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 cholic acid CoA ligase activity of rat liver was quantitatively inactivated by proteolysis with pronase, chymotrypsin, subtilisin, or proteinase K in intact microsomal vesicles. Under the conditions employed, less than 14% of the lumenal mannose-6-phosphate phosphatase activity was lost, and the mannose-6-phosphate phosphatase activity remained highly latent. After microsomal integrity was disrupted with sodium deoxycholate, protease treatment resulted in a loss of greater than 74% of the mannose-6-phosphate phosphatase activity. Cholic acid CoA ligase activity was unaffected by preincubation of microsomes with sodium taurocholate under conditions that led to the complete expression of latent mannose-6-phosphate phosphatase activity. The data suggest that cholic acid CoA ligase activity is located on the cytoplasmic surface of hepatic microsomal vesicles.
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PMID:Evidence that cholic acid CoA ligase is located asymmetrically on the cytoplasmic surface of hepatic microsomal vesicles. 43 52

The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER). Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991). To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of [35S]methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product. It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes. This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent. These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.
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PMID:Topology of ER processing alpha-mannosidase of Saccharomyces cerevisiae. 142 58

Mild proteolysis of rat liver microsomes with increasing concentrations of proteinase K caused a marked decrease in the levels of microsomal cytochrome P-450 reductase (Fp) without having any significant effect on the cytochrome P-450s. About 20% of the microsomal cytochrome b5 was susceptible to proteolysis at low concentrations of proteinase K, while the remaining 80% was resistant to proteolysis, even at significantly higher proteinase K concentrations. Low concentrations of the proteases released about 30% of Fp from microsomes isolated from both uninduced and phenobarbital-induced rats, but did not affect the rates of benzphetamine bital-induced rats, but did not affect the rates of benzphetamine demethylation significantly. Further depletion of microsomal Fp at higher concentrations of proteinase K resulted in reductions of the rates of benzphetamine demethylation. However, even at higher protease concentrations, the decrease in the rate of the demethylation reaction was significantly less than the loss of Fp. Similar results were observed for the metabolism of two other substrates, 7-ethoxycoumarin and p-nitroanisole, suggesting that the P-450s, not the Fp, were the rate-limiting components in the metabolism of these xenobiotics by microsomes. It is clear that the decreases in the P-450-dependent oxidations were due to depletion of the NADPH-cytochrome P-450 reductase since reconstituting the protease-treated microsomes with native Fp restored the oxidation reactions. The amount of Fp required to completely restore the oxidation of benzphetamine only partially restored the oxidation of 7-ethoxycoumarin and p-nitroanisole.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential effects of proteinase K on the components of the liver microsomal cytochrome P-450 mixed function oxidase system. 168 5

The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.
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PMID:The role of cytosolic and membrane factors in processing of the human beta-2 adrenergic receptor following translocation and glycosylation in a cell-free system. 169 24

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.
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PMID:Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis. 170 26

The contributions of the hepatic microsomal and cytosolic fractions in the metabolic activation of the promutagen 2-aminofluorene into mutagenic intermediates in the Ames test were investigated. Rat hepatic postmitochondrial, microsomal and cytosolic preparations could convert 2-aminofluorene to mutagens, the postmitochondrial preparation being the most and cytosol the least efficient. Pretreatment of the rats with Aroclor 1254 markedly enhanced the cytosol-mediated mutagenicity of the amine but increased microsomal- and postmitochrondrial-mediated mutagenicity only modestly. The cytosol-mediated mutagenicity of 2-aminofluorene was abolished by heat treatment and by incubation with proteinase K, but was unaffected by dialysis emphasising the protein nature of the cytosolic activation system. Oxygen radical generating systems and oxygen radical scavengers did not significantly influence the cytosol-mediated mutagenic response. Similarly incorporation of xanthine or allopurinol into the cytosolic activation system did not modulate the mutagenic response indicating no role for the molybdenum oxygenases. The cytosolic activation of 2-aminofluorene differed from that mediated by the microsomes in cofactor requirement, substrate specificity and sensitivity to DMSO and (+)-catechin. Further centrifugation of the cytosolic fraction to remove any microsomal contamination did not decrease the cytosolic activation of 2-aminofluorene. It is concluded that the hepatic cytosol contains an oxygenase activity capable of activating certain aromatic amines.
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PMID:A cytosolic oxygenase activity involved in the bioactivation of 2-aminofluorene. 172 70

The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products. These clones also yielded low amounts of the S-COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon. In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon. The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence.
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PMID:Cell-free synthesis of rat and human catechol O-methyltransferase. Insertion of the membrane-bound form into microsomal membranes in vitro. 176 63

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.
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PMID:The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated. 216 4

Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [acetyl-14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Topographic studies of microsomal and pure prostaglandin H synthase. 249 19

1. Limited proteolytic digestion of rat liver microsomes (microsomal fractions) with trypsin (5 micrograms/ml), proteinase K (1.0 microgram/ml) and Pronase (20 micrograms/ml final concns.) resulted in abolition of GTP-dependent vesicle fusion. 2. Vesicle fusion could be partially restored to microsomes which had undergone limited tryptic digestion, by the addition of untreated microsomal vesicles. 3. GTP-dependent Ca2+ efflux from rat liver microsomes was also observed to be inhibited by limited proteolysis with trypsin and proteinase K. 4. Limited proteolysis of rat liver microsomes had no effect on subsequent GTP-dependent phosphorylation of polypeptides of Mr 17,000 and 38,000, and thus it is unlikely that the phosphorylation of these proteins is involved in GTP-dependent Ca2+ efflux and GTP-dependent vesicle fusion. 5. GTP binding by Gn proteins [proteins which bind GTP after transfer to nitrocellulose, as defined by Bhullar & Haslam (1986) Biochem. J. 245, 617-620] was inhibited by pre-treatment of microsomes with trypsin, proteinase K and Pronase at concentrations similar to those which abolished GTP-dependent Ca2+ efflux and vesicle fusion. 6. We suggest that one or more of the Gn proteins may be involved in the molecular mechanisms of GTP-dependent vesicle fusion and Ca2+ efflux in rat liver microsomes and that limited proteolytic digestion may be a useful tool in further investigation of these processes.
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PMID:The effect of limited proteolysis on GTP-dependent Ca2+ efflux and GTP-dependent fusion in rat liver microsomal vesicles. 249 9


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