Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteasome (the multicatalytic endoproteinase complex) in mammalian tissues hydrolyzes proteins and several types of peptides. When this structure was isolated rapidly from rabbit skeletal muscle in the presence of glycerol, its various peptidase and protease activities showed a large reversible activation by physiological concentrations of ATP (Ka = 0.3-0.5 mM). Hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) was stimulated up to 12-fold by ATP, whereas degradation of casein and bovine serum albumin increased 4- to 7-fold. Neither ADP nor AMP had any effect. CTP, GTP, UTP, and the nonhydrolyzable analogs adenosine 5'-[beta,gamma-imino]triphosphate (AMPP[NH]P) and adenosine 5'-[alpha,beta-methylene]triphosphate (AMP[CH2]PP) increased peptide hydrolysis as well as ATP did. However, only ATP stimulated casein breakdown and only in the presence of Mg2+. Thus, nucleotide binding allows activation of the peptidase functions, but ATP hydrolysis seems necessary for enhanced degradation of proteins. The ATP effect on proteolysis was reversible and did not require ubiquitin. Sensitivity to ATP was labile, and with storage at 4 degrees C the enzyme became fully active in the absence of ATP or Mg2+. The ATP-activated form closely resembles the proteasome complex described previously, which did not show ATP dependence: both have molecular masses of 650 kDa, contain the same 8-10 subunits, and are precipitated by the same antibodies. A similar ATP-activated form was found in rabbit liver but not in rabbit reticulocytes. The proteasome seems to represent a ubiquitin-independent, ATP-stimulated proteolytic activity within nucleated mammalian cells.
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PMID:Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin. 253 33

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.
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PMID:An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts. 284 2

The role of proteasomes, particles with latent multicatalytic proteinase, in ATP-dependent proteolysis in rat reticulocyte extracts was examined. Removal of proteasomes from the extracts by immunoprecipitation caused almost complete inhibition of ATP-dependent degradation of [3H]methylcasein, without affecting ATP-dependent proteolysis. Peptide fragments of [3H]casein, obtained by cyanogen bromide cleavage, were rapidly degraded in an ATP-independent fashion and this activity was not affected by removal of the proteasomes. These results suggest that proteasomes are involved in ATP-dependent proteolysis in the extracts and that they catalyze the initial cleavage of large proteins.
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PMID:Involvement of proteasomes (multicatalytic proteinase) in ATP-dependent proteolysis in rat reticulocyte extracts. 304 62

Lipoxygenase purified from rabbit reticulocyte lysate has a molecular mass of 68 kDa on SDS gel and a pI of 5.97. Lipoxygenase is inhibited by nordihydroguaiaretic acid (NDGA), 3-amino-1-(m-(trifluoromethyl)phenyl)-2-pyrazoline (BW755C), 5,8,11,14-eicosatetraynoic acid (ETYA), salicylhydroxamate (SHAM) or hemin. Metal ions or nucleotides do not affect its activity. The addition of certain of these inhibitors to the reticulocyte extract also inhibited the ATP-dependent proteolysis of casein, one of the distinct characteristics of reticulocytes. No clear correlation between lipoxygenase activity and ATP-dependent proteolysis could be detected. Hemin and NDGA inhibited both processes, but the concentrations necessary for inhibition were quite different. SHAM completely inhibited lipoxygenase, but not proteolysis. o-Phenanthroline inhibited ATP-dependent proteolysis, but had no effect on lipoxygenase activity. We have also purified a high-molecular-mass protease, ingensin, from reticulocyte extract. This protease accounted for more than 90% of the casein-degrading activity in reticulocyte extract. NDGA inhibited ingensin at the same concentrations required for inhibition of ATP-dependent proteolysis. These results suggest that lipoxygenase is not indispensable for the ATP-dependent proteolysis and the novel high-molecular-mass protease, ingensin, may be involved in the process.
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PMID:Reticulocyte lipoxygenase, ingensin, and ATP-dependent proteolysis. 308 25

A transformable strain of Bacillus subtilis 6160, a derivative of B. subtilis 168, produces three kinds of casein hydrolytic enzymes (alkaline protease, neutral protease, and esterase) in a culture medium. B. natto IAM 1212 produces 15 to 20 times as much total proteolytic activity as does B. subtilis. Extracellular proteases produced by the two strains were separated into each enzyme fraction by diethylaminoethyl-Sephadex A-50 column chromatography. The difference in the total protease activities of extracellular proteases between the two strains was due to the amount of neutral protease. The ratios of neutral protease activity to alkaline protease activity (N/A) were 1.1 in B. subtilis 6160 and 13.0 in B. natto IAM 1212. Enzymological and immunological properties of alkaline protease and neutral protease obtained from the two strains were quite similar or identical, respectively. Specific activities measured by an immunological analysis of the two neutral proteases against casein were also equal. A genetic character of high protease productivity in B. natto IAM 1212 was transferred to B. subtilis 6160 by the deoxyribonucleic acid-mediated transformation. Among 73 transformants that acquired high protease productivity, 69 produced a higher amount of neutral protease and the ratios of N/A were changed to 15 to 60. Three other strains were transformed in the productivity of neutral protease and alpha-amylase simultaneously, and one showed considerable change in the production of alkaline protease and neutral protease. The specific activities (casein hydrolytic activities/enzyme molecules) of neutral proteases from the representative four transformants were equal to those of the two parental strains. These results suggested the presence of a specific gene(s) that participated in the productivity of neutral protease in B. subtilis.
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PMID:Regulation of neutral protease productivity in Bacillus subtilis: transformation of high protease productivity. 420 70

