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)

1. Trypsin digestion of perchloric acid precipitated horse plasma yielded polypeptides with inhibitory properties for trypsin, chymotrypsin and, to a small extent, kallikrein. 2. The Mr of the inhibitory polypeptides were 73,000 and 24,000. 3. The number, enzyme specificity and Mr of the inhibitory polypeptides differed from the values known for the human being. 4. The inhibitory polypeptides were purified by affinity chromatography on Sepharose-trypsin and by gel filtration through Sephadex G-75. 5. Protease inhibitory polypeptides were generated in the same manner by chymotrypsin, elastase, proteinase K, pronase, collagenase, papain and subtilisin. 6. The number and electrophoretic migration of the inhibitory polypeptides obtained with the different enzymes were variable. 7. The enzyme specificity was constant since all polypeptides inhibited only trypsin, chymotrypsin and kallikrein to a small extent. 8. None of the inhibitory polypeptides were immunologically related to native plasma proteins or plasma protease inhibitors.
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PMID:Acid-stable protease inhibiting polypeptides formed from denatured horse plasma by proteolysis. 367 4

The first step in virus replication is attachment of the virus to the host cell surface. To investigate this process, the binding of TC-83 (the attenuated strain of Venezuelan equine encephalomyelitis virus) to BW-J-M (a macrophage-like murine cell culture line) has been characterized. The binding of radiolabelled virus can be blocked by excess unlabelled virus and has a pH optimum in the physiological range. Binding is saturable; analysis using Scatchard plots or the computerized binding data analysis program, LIGAND, yielded estimates of a single class of 4 X 10(5) binding sites per cell and an equilibrium binding constant of 2.0 +/- 0.7 X 10(12) M-1. Virus bound to the cell could be visualized by transmission electron microscopy and was localized primarily in coated regions of the membrane. Virus, bound under optimum conditions at 0 degrees C, was internalized upon warming and infected cells productively. Treatment of BW-J-M cells with low concentrations (1 to 10 micrograms/ml) of the proteolytic enzymes trypsin, Pronase or proteinase K caused a dose-dependent reduction in binding capacity. Trypsin-treated cells, upon return to culture, progressively regained their binding capacity within 24 h. As a further characterization of the virus binding site, several lectins were studied for their ability to inhibit TC-83 binding to BW-J-M cells. Canavalia ensiformis agglutinin, Glycine max agglutinin and Triticum vulgaris agglutinin were potent inhibitors of virus binding. This evidence suggests that TC-83 binds to a specific receptor on the BW-J-M cell and that the receptor may be glycoprotein.
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PMID:Characterization of the binding of the TC-83 strain of Venezuelan Equine encephalomyelitis virus to BW-J-M, a mouse macrophage-like cell line. 633 32

We have distinguished three fractions of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo marmorata electric organs, according to their solubilization characteristics. The low-salt-aggregating collagen-tailed forms are soluble in high-salt buffers; their hydrodynamic properties ae not modified in the presence of detergents. They constitute the A fraction, which amounts to about a third of the tissue's AcChoE activity. The low-salt-soluble (LSS) and detergent-soluble (DS) fractions are not sensitive to ionic strength and collagenase. In the presence of nonionic detergents or bile salts, both fractions behave as a monodisperse "6.3S" form, the properties of which have been investigated mostly in the case of Triton X-100. Disulfide bond reduction dissociates the detergent form into a smaller "5S" form. These two forms are thought to be, respectively, detergent-associated dimers and monomers. In the absence of detergent, the LSS fraction is polydisperse: it contains a major 8S component, 11S and 14S components, and faster-sedimenting aggregates, which appear to represent dimers, tetramers, and higher polymers. The heterogeneity of the 8S component in gel filtration suggests that it also contains variable noncatalytic elements. Upon removal of the detergent the DS fraction forms ill-defined aggregates. Trypsin induces quaternary rearrangements of part of the 8S component into 11S and 14S components, which are still convertible into the detergent form; therefore trypsin probably digests noncatalytic elements. Pronase and proteinase K, on the other hand, convert the enzyme into a dimeric form, G2, that does not interact with detergents, probably by cleaving a minor fragment of the subunit that is involved in hydrophobic interactions.
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PMID:Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organ. 693 97

Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.
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PMID:Modulation of lipopolysaccharide binding to human granulocytes. 753 36

