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 multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Originally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so-called trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, t-butoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-Val-Tyr-NH-MEc (Suc, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-Leu-Glu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-amino-ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133-4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.
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PMID:Use of serine-protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex. 142 69

Relative biological values (BV) of 36 feed phosphates were determined with female turkeys in bioassays of 21-day duration using three response criteria: weight gain, tibia ash percentage, and gain:feed ratio. Calcium phosphate, dibasic dihydrate (United States Pharmacopeia) was the reference standard. Nine mono-dicalcium phosphates (M-DCP, 21.0% phosphorus), 13 di-monocalcium phosphates (D-MCP, 18.5% phosphorus), and 14 defluorinated phosphates (DFP, 18.0% phosphorus) were evaluated. The average relative BV for M-DCP, D-MCP, and DFP samples were 97.6, 94.6, and 90.8%, respectively. Solubility of phosphates was determined by four recognized methods. The solvents were water, .4% HCl, 2.0% citric acid (CA), and neutral ammonium citrate (NAC). Water solubility of M-DCP samples was greater (67.5%) than that of D-MCP (38.8%) and DFP (8.9%) samples. Correlation of water solubility of phosphates to their relative BV was quite low, and water solubility was a poor indicator of BV. When .4% HCl was the solvent, correlation coefficients (r) were .55, .33, and .72 for M-DCP, D-MCP, and DFP, respectively. Based on these results and prediction equations, .4% HCl solubility would be inappropriate for estimating BV of M-DCP and D-MCP samples. Solubility of feed phosphates (mainly D-MCP and DFP) in 2.0% CA or NAC was positively correlated with BV; the r values were .87 to .95. Both of these solubility tests provided a good index of BV. However, it would seem inappropriate and risky to replace bioassays totally with these tests. Feed phosphate users could perform either the 2.0% CA or NAC solubility test easily as a screen for BV along with other quality control procedures (i.e., phosphorus, calcium, sodium, and fluoride determinations).
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PMID:Correlation of biological value of feed phosphates with their solubility in water, dilute hydrogen chloride, dilute citric acid, and neutral ammonium citrate. 147 May 90

To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.
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PMID:Processing of an NH2-terminally extended thermostable alpha-amylase by Bacillus subtilis alkaline protease. 212 90

The multicatalytic proteinase complex (MPC; proteasome) can be isolated in a latent form which then can be activated for protein hydrolysis by physiological and nonphysiological treatments, including high temperature. In this study, the temperature dependency profiles for the hydrolysis of Cbz-Gly-Gly-Leu-pNA and Cbz-Val-Gly-Arg-pNA by bovine lens MPC are found to be those expected for a thermostable enzyme, with single optima above 50 degrees C. In contrast, hydrolyses of Cbz-Leu-Leu-Glu-2NNp and alpha 2-crystallin, a lens structural protein, show two temperature transitions, indicating that hydrolysis of these substrates can be activated by elevated temperature. Temperature dependency profiles of peptidase activity in Tris-HCl compared to Hepes buffer suggest that Tris decreases the thermal stability of MPC. After 10 min preincubation in Tris-HCl at 53 degrees C, lens MPC activities are reduced by 50-60% and loss of the major MPC band can be seen on nondenaturing gels. The presence of alpha 2-crystallin during preincubation partially prevents the loss of activity. Although alpha-crystallin has been reported to function as a molecular chaperone, similar protection by other MPC substrates suggests that alpha 2-crystallin stabilized the MPC as a substrate. Our findings indicate both activation and inactivation of the enzyme at elevated temperatures. It is proposed therefore that high temperature activates the MPC but to a more labile form which can be partially stabilized by protein substrates.
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PMID:Thermal stability and activation of bovine lens multicatalytic proteinase complex (proteasome). 823 52

Within 1 h after slaughter, two 10-g samples of longissimus muscle were obtained from four crossbred beef cattle. Samples were homogenized in three or six volumes of extraction solution that consisted of 50 mM Tris base, 10 mM EDTA, and 10 mM 2-mercaptoethanol, pH adjusted to 8.3 with 6 N HCl. After centrifugation the supernatant from the three-volume extract was fractionated by addition of solid (NH4) 2SO4. Proteins that precipitate between 40 and 65% (NH4) 2SO4 were dialyzed and then loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 100 to 400 mM (125 mL of each; Method A). The six-volume extract was loaded onto a DEAE-Sephacel column and eluted with a continuous gradient of NaCl from 0 to 350 mM (250 mL of each; Method B). Total peptidase activity eluted from the column was determined using the synthetic peptide N-CBZ-Gly-Gly-Leu-p-nitroanilide. Method B yielded greater multicatalytic proteinase complex (MCP) activities (picomoles of p-nitroaniline released/hour-1) per gram of muscle (1,538.25 +/- 105.15) than did Method A (1,195.05 +/- 86.55; P < .05). In addition, Method B permitted the quantification of calpain activity from the same fractions eluted. The relationship between enzyme activity and assay time (up to 45 min) and protein concentration (up to 10 micrograms) in the assay was linear. Studies indicated that the optimum temperature is in the range of 50 to 60 degrees C and the optimum pH in the range of 7.5 to 8.5.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Technical note: quantification of multicatalytic proteinase complex (proteasome) activity by ion-exchange chromatography. 829 81

