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 present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.
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PMID:Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K? 765 31

We have applied the technique of lipopolysaccharide (LPS) profiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the typing of 124 isolates of 12 gram-negative species from suspected outbreaks of infection. LPS was prepared by proteinase K digestion or micro-phenol-water extraction. A total of 11 of the 12 species gave clear ladder band profiles, the exception being Acinetobacter baumannii. When compared with conventional typing for Enterobacter cloacae, Pseudomonas aeruginosa, and Serratia marcescens, LPS profile type alone was sufficient to allow relatedness or distinguishability of isolates to be established, and this was corroborated by serotype and phage type data. Serologically nontypeable isolates invariably lacked O repeating units and thus could not be classified by their silver stain profile. We conclude that LPS profiling is useful for the epidemiological investigation of small clusters of isolates in order to determine whether or not cross-infection between patients has occurred.
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PMID:Lipopolysaccharide profile typing as a technique for comparative typing of gram-negative bacteria. 768 51

Lipopolysaccharides (LPS) were extracted from seven Bacteroides strains by three different techniques: the phenol-water (PW), phenol-chloroform-petroleum (PCP) and Triton-Mg2+ methods. The strains selected included two different B. fragilis strains, one of which was grown in two different media. Yields varied between the strains, growth media and extraction technique, but generally the highest yield by weight was from the PCP method and the lowest from the PW method. The PW method was selected for the greatest amounts of carbohydrate and KDO, and the PCP method for the least. Phosphorus levels were more uniform among all extraction methods. Protein contamination was found in all Bacteroides LPS extracts, with extremely low levels in PW-LPS and the highest levels in material extracted by the PCP and Triton-Mg2+ techniques. No protein contamination could be detected after proteinase K treatment. After silver staining LPS PAGE profiles showed ladder patterns characteristics of smooth LPS for B. vulgatus, B. thetaiotaomicron and the control Escherichia coli O18:K- strains, whereas the other Bacteroides strains showed mainly rough and low M(r) material only. The PCP method did not select for high M(r) material in the B. fragilis strains; otherwise the LPS profiles for all extraction methods were identical. The biological activities of native and sodium salt form LPS were investigated on a weight for weight basis and compared to that of E. coli O18:K- PW-LPS. Amongst the LPS from Bacteroides strains, those prepared by the PW method were found to have a significantly higher activity in a galactosamine mouse lethality model, in induction of TNF and the Limulus amoebocyte lysate (LAL) assay, than LPS extracted by the PCP or Triton-Mg2+ methods. LPS from Bacteroides strains extracted by the PCP method had consistently low activity in all assays. Comparing PW-LPS from Bacteroides strains with that from E. coli O18:K- in the galactosamine mouse model, the E. coli O18:K- LPS was c. 5000-fold more active than the most active bacteroides LPS. However, in the LAL assay native PW-LPS from both the B. fragilis strains, and B. caccae had higher activities (up to 30-fold) than E. coli O18:K- LPS, with the PW-LPS from the other Bacteroides spp. being up to 15-fold less active than the E. coli O18:K- PW-LPS. In the TNF induction assay, E. coli O18:K- PW-LPS was 4-50-fold more active than bacteroides PW-LPS.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A re-appraisal of the biological activity of bacteroides LPS. 786 45

Subtilisins (subtilopeptidase A, nagarse) and proteinase K were able to catalyze the synthesis of taurine-containing peptides from various N-acylated amino acid or peptide esters and nonprotected taurine. The synthesis was optimized using a model reaction between Boc-Tyr-OMe and taurine. The best results were obtained under strongly alkaline conditions in acetonitrile with low water content as the reaction medium. The choice of the base added to the reaction medium had a substantial effect on the product yield. A preparative synthesis of Tyr-Tau and Ala-Phe-Tau is described.
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PMID:Enzymatic approach to the synthesis of taurine-containing peptides. 789 5

A method for the detection of Pneumocystis carinii by polymerase chain reaction using specimens obtained by scraping bronchoalveolar lavage or tissue impression smears is described. The smears were scraped into water and then absorbed onto a glass-fiber filter. After fixing with methanol, the specimen on the filter was digested with proteinase K. The digestion mixture was then clarified, and a portion of the clarified supernatant was used as a template for the amplification of a portion of the mitochondrial rRNA gene of P. carinii. Using this method of sample preparation, we were able to amplify P. carinii DNA from both unstained and Giemsa stained smears.
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PMID:Amplification of Pneumocystis carinii DNA on specimens scraped from slides. 792 13

The reliability and applicability of quantitative and qualitative diatom analysis by an enzymatic digestion method in the diagnosis of drowning of putrified bodies has been evaluated. The authors report the analysis of water and organ samples of 12 immersion cases using light microscopy. This study included control organ samples from the bodies of persons who died from causes other than drowning. Organ samples were treated by both chemical and enzymatic methods, the first one using concentrated nitric acid and the second proteinase K. Diatoms were present in most organ samples of the immersed corpses; no diatoms could be found in the control samples. Our experience was that the enzymatic method seemed to be more convenient in terms of rapidity, safety and environmental protection than chemical digestion. The number of diatoms recovered with both methods was similar. Qualitative and quantitative analysis of both water and organ samples of immersion cases supported the diagnosis of death by drowning in 41.6% of the putrified cases studied. The authors suggest that diatom analysis using enzymatic digestion of organs can be used as a criterion for positive diagnosis of drowning in cases involving putrified bodies.
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PMID:Application of a simple enzymatic digestion method for diatom detection in the diagnosis of drowning in putrified corpses by diatom analysis. 799 44

