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)

Both streptozotocin and chlorozotocin, the 2-chloroethyl analogue of streptozotocin, are diabetogenic chemicals in rodents. Although these chemicals are similar structurally, they appear to act on pancreatic B cells via different mechanisms. In studies here, damage and repair of DNA after exposure of an insulin-secreting cell line to streptozotocin and chlorozotocin were assessed by nucleoid sedimentation and alkaline elution. Equitoxic concentrations of streptozotocin and chlorozotocin caused significant single-strand breakage of DNA (p less than 0.005). These lesions were repaired in a time-dependent manner, with most repair completed by 24-h post-exposure to chemicals. Additionally, chlorozotocin caused DNA-DNA and DNA-protein crosslinks in insulinoma cells. When proteinase K was included in the crosslinking assay, a substantial proportion of the chlorozotocin-associated crosslinks proved to be DNA interstrand in nature. Analysis of the amount of interstrand crosslinking in insulinoma cells after exposure to chlorozotocin for 1 h showed that formation of interstrand crosslinks was slow. Increasing amounts appeared over a 24-h period. These results suggest that the formation of irreversible DNA interstrand crosslinks may be a critical factor in cytotoxicity and diabetogenicity caused by chlorozotocin.
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PMID:Comparative interactions of streptozotocin and chlorozotocin with DNA of an insulin-secreting cell line (RINr). 293 99

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.
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PMID:Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1. 316 11

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.
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PMID:Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor. 335 Aug 13

Pronase, proteinase K, carboxypeptidases A and B, aminopeptidase M, intestinal mucosa exopeptidases and prolidase, immobilized to derivatized controlled-pore glass beads, were used in a study of total enzymic hydrolysis of proteins. The combined use of immobilized enzymatic and acid hydrolysis, for assessment of protein quality, will give a more accurate chemical score than that afforded by acid hydrolysis alone. Amino acid analysis of enzymic hydrolysates of native protein substrates (beta-lactoglobulin and insulin) yielded 92% of the theoretical values and 103% of the values observed for standard acid hydrolysates. These results suggest that using a combination of immobilized proteases in concert gives essentially total hydrolysis of protein substrates in a time period (18-24 h) comparable to conventional acid hydrolysis methods.
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PMID:Compositional analysis of proteins following hydrolysis by immobilized proteases. 644 Aug 87

A proteinase secreted by the sapstaining fungus Ophiostoma piceae is thought to be necessary for the primary retrieval of nitrogen from wood proteins. By using mass spectrometry (MS) techniques, we have established the cleavage specificity of this subtilisin-like serine proteinase. This work demonstrated the potential of MS in determining cleavage specificities of newly isolated proteinases in a relatively short time frame, and determined that the O. piceae proteinase showed a substrate specificity similar to that of proteinase K. Primary cleavage of the insulin B-chain occurred between Leu15 and Tyr16. In addition numerous secondary cleavage sites occurred after hydrophobic, polar, and charged amino acids indicating a broad specificity.
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PMID:Characterization of the cleavage specificity of a subtilisin-like serine proteinase from Ophiostoma piceae by liquid chromatography/mass spectrometry and tandem MS. 758 36

A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.
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PMID:Macluralisin--a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid. 776 35

The functional significance of the insulin receptor on bovine aorta endothelial (BAE) cells is not well defined. The insulin receptor expressed on BAE cells does not mediate insulin hormonal effects and does not mediate the transcytosis of insulin from the apical to the basolateral domain of the cell monolayer. To assess the role of the insulin receptor on BAE cells, the physical characteristics of the BAE cell receptor were investigated, and the time-dependent interaction of insulin and insulin degradation products with BAE cell monolayers was quantitated. The BAE cell insulin receptor was found to be highly resistant to the proteolytic action of trypsin, pronase, and proteinase K at either 4 degrees C or 37 degrees C. This resistance may permit the receptor to maintain insulin binding capabilities in spite of the high concentrations of proteases which are normally present in blood. Scatchard analysis of cell-surface and total cellular insulin receptor demonstrated dissociation constants similar to values obtained with other cells and tissues. However, whereas other cells and tissues contain an intracellular pool of receptor that ranges from 20-40% of the total cellular receptor content, no intracellular population of insulin receptors was detected in BAE cells. Upon incubation of intact BAE cell monolayers with insulin, no endocytosis of cell-surface insulin receptor could be demonstrated. However, insulin degradation by the BAE cells was readily quantitated, at a rate of 16.3 fmol/10(6) cells/h at an insulin concentration of 2 nM. This rate of degradation was not inhibited by chloroquine, which inhibits insulin degradation in fibroblasts, hepatocytes, and adipocytes, nor by phenylarsine oxide, which inhibits endocytosis. Bacitracin inhibited insulin binding to the cell monolayers and inhibited insulin degradation with identical IC50 values (80 microM). These data suggest that in BAE cells, insulin degradation occurs in the absence of receptor-mediated endocytosis and is mediated by binding of insulin to its receptor. Therefore, it is concluded that the functional role of the insulin receptor expressed in BAE cells is to bind blood-borne insulin at the plasma membrane of the cell and thereby facilitate the degradation of insulin at the BAE cell plasma membrane.
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PMID:Insulin receptor characterization and function in bovine aorta endothelial cells: insulin degradation by a plasma membrane, protease-resistant insulin receptor. 822 65