The multicatalytic proteinase complex (proteasome) contains at least four distinct active sites catalyzing the degradation of selected chromogenic substrates (trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolyzing activities) and proteins such as beta-casein. Oxidized insulin B chain was recently proposed as a model substrate for protein degradation by the multicatalytic proteinase complex (Dick, L. R., Moomaw, C. R., DeMartino, G. N., and Slaughter, C. A. (1991) Biochemistry 30, 2725-2734). We studied the dialysis-induced activation of the hydrolysis of oxidized insulin B chain by this enzyme. Removal of EDTA from purified preparations of bovine pituitary multicatalytic proteinase complex by dialysis against Tris-HCl buffers led to marked changes in the catalytic properties and structure of the enzyme. Dialysis produced a time-dependent activation of oxidized insulin B chain hydrolysis with predominant cleavage at the Glu13-Ala14 bond. A new chromogenic assay was developed for measurement of this activity. Activation was accompanied by a virtually total inactivation of the chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a loss of the 24-kDa subunit and the appearance of a new band at 21 kDa. Amino-terminal amino acid analysis established that the 21-kDa band was autolytically derived from the 24-kDa subunit. Evidence for partial dissociation and/or aggregation indicated that autolysis destabilizes the complex. By altering the profile of catalytic activities of the multicatalytic proteinase complex, autolysis may serve as a mechanism for regulation of this macromolecule.
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PMID:Changes in the structure and catalytic activities of the bovine pituitary multicatalytic proteinase complex following dialysis. 842 Sep 77

Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its activity can be stimulated by SDS, Mg2+, and also guanidine HCl, but not urea. The enzyme was strongly inhibited by novel peptide aldehydes with hydrophobic P4 residues, and was rapidly inactivated by 3, 4-dichloroisocoumarin (DCI). DCI modified the N-terminal threonine of the catalytic beta-subunit, the presumed active site nucleophile. To define how proteins are degraded, casein was derivatized with fluorescein isothiocyanate to facilitate detection of released products by the proteasome. Many fluorescent peptides were generated, but the relative amounts of different peptides were independent of the duration of the reaction. The rate of disappearance of protein substrates paralleled the rate of appearance of small products. Unlike conventional proteases, proteasome degrades proteins processively without release of polypeptide intermediates. Upon activation by SDS, guanidine, heat (55 degrees C), or partial inhibition with DCI, proteasomes still functioned processively, but generated a different pattern of peptides under each condition. Thus, processivity is an inherent feature of the 20 S proteasome, not requiring all active sites or ATP hydrolysis.
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PMID:Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum. 899 62

Fresh raw milks, with low (3.1 x 10(4) cell/ml) and high (1.1 x 10(6) cells/ml) somatic cell count (SCC), were standardized to 3.25% fat, and from each a preserved (with 0.02% potassium dichromate) and an unpreserved portion were prepared. Subsamples of each portion were carbonated to contain 0 (control, pH 6.9) and 1500 (pH 6.2) ppm added CO2, and HCl acidified to pH 6.2 Milk pH was measured at 4 degrees C. For the preserved low- and high-SCC milks, two additional carbonation levels, 500 (pH 6.5) and 1000 (pH 6.3) ppm, were prepared. Milks were stored at 4 degrees C and analyzed on d 0, 7, 14, and 21 for microbial count, proteolysis, and lipolysis. The addition of 1500 ppm CO2, but not HCl, effectively delayed microbial growth at 4 degrees C. In general, in both the low- and high-SCC unpreserved milks, there was more proteolysis and lipolysis in control and HCl acidified milks than in milk with 1500 ppm added CO2. Higher levels of proteolysis and lipolysis in the unpreserved milks without added CO2 were related to higher bacteria counts in those milks. In preserved low- and high-SCC milks, microbial growth was inhibited, and proteolysis and lipolysis were caused by endogenous milk enzymes (e.g., plasmin and lipoprotein lipase). Compared with control, both milk with 1500 ppm added CO2 and milk with HCl acidification had less proteolysis. The effect of carbonation or acidification with HCl on proteolysis in preserved milks was more pronounced in the high SCC milk, probably due to its high endogenous protease activity. Plasmin is an alkaline protease and the reduction in milk pH by added CO2 or HCl explained the reduction in proteolysis. No effect of carbonation or acidification of milk on lipolysis was observed in the preserved low- and high-SCC milks. The CO2 addition to raw milk decreased proteolysis via at least two mechanisms: the reduction of microbial proteases due to a reduced microbial growth and the possible reduction of endogenous protease activity due to a lower milk pH. The effect of CO2 on lipolysis was mostly due to a reduced microbial growth. High-quality raw milk (i.e., low initial bacteria count and low SCC) with 1500 ppm added CO2 can be stored at 4 degrees C for 14 d with minimal proteolysis and lipolysis and with standard plate count < 3 x 10(5) cfu/ml.
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PMID:Effect of CO2 addition to raw milk on proteolysis and lipolysis at 4 degrees C. 1277 72

