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)

The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.
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PMID:Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. 819 50

We previously demonstrated that Treponema pallidum cells incubated in vitro in the presence of heat-inactivated normal rabbit serum (HINRS) synthesize, in very small quantities, several pathogen-specific, low-molecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically radiolabel T. pallidum cells to high specific activity to further characterize these antigens. We found that the low-molecular-mass proteins are not related to the 15- and 17-kDa detergent-phase proteins (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard, Infect. Immun. 56:490-498, 1988). The low-molecular-mass proteins did not incorporate 3H-labeled fatty acids and were not precipitated by rabbit immunoglobulin G (IgG) antibodies directed against glutathione S-transferase fusions to the nonlipidated 15- and 17-kDa proteins. We prepared polyclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG antibodies present in the sera of both rabbits precipitated a 21.5-kDa protein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an additional 15.5-kDa low-molecular-mass protein only from solubilized extracts of supernatant. While investigating the effect of eliminating HINRS from the extraction medium, we observed that the low-molecular-mass proteins remained associated with treponemal cells that were incubated in the absence of HINRS. These proteins could be eluted from the cells by the addition of HINRS or rabbit serum albumin, suggesting that they are located on or near the treponemal cell surface. The 15.5- and 21.5-kDa low-molecular-mass proteins were not washed off treponemal cells with buffer containing 1 M KCl. Experiments employing selective solubilization of the T. pallidum outer membrane with 0.1% Triton X-114 and proteinase K accessibility indicated that the 15.5-kDa protein, but not the 21.5-kDa protein, is cell surface exposed.
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PMID:Characterization of the low-molecular-mass proteins of virulent Treponema pallidum. 826 39

Outer membrane proteins of Aeromonas hydrophila A6 were isolated by affinity chromatography on the basis of their reactivity with trisaccharide structures analogous to the terminal trisaccharide of the H antigen of the human ABO(H) blood group system and were characterized by using antisera raised against the isolate. The outer membrane extract for affinity chromatography was prepared from pressure-disrupted outer membranes by differential centrifugation, followed by solubilization of outer membrane components in a nondenaturing, nonionic detergent. Carbohydrate-reactive outer membrane proteins (CROMPs) were then purified by affinity chromatography on two different affinity matrices composed of trisaccharides resembling the terminal trisaccharide of the H antigen, attached to inert silica beads. The relative efficiencies of H type 1 and 2 terminal trisaccharides as affinity adsorbents were established. Reactive proteins were eluted under alkaline conditions (pH 11.0) and in the presence of soluble H substance prepared from group O secretor saliva, but not by 60 mM alpha-L-fucose or under acid conditions (pH 3.0). The eluate contained at least three components (M(r)s, 43,000, 40,000, and < 14,000), as detected by immunoblot analysis with a polyvalent, polyspecific rabbit antiserum to A. hydrophila A6 (serum 3/83). A specific antiserum (serum 3/91) prepared in a rabbit by repeated immunizations with nitrocellulose containing the 43,000-Da band reacted with three bands (M(r)s, 43,000, 40,000, and < 14,000) in immunoblot analysis of solubilized outer membranes of A. hydrophila A6, suggesting that the 40,000- and < 14,000-Da elements are immunologically related to components of the 43,000-Da protein. Furthermore, pretreatment of A. hydrophila A6 with serum 3/91 reduced the strength of bacterial hemagglutination. The purified CROMPs did not agglutinate human group O erythrocytes. The reactivity of isolated CROMPs with a second CROMP-specific antibody (lipopolysaccharide-absorbed serum 3/83) was investigated. CROMPs, proteinase K-treated CROMPs, and bovine serum albumin were bound to latex beads and reacted with lipopolysaccharide-absorbed serum 3/83. Antibodies eluted from CROMP-latex inhibited hemagglutination of human erythrocytes by A. hydrophila A6 to a titer of 4. Antibody eluted from proteinase K-treated CROMP-latex beads showed hemagglutination inhibition activity only when undiluted. There was no hemagglutination inhibition antibody activity detectable in the eluate from bovine serum albumin-latex beads. These results show that antibodies which react with the isolated CROMPs also react with an H-antigen-reactive hemagglutinin of A. hydrophila A6. The possibility that CROMPs act as an adhesin, or adhesins, and contribute to the virulence of this organism is discussed.
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PMID:Isolation of carbohydrate-reactive outer membrane proteins of Aeromonas hydrophila. 838 Jul 92