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, catalyzed by various proteases, was studied by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The end-labeled peptides and the Cy3 diacid internal standard were separated on a 20-microns x 27-cm capillary with LIF detection (emission, 580 nm) using a frequency-doubled solid-state diode laser emitting at 532 nm or a He-Ne laser emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, catalyzed by proteinase K, is a sequential process beginning from the C-terminal of the peptide, instead of from random cleavages. Trypsin catalyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anticipated. Using a combination of endopeptidase and carboxypeptidases, the remnant of the labeled species was characterized by CE-LIF. The method provides a general tool for studying the mechanism of protease-catalyzed hydrolysis of peptide.
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PMID:Characterization of protease-catalyzed hydrolysis of cyanine-labeled angiotensin using capillary electrophoresis with laser-induced fluorescence detection. 776 1

The 26S protease responsible for the selective degradation of ubiquitinated proteins is composed of a regulator complex and the 20S proteosome which is the catalytic core. In the absence of ATP the 26S protease dissociates to free regulator complex and 20S proteosome, and this process can be reversed in vitro in the presence of ATP. Trypsin, chymotrypsin or proteinase K digestion selectively removes several subunits of the free regulator complex of Drosophila 26S protease generating a well-defined new subparticle. Three subunits highly sensitive in the free regulator complex, however, were selectively protected within the in vitro reconstituted 26S protease, indicating that the ATP-dependent association of the 20S proteosome in the regulator complex selectively shields these subunits. In the same concentration range the 20S proteosome was completely resistant for proteolytic degradation.
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PMID:Dissection of the regulator complex of the Drosophila 26S protease by limited proteolysis. 860 38

Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells. Streptococcus dysgalactiae (n = 60) isolated from bovine intramammary infections were examined for expression of SH after growth in Todd-Hewitt broth (THB) and THB supplemented with skim milk, whey, lactose, and casein. Strains were significantly more hydrophobic after growth in THB and THB plus whey and more hydrophilic after growth in THB plus skim milk. Both trypsin and proteinase K abolished SH in three strains tested. Mild pepsin treatment had little effect on SH, while heat treatment at 70 degrees or 80 degrees C abolished SH in two strains tested. A hydrophilic strain of S. dysgalactiae did not adhere as well to bovine mammary epithelial cells as a hydrophobic strain. Trypsin treatment significantly reduced adherence of a hydrophobic strain of S. dysgalactiae to epithelial cells while adherence of a hydrophilic strain remained unaltered. A hydrophilic strain of S. dysgalactiae was significantly more resistant to phagocytosis by bovine mammary gland macrophages than a hydrophobic strain. Differences in expression of SH may play an important role in determining the ability of S. dysgalactiae to establish successfully within the mammary gland.
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PMID:Influence of Streptococcus dysgalactiae surface hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary macrophages. 877 7

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.
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PMID:Assaying for structural variation in the parvovirus capsid and its role in infection. 977 Apr 25

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (< 14.4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin-proteinase K complex and its sequence determined to be Val-Ala-Gln-Gly-Gly-Ala-Ala-Gly-Leu-Ala. Circular dichroism studies indicated a high alpha-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.
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PMID:Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K. 1019 76

The protein assumed to be associated with bacteriochlorophyll (BChl) a in chlorosomes from the photosynthetic green filamentous bacterium Chloroflexus aurantiacus was investigated by alkaline treatment, proteolytic digestion and a new treatment using 1-hexanol, sodium cholate and Triton X-100. Upon alkaline treatment, only the 5.7 kDa CsmA protein was removed from the chlorosomes among six proteins detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, concomitantly with the disappearance of BChl a absorption at 795 nm. Trypsin treatment removed two proteins with molecular masses of 11 and 18 kDa (CsmN and CmsM), whereas the spectral properties of BChl a and BChl c were not changed. By the new hexanol-detergent (HD) treatment, most BChl c and all of the detected proteins except CsmA were removed from the chlorosomes without changing the BChl a spectral properties. Subsequent proteinase K treatment of these HD-treated chlorosomes caused digestion of CsmA and a simultaneous decrease of the BChl a absorption band. Based on these results, we suggest that CsmA is associated with BChl a in the chlorosomes. This suggestion was supported by the measured stoichiometric ratio of BChl a to CsmA in isolated chlorosomes, which was estimated to be between 1.2 and 2.7 by amino acid analysis of the SDS-PAGE-resolved protein bands.
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PMID:Association of bacteriochlorophyll a with the CsmA protein in chlorosomes of the photosynthetic green filamentous bacterium Chloroflexus aurantiacus. 1055 29


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