The role of cardiac lysosomal and nonlysosomal protease alterations in the development of the cardiomyopathy that occurs in genetically diabetic C57BL/KsJ db/db mice has been examined. The db/db mice and age-matched controls were sacrificed between 7 and 24 weeks of age. Cathepsin D activity, myofibrillar alkaline protease (MAP) activity (including serine protease activity), and Ca2+-activated protease activity were determined by using [3H]acetyl-casein as substrate. There is a significant decrease in cathepsin D, MAP, and serine protease activities in the myocardium of 7- to 20-week old diabetic mice with a rebound of these activities toward normal levels by 24 weeks of age. Cathepsin D and MAP activities are inversely related to heart weight in diabetic mice with the higher levels being recorded in association with the most pronounced decrease in heart weight. In contrast, Ca2+-activated protease activity in the hearts of diabetic mice does not differ significantly from controls throughout the period of observation. The results suggest that both lysosomal cathepsin D and nonlysosomal MAP may mediate the accelerated cardiac muscle degradation that occurs in the late stage of diabetic cardiomyopathy in the db/db mice.
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PMID:Lysosomal and nonlysosomal proteolytic activities in experimental diabetic cardiomyopathy. 632 62

Two-kidney, one clip Goldblatt hypertension of 2, 4 and 8 weeks duration was induced in 100-g male Wistar-Kyoto rats. Nucleic acid content was determined in the isolated cardiac muscle cells from the left ventricle. The profile for several major proteolytic activities in either isolated cardiac muscle cells or left ventricle preparations was also studied, using [3H]acetyl-casein as substrate. From the soluble fraction of the tissue or cell preparation, a pH 6 proteolytic activity, two forms of calcium-activated protease as well as cathepsin D were identifiable by inhibitor assay or DEAE-cellulose chromatography. From the myofibrillar fraction of the same preparation, two kinds of proteolytic activity were detected at alkaline pH: a phenylmethylsulfonyl fluoride (PMSF) inhibitable activity that was serine protease-like and the other a N-ethylmaleimide (NEM) inhibitable activity that resembled Ca2+-activated protease. At 2 weeks of hypertension, there was a significant increase in the pH 6 proteolytic activity as well as the calcium-activated protease I and the NEM-inhibitable alkaline protease activities, while the other identifiable proteolytic activities remained unchanged. Lysosomal cathepsin D showed a rise in activity only after 8 weeks of hypertension. These results may be related to the development of myocyte necrosis and lysis that occur in this model of hypertensive cardiomyopathy.
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PMID:Proteolytic activities in hypertensive cardiomyopathy of rats. 634 96

The chromatin fraction of rat liver exhibited proteolytic activity caused by serine proteases, with maximal activity at pH 8 or 10. They were analyzed by affinity labeling with [3H]diisopropylfluorophosphate followed by SDS-polyacrylamide gel electrophoresis, and partially purified by gel filtration through Sepharose 6B after selective extraction from the chromatin fraction. The following results were obtained. 1. The chromatin fraction contained three DFP-binding proteins with molecular weights of 52,000, 25,000, and 15,000 daltons, and they were tentatively designated proteins A, B, and C, respectively. Unlike proteins A and B, protein C reacted with DFP more strongly at pH 10 than at pH 8. A greater part of protein B was present in the nucleoli, while the others were predominantly distributed in extra-nucleolar chromatin. 2. Proteins A and B were extracted from the chromatin fraction by 5 M urea and 0.7 M NaCl, respectively; while protein C was not extractable by either solution. Proteins A and B were further purified by gel filtration through Sepharose 6B. 3. Protein B was a neutral protease with a maximal activity for 3H-labeled ribosomal proteins at pH 8 and showed high specificity for basic proteins, such as histone and ribosomal proteins. Protein A was an alkaline protease with a maximal activity at pH 10 and showed proteolytic activity not only for basic proteins but also for hydrophobic proteins, such as casein and non-histone proteins of chromatin. 4. Protein A was activated at pH 8 by high concentrations of NaCl, suggesting the presence of some inhibitor(s). Protein A was converted to protein C at pH 10, and also at pH 8 with high concentrations of NaCl. Thus, protein A is suggested to be the complex of protein C and unknown inhibitor(s). 5. When the chromatin fraction was incubated at pH 10, non-histone proteins were degraded much faster than at pH 8, although H1 histone was degraded at similar rates at both pHs. These results indicate that the chromatin fraction of rat liver contains at least two kinds of serine proteases, B and C, and that protease B is probably involved in the metabolism of basic protein, especially H1 histone. Protease C, the greater part of which associates with some inhibitors to form protein A, may play its main role in the metabolism of non-histone proteins.
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PMID:Studies on the serine proteases associated with rat liver chromatin. 675

The aim of this work was to study the influence of medium composition and the effect of the operative conditions on alkaline protease production. Several nitrogen sources (in amount of 10 g/l) were tested and compared with bacto peptone Difco: Casein was the best. The addition of "Tween 80" 0,5% resulted in a marked increase in yields of the alkaline protease. The maximum alkaline protease production (3.342.290 UAPAM/g) was achieved using the following medium: Lactose 20 g/l; casein 10 g/l; NaCl 1,5 g/l; MgSO4.7H2O 0,15 g/l; CaCl2.2H2O 0,06 g/l; MnCl2.4H2O 0,01 g/l; "Tween 80" 5,0 g/l; EDTA 1,5 X 10(-4)M; KH2PO4 1,5 g/l; K2H PO4 1,5 g/l; Na2SO4 1,5 g/l. It was found that a volumetric relation of 0,1 ml, vol. of medium/vol. of flask at 25 degrees C of temperature was the best process condition.
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PMID:[Production on alkaline protease by Bacillus subtilis NRRL 3411]. 681 11

It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 000-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.
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PMID:Purification and characterization of alkaline protease and neutral protease from chromatin of rats. 702 44


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