Binding of outer membrane (OM) preparations of the thermophilic Campylobacter species C. jejuni to epithelial cell membranes and extracellular matrix proteins were studied in an in vitro model system using enzyme-linked immunosorbent assay. The OM preparations exhibited significant binding to INT 407 intestinal cell membranes. The process of adhesion was modulated by enzymatic, chemical or immunological pretreatment of the bacteria. Following oxidation of the lipopolysaccharide (LPS) with sodium meta-periodate, the OM preparations essentially retained their binding properties. After pretreatment with proteinase K, the OM preparations lost their binding capacity and the apparent molecular mass of the major OM protein shifted from 42 to 24 kDa. Preincubation of C. jejuni bacteria with C. jejuni-specific antiserum reduced adhesion significantly; preincubation with LPS-specific monoclonal antibodies only to a minimal extent. The OM preparations also bound significantly to the extracellular matrix proteins collagen and fibronectin; however, they bound virtually no bovine serum albumin or horse serum.
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PMID:Binding of outer membrane preparations of Campylobacter jejuni to INT 457 cell membranes and extracellular matrix proteins. 857 16

Estrogen-mediated accumulation of the avian apolipoprotein (apo) II mRNA is in part due to its stabilization. To identify the biochemical activity responsible for this effect, radiolabeled, capped, and polyadenylated apoII mRNA was incubated in vitro in liver cytosolic extracts from roosters who received either estrogen (estrogen-treated extract) or the vehicle (control extract) parenterally. The mRNA was very stable in estrogen-treated extract but was rapidly degraded in control extract. The RNA was degraded predominantly by endonuclease rather than exonuclease activity. The addition of the estrogen-treated extract to the control extract prevented the degradation of the mRNA in trans. This biochemical activity was heat labile and was also destroyed by proteinase K but not by micrococcal nuclease, indicating that estrogen treatment resulted in the expression of a protein in the liver that stabilized the apoII mRNA by inhibiting its nucleolytic degradation. This mRNA stabilization factor was labile around 60 degrees C, whereas the RNase remained stable up to 80 degrees C. Studies on mRNA protein interaction showed that both control and estrogen-treated extracts contain mRNA-binding (mRNP) proteins that bind apoII mRNA. An increased binding to apoII mRNA by a subset of these proteins was observed with estrogen-treated extract as compared with the control extract. This activity, although it afforded complete protection from nucleolytic degradation to apoII and apo A1 mRNAs, appeared to provide less protection to mRNAs encoding chicken serum albumin and vitellogenin, suggesting differential stabilization of mRNAs. These studies indicate that a cytosolic mRNA-stabilization factor, providing apoII mRNA complete protection from nucleolytic degradation, is expressed in the avian liver upon estrogen treatment. This appears to be the first time that a biochemical activity responsible for hormone-mediated stabilization of mRNAs and estrogen induction of mRNA binding by specific mRNPs have been identified and partially characterized in vitro.
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PMID:In vitro characterization of an estrogen-regulated mRNA stabilizing activity in the avian liver. 877 38

Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase resulted in fluorescence enhancement of 4300%. Both alpha-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 x 10(-6) units for I ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 10(-3) units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.
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PMID:Detection of protease activity using a fluorescence-enhancement globular substrate. 882 59

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.
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PMID:Determination of the extent of protein biotinylation by fluorescence binding assay. 902 42