Macrophages play a major role in the pathogenesis of insulin-dependent diabetes mellitus in animals. These cells are the first to invade the pancreas and macrophage-eradicating treatments reduce the incidence of the disease. In humans, however, their role is less clear. In this study we investigated the hypothesis that the pancreatic environment per se could activate macrophages. Tissue culture supernatants from human islets of Langerhans were tested for chemotactic activity and oxidative burst response in monocytes isolated from healthy adults. Preincubation with the supernatants enhanced the oxidative burst response evoked by fMLP (up to 379%) and opsonized zymosan (up to 173%). The activity decreased by dilution and was no longer detectable at 1:16. No increased activity was seen in supernatants from a number of other human endocrine and non-endocrine primary cells, suggesting a factor specific for islet tissue. The increased oxidative burst response could partially be eliminated by heat- and proteinase K treatment, suggesting that the activity could be of polypeptide nature. The factor could not be absorbed by polyvalent rabbit antibodies directed towards a variety of cytokines not by a mixture of high-titer anti-cytokine antibodies. It is possible that islet factors could also promote such monocyte activation in vivo in monocytes attracted to the islets of Langerhans by other means. This could contribute to the development of insulin-dependent diabetes in humans.
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PMID:Tissue culture supernatants from human islets of Langerhans activate the oxidative burst response of human monocytes in vitro. 861 55

To improve the sequence ions of a protein in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), proteinase K was used to digest the protein followed by MALDI-MS characterization of the peptide fragments. The primary structures of three proteins, insulin B chain, cytochrome c and lysozyme, were determined by this method. A series of peptide fragments including those differentiated by one residue can be produced from the protein by using proteinase K digestion, thus providing support to the protein sequence. The peptide fragments liberated from proteinase K proteolysis of the insulin B chain allow the protein to be partially sequenced. Furthermore, some of the residues are double or triple checked by generating a variety of fragments. The same method was used to investigate cytochrome c and lysozyme denaturated in 3 M guanidine hydrochloride. The success of the method relies on the intrinsic properties of proteinase K and accurate determination of the peptide fragments by MALDI-MS.
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PMID:Primary structures of proteins characterized by proteinase K digestion and matrix-assisted laser desorption/ionization mass spectrometry. 940 26

Patients with cancer cachexia exhibit increased glucose flux and lactate production in skeletal muscle. The aim of this study was to examine the direct effect of cancer cell-conditioned media on glucose metabolism in L6 myoblasts. Media from PANC-1 and Colo 320 cells caused a marked time-dependent and concentration-dependent increase of 2-deoxyglucose uptake in GLUT-4 transfected L6 myoblasts. This effect was greater than maximal acute stimulation by insulin and the effect of insulin was additive. Glucose utilization and lactate production increased in parallel to glucose uptake. The effect was inhibited by the protein synthesis inhibitor, cycloheximide and the glucose transport inhibitor, cytochalasin B. The bioactive factor had a molecular weight of approximately 5,000 and the biological activity was destroyed by proteinase K digestion. Radioimmunoassay and immunoneutralization studies indicated the major factor involved is not TNFalpha, IL-1beta, insulin, IGF-I or IGF-II. Further purification and characterization are needed to reveal the identity of this novel factor or factors which may have other metabolic effects that contribute to the cancer cachexia and insulin resistance.
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PMID:A factor from pancreatic and colonic cancer cells stimulates glucose uptake and lactate production in myoblasts. 1040 17


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