Mycoplasmal products may exert a number of diverse in vitro effects on cells of the immune system. A macrophage-activating substance from Mycoplasma fermentans was described in this laboratory and named mycoplasma-derived high-molecular-weight material (MDHM). Using synthesis of nitric oxide by peritoneal cells from endotoxin low-responder mice as an assay system, MDHM was purified as follows. After freeze-thawing of M. fermentans, MDHM activity was sedimented with the membrane fraction. Membranes were delipidated with chloroform-methanol, and MDHM activity was extracted with octyl glucoside. Coextracted proteins were degraded by proteinase K. MDHM was further purified by reversed-phase high-pressure liquid chromatography and eluted in one major and one minor peak of activity. Neither carbohydrates nor amino acids were found as constituents. MDHM had the following properties: it partitioned into the phenol phase upon phenol-water extraction and into the Triton phase after extraction with Triton X-114. MDHM was not inactivated by either phospholipase A2 or triglyceride lipases. However, mild periodate treatment led to a > 95% loss of activity. Also, alkaline hydrolysis at 25 degrees C completely abolished MDHM activity with a half-life of 2 min. MDHM activity was spread out over a wide molecular weight range upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes, whereas after proteinase treatment MDHM activity migrated close to the front. These features of MDHM, taken together, speak in favor of an amphiphilic molecule with a lipid moiety carrying fatty acids in ester linkage and a polyol moiety of unknown character. MDHM was active in the nanogram-per-milliliter range, activating macrophages to release nitric oxide, interleukin-6, and tumor necrosis factor.
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PMID:Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6. 806 96

Proteinase K from the fungus Tritirachium album Limber binds two Ca2+ ions, one strongly (Ca 1) and the other weakly (Ca 2). Removal of these cations reduces the stability of proteinase K as shown by thermal denaturation, but the proteolytic activity is unchanged. The x-ray structures of native and Ca(2+)-free proteinase K at 1.5-A resolution show that there are no cuts in the polypeptide backbone (i.e. no autolysis), Ca 1 has been replaced by Na+, while Ca 2 has been substituted by a water associated with a larger but locally confined structural change at that site. A small but concerted geometrical shift is transmitted from the Ca 1 site via eight secondary structure elements to the substrate recognition site (Gly100-Tyr104, and Ser132-Gly136) but not to the catalytic triad (Asp39,His69,Ser224). This is accompanied by positional changes of localized waters.
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PMID:Crystal structure of calcium-free proteinase K at 1.5-A resolution. 808 13

Glycophorin A (GPA) has been reconstituted into dimyristoylphosphatidylcholine vesicles and digested with proteinase K to identify the membrane domain and to characterize its structure and orientation. After digestion of the inner and outer domain of GPA by protease action restricted to the aqueous phase, a protected peptide migrates on an electrophoresis gel as a 7.5-kDa dimer (His66-Ile95). The secondary structure and orientation in a lipid bilayer of the 7.5-kDa dimer have been studied by Fourier transform infrared spectroscopy. Our proteolytic and spectroscopic data are in agreement with a topological model in which the His66-Glu72 peptide adopts a beta-sheet conformation and is oriented parallel to the lipid-water interface and the Ile73-Ile95 domain is helical and oriented parallel to the lipid acyl chains, in a transmembrane configuration. Digestion of the domain protruding to the outside of the liposome generates "head-head" and "head-tail" dimers of 16 and 38 kDa, respectively. This observation is discussed in terms of the specificity of the dimer formation process.
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PMID:Sequence and structure of the membrane-associated peptide of glycophorin A. 820 24

The crystal structure of a transition state/product complex formed by the interaction between proteinase K and the substrate analogue N-Ac-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2 has been determined at a resolution of 2.2 A and refined to an R-factor of 0.165 for 12,725 reflections. The inhibitor forms a stable complex through a series of hydrogen bonds with protein atoms and water molecules. The inhibitor is hydrolyzed between Phe 4I and D-Ala5I (I indicates inhibitor). The two fragments are separated by a distance of 3.07 A between the carbonyl carbon and the main chain nitrogen. Both fragments remain bound to the protein. The N-terminal fragment occupies subsites S5 to S1, whereas the C-terminal part is bound in S1' and S2', the first time that electron density for a substrate analogue has been observed in the P1' and P2' sites of a subtilisin-like enzyme. The flexible segments of the substrate recognition sites Gly100-Tyr104 and Ser132-Gly136 move appreciably to accommodate the inhibitor. Biochemical results indicate an inhibition by this specifically designed peptide of 95%.
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PMID:Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution. 834 Apr 10


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