Early diagnosis of Pneumocystis carinii pneumonia, a life-threatening complication in immunosuppressed patients, may lower morbidity and mortality. We have developed a one-tube nested PCR assay for the detection of P. carinii in respiratory specimens. Four primers were selected from the sequence of the small-subunit rRNA gene of P. carinii to amplify a 265-bp fragment, and their specificities for P. carinii were confirmed by both theoretical evaluations (by computer-assisted comparison with the sequences in GenBank) and empirical evaluations (with DNA from medically important fungi and diagnostic samples). The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (digestion with proteinase K directly in PCR buffer at room temperature in the presence of 10% Chelex 100 and no further purification steps). Bovine serum albumin (1 mg/ml) and glycerol (10%) in the amplification buffer reduced the number of samples inhibitory to the PCR, as assessed by control reactions containing a size-modified target. A total of 749 clinical specimens (312 bronchoalveolar lavage, 403 sputum or induced sputum, and 34 other specimens) from 507 patients (295 human immunodeficiency virus [HIV]-infected and 164 non-HIV-infected patients and 48 patients whose HIV status was unknown) were tested by PCR, and the results were compared with those of an indirect immunofluorescence assay (IFA). Concordant results were obtained for 732 samples (646 negative and 86 positive). There were 17 discrepant results: 12 were PCR positive and IFA negative, and 5 were PCR negative and IFA positive. After resolution of the discrepant results by review of the patients' clinical data, the sensitivity and specificity were 94.8 and 99.1%, respectively, for PCR and 93.8 and 100%, respectively, for IFA. In conclusion, the short procedure time and the technical ease of this PCR assay render it suitable for implementation in routine diagnostic laboratories.
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PMID:Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens. 919 75

RNase A, which is routinely added during DNA purification to reduce contaminating RNA, causes shifting of DNA bands in agarose gels. DNA band sizes on agarose gels increase as much as 10%-20% when RNase A is present. The low concentrations of RNase A typically used to purify DNA cause shifting of select DNA bands, in contrast to higher concentrations of RNase A where all band are shifted and smeared. The binding of RNase A to the DNA is specific and the degree of the shift varies; not all DNA bands are retarded, and the deviation is more pronounced in certain buffers. Other proteins, such as bovine serum albumin or proteinase K, do not induce DNA band shift, suggesting the interaction is specific. The binding of RNase A to DNA is reversible. The formation of RNase:DNA complexes may affect experiments involving DNA:protein interactions such as gel shift, footprinting and filter binding assays. Researchers performing DNA characterization from miniprep protocols should be aware that RNase may cause the apparent sizes of DNA fragments to be altered and obscure the presence of very small cloned fragments.
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PMID:Presence of RNase A causes aberrant DNA band shifts. 923 44

Whey protein was digested with one of seven kinds of proteases at 37 degrees C (trypsin, proteinase K, actinase E, thermolysin, or papain) or at 25 degrees C (pepsin or chymotrypsin) for 24 h. The digested samples were assayed for the inhibitory activity of angiotensin-converting enzyme and for changes in the systolic blood pressure caused in spontaneously hypertensive rats after gastric intubation. The strongest depressive effect on the systolic blood pressure (-55 mm Hg) was observed at 6 h after gastric intubation of the whey protein that was digested by proteinase K. Finally, six peptides were chromatographically isolated from the proteinase K digest by a combination of hydrophobic reversed-phase HPLC and gel filtration. The amino acid sequences and their origins were clarified as follows: Val-Tyr-Pro-Phe-Pro-Gly [beta-casein (CN); f 59-64], Gly-Lys-Pro (beta 2-microglobulin; f 18-20), Ile-Pro-Ala (beta-lactoglobulin; f 78-80), Phe-Pro (serum albumin; f 221-222; beta-CN, f 62-63, f 157-158, and f 205-206), Val-Tyr-Pro (beta-CN; f 59-61), and Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro (beta-CN; f 80-90). Chemical synthesis of these six peptides confirmed that all peptides, except an undecapeptide, have antihypertensive activity in spontaneously hypertensive rats. The synthetic tripeptide Ile-Pro-Ala, originating from beta-lactoglobulin, showed the strongest antihypertensive activity (-31 mm Hg).
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PMID:Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. 989 Dec 60


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