Ruminants fed high-grain diets often are subjected to ruminal acidosis, which can lead to excessive absorption of lactate into the blood stream, thereby causing metabolic acidosis. Metabolic acidosis leads to body protein loss, mainly due to increased skeletal muscle degradation. Our objective was to determine the effects of metabolic acidosis on the messenger RNA (mRNA) abundance of genes encoding components of the ubiquitin-mediated proteolytic pathway in the skeletal muscle of lactating Holstein cows. Cows (n = 20) were assigned to one of two treatments: 1) control; or 2) NutriChlor 18-8, an HCl-treated supplement, which was fed to induce chronic metabolic acidosis. The longissimus muscle was biopsied before and after 10 d of treatments. Total RNA isolated from muscle tissue was hybridized with (32)P-labeled cDNA probes encoding for 14-kDa ubiquitin carrier protein E2 (14-kDa E2), ubiquitin, and C8 and C9 subunits of the 20S proteasome. Induction of metabolic acidosis increased (P < 0.05) skeletal muscle mRNA levels for ubiquitin (25%), 14-kDa E2 (34%), and the C8 subunit (20%); however, mRNA abundance for the C9 subunit was unaffected (P > 0.05). These results suggest that up-regulation of the ubiquitin-proteasome pathway is the mechanism by which metabolic acidosis stimulates muscle wasting in ruminants.
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PMID:Chronic metabolic acidosis increases mRNA levels for components of the ubiquitin-mediated proteolytic pathway in skeletal muscle of dairy cows. 1498 46

In the present study, several nasal absorption enhancers, used in metoclopramide hydrochloride (MCP HCl) nasal solutions, have been screened for their possible damaging effect in the in vitro human erythrocytes lysis experiment. Moreover, the in vivo leaching of biological markers from the rat nasal epithelium was used as a quantitative assessment for possible nasal mucosal irritation whereby the extent of release of total protein and lactate dehydrogenase (LDH) in the nasal lavage fluid was determined. Results showed that insignificant hemolysis from normal saline (P<0.05) occurred with the enhancer protamine sulphate while poly-l-arginine and sodium cholate demonstrated very low (<15%) hemolysis and caused insignificant protein and LDH release from the rat nasal mucosa. Conversely, sodium deoxycholate and chitosan polymers (either of low or high molecular weight) showed high (>60%) hemolysis in vitro and the release of the biological markers in vivo was significantly higher (P<0.05) than the control solution (no enhancer). A significant correlation (P<0.05) existed between the enhancement effect of MCP HCl nasal absorption and the amounts of protein (r=0.85) and LDH (r=0.88). Furthermore, the pharmacokinetics of MCP HCl was determined after intravenous (IV), per-oral and intranasal administration of 10mg drug dose in rabbits. The application of a nasal spray (NS) solution containing 0.5% sodium cholate resulted in a significant improvement (P<0.05) in both the rate and extent of absorption of MCP HCl where the T(max) achieved was 23.3min as compared to 50min in case of the oral solution while the area under the serum concentration-time curve (AUC(0-infinity)) were 506.1, 434.9 and 278.7microg/mlmin for IV, NS and oral solutions, respectively. These values corresponded to absolute bioavailabilities of 87.21 and 55.61% for the NS and oral solutions, respectively. It could thus be concluded that NS of MCP HCl represents a viable approach to achieving rapid and high systemic drug absorption during the emergency treatment of severe emesis.
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PMID:Rapid-onset intranasal delivery of metoclopramide hydrochloride Part II: Safety of various absorption enhancers and pharmacokinetic evaluation. 1695 53


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