemotactic activity. To aid in the development of a purification scheme for MEF we have employed biophysical characterization techniques to define its physical properties. Using the technique of velocity sedimentation in isokinetic sucrose gradients, the S20w for MEF was determined to be 2.25. The Stoke's radius for MEF was determined by Sephadex G-100 gel filtration to be 28.9 A. From these measurements the D20w was calculated to be 7.55 x 10(-7) cm2/sec, the mol. wt was calculated to be 28,000, the frictional ratio (f/f0) was calculated to be 1.45, the axial ratio was calculated to be 1:8, and the dimensions of the molecule were estimated to be 20 x 160 A. Using the technique of isoelectric focusing in liquid density gradients, the isoelectric point for MEF was estimated to be 8.8. We have also determined by enzyme treatment that MEF is resistant to DNase and RNase and susceptible to proteinase K and L-fucosidase. In addition, MEF partitioned to the aqueous phase during methanol-chloroform extraction procedures. MEF was inactivated at pH 12; at 100 degrees C MEF was stable for 10 min but was inactive after 1 hr. Collectively, these data will facilitate the development of a purification scheme for MEF which will ultimately permit the analysis of the molecule and its function.
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PMID:Biophysical characterization of macrophage migration enhancement factor (MEF). 343 51

Pregnant rats were deprived of paradoxical sleep for 3 days starting on the 18th gestational day. The condition of PS-D was imposed by confinement on a small platform surrounded by water or by daily injections of clomipramine. Four hours before the killing rats received a s.c. injection of [3H]-thymidine. The amount of radioactive DNA determined by autoradiography in several regions of fetal brain was found to be markedly increased under both experimental conditions in comparison with the control fetal brain. Considerably more limited effects were observed in kidney. Comparable changes of lower magnitude were obtained by comparing the specific radioactivity of DNA samples purified by chlorophorm extraction and digestion with RNase and proteinase K. The results fully confirm our previous data obtained under similar experimental conditions but based on the analysis of an acid-washed DNA fraction.
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PMID:Paradoxical sleep deprivation of the mother enhances DNA synthesis in fetal rat brain: autoradiographic and biochemical evidence. 345 82

N-(p-Azido[3,5-3H]benzoyl)daunorubicin ([3H]NABD), a radioactive photoactive anthracycline analogue, was used to photoaffinity label anthracycline binding polypeptides in P388 murine leukemic cell lines. Whole cell homogenates were mixed with 6 X 10(-8) M [3H]NABD, exposed to ultraviolet light, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radiolabel incorporation. Autoradiofluorography showed incorporation of radioactivity into a Mr 18,000 component independent of polypeptides prominently stained with Coomassie blue. Photolabeling of subcellular fractions showed predominant mitochondrial localization of the Mr 18,000 radiolabel. The protein composition of the photolabeled constituents was confirmed by treatment with proteinase K, DNase and RNase, or by lipid extraction with organic solvent. [3H]NABD photolabeling of homogenates from anthracycline sensitive and resistant cells resulted in Mr 18,000 radiolabel incorporation of 3,966 +/- 355 and 6,487 +/- 533 dpm per 50 micrograms cellular protein for anthracycline sensitive and resistant cells, respectively (P less than 0.005). These studies characterize the photoaffinity labeling of a low molecular weight mitochondrial polypeptide using a photoactive anthracycline analogue. The role for this polypeptide as a mediator of anthracycline activity remains to be determined.
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PMID:Anthracycline photoaffinity labeling of a mitochondrial polypeptide in P388 murine leukemic cell lines. 346 35

Tumours produce substances that inhibit the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity in mice. Phenol-saline extracts of bovine ocular squamous cell carcinoma (BOSCC) which have immunotherapeutic activity in cattle were able to immunize mice against this depressive effect. Such immunization was effective against products of BOSCC, a spontaneous rat tumour, three of four human tumour cell lines and (in other experiments) mouse tumours. Phenol-saline extracts of mouse tumour cell lines were immunogenic (protective against depression of delayed-type hypersensitivity) in mice. Fractions of BOSCC phenol-saline extracts which were immunotherapeutically active in cattle were generally also protective in mice. The protective activity was lost after treatment with proteinase K, and was present in the supernatant after precipitation with 55% ammonium sulphate. It was not affected by treatment with RNase or DNase or by heating to 50 degrees C for 2 h. It was present in gel filtration fractions with an apparent molecular weight of 10,000-37,000 daltons. The immunogenic factor in mice and the immunotherapeutic factor in cattle may be related to each other.
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PMID:Depression of cell-mediated immunity by tumour cell products: induction of resistance by immunotherapeutically active extracts of bovine ocular squamous cell carcinoma. 359 86

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.
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PMID:Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis. 618 37

An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 degrees C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.
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PMID:Isolation and characterization of a translation inhibitor from human term placenta. 620 28

Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving a small peptide on the RNA, this peptide could also be removed by the enzyme. If the RNA was degraded with T1 RNase, leaving VPg attached to a nonanucleotide, the enzyme still would cleave off VPg, although incompletely. If the RNA was degraded completely, leaving either pUp or pU attached to VPg, the enzyme would not remove the nucleotides from the protein. Thus, for the enzyme to be active requires some length of polynucleotide attached to the protein but only a short peptide need be present for the enzyme to act.
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PMID:Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA. 624 32

We identified, by anticomplement immunofluorescence, a nuclear antigen (hepatitis B virus-associated nuclear antigen [HBNA]) in two human hepatoma cell lines containing integrated hepatitis B virus DNA but not in three hepatoma cell lines lacking it. The antigen resembled neoantigens associated with the oncogenesis of certain papovaviruses, adenoviruses, and herpesviruses. Antibody to the antigen (anti-HBNA) was found in 7.3% of hepatitis B surface antigen-positive sera from patients with hepatocellular carcinoma but not in surface antigen-negative sera. The staining of HBNA was characterized by two patterns, reticular nuclear fluorescence and nucleolar fluorescence. The expression of HBNA did not parallel the production of extracellular hepatitis B surface antigen. Treatment of cells with proteinase K, RNase, DNase, or cycloheximide significantly diminished the staining of HBNA.
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PMID:Nuclear antigen detected in hepatoma cell lines containing integrated hepatitis B virus DNA. 630 93

A bacteriocin produced by Bacteroides fragilis 1356 was purified from culture medium and characterized. The spectrum of the inhibitory activity of this bacteriocin was species specific. The bacteriocin was recovered from the initial stages of purification as a complex, greater than 2 X 10(7) daltons in mass, containing protein, lipid, and carbohydrate. The dissociation of this complex by 6.0 M guanidine hydrochloride permitted further purification of the bacteriocin by removal of lipid and carbohydrate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified bacteriocin was homogeneous, with a relative molecular weight of 5,000. The activity of the purified bacteriocin was not affected by RNase, DNase, phospholipase A, pancreatic lipase, or dextranase, but was destroyed by trypsin, proteinase K, heat (80 degrees C, 30 min), or a pH below 5 or above 8. Amino acid analysis indicated a predominance of acidic and polar amino acids.
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PMID:Purification and characterization of a bacteriocin from Bacteroides fragilis. 635 Feb 64


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