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)

Porcine and ovine 19-S thyroglobulins prepared from frozen glands in several buffers using slice extraction or homogenization, ammonium sulfate precipitation and DEAE-cellulose chromatography or Sepharose 6B gel filtration were contaminated with protease activity of pH optima 4.5 and 8.6, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum temperatures of autodigestion were 37 degrees C at pH 4.5 and 25 degrees C at pH 8.6. Thyroglobulins prepared from unfrozen glands pH 7.2 in 0.1 M sodium phosphate using slice extraction, ammonium sulfate precipitation and Sepharose 6B gel filtration were devoid of acid proteolytic activity but still underwent autodigestion at pH 8.6. Diisopropylfluorophosphate was a potent inhibitor of the alkaline protease activity of ovine thyroglobulin preparations. In contrast to thyroglobulin obtained from frozen glands the proteins purified from fresh unfrozen glands at pH 7.2 only showed the 19-S and the 12-S species by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Very few bands migrating faster than 12-S were visible. After full reduction and S-alkylation of porcine and ovine thyroglobulins, no qualitative changes were observed in the gel electrophoresis pattern as compared to the unmodified proteins. Species of apparent mol. wt. corresponding to the native 12 S were the major component, strongly suggesting a mol. wt. of about 330 000 for the elementary peptide chains of pig and sheep thyroglobulins.
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PMID:Endogenous proteolytic activity and constituent polypeptide chains of sheep and pig 19 S thyroglobulin. 0 35

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20-50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, PH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60 degrees C, pH 10 for alkaline protease and 50 degrees C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the Km values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60 degrees C. Other peptide hydrolases, beta-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.
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PMID:Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde. 2 75

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

For a relaxed (rel-), protease producing (A-type) and a stringent (rel+), not-protease producing (B-type) variant of Bacillus licheniformis we determined fermentation patterns and products, growth parameters and alkaline protease-production (if any) in anaerobic, glucose-grown chemostats and batch-cultures. Glucose is dissimilated via glycolysis and oxidative pentose phosphate pathway simultaneously; the relative share of these two routes depends on growth phase (in batch) and specific growth rate (in chemostat). Predominant products are lactate, glycerol and acetaldehyde for A-type batches and acetaldehyde, ethanol, acetate and lactate for B-type batches. Both types show a considerable acetaldehyde production. In chemostat cultures, the fermentation products resemble those in batch-culture. From the anaerobic batches and chemostats, we conclude that the A-type (with low ATP-yield) will have a YATPmax of probably 12.9 g/mol and the B-type (with high ATP-yield) a YATPmax of about 10.1 g/mol. For batch-cultures, both types have about the same, high Yglucose (12 g/mol). So, the slow-growing A-type has a relatively high efficiency of anaerobic growth (i.e. an efficient use of ATP) and the fast-growing B-type a relatively low efficiency of anaerobic growth. In aerobic batch-cultures, we found 48, respectively 41% glucose-carbon conversion into mainly glycerol and pyruvate, respectively acetate as overflow metabolites in the A- and B-type. In both aerobic and anaerobic batch-cultures of the A-type, protease is produced predominantly in the logarithmic and early stationary phase, while a low but steady production is maintained in the stationary phase. Protease production occurs via de novo synthesis; up to 10% of the total protease in a culture is present in a cell-associated form. Although anaerobic protease production (expressed as protease per amount of biomass) is much higher than for aerobic conditions, specific rates of production are in the same range as for aerobic conditions while, most important, the substrate costs of anaerobic production are very much higher than for aerobic conditions.
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PMID:Formation of fermentation products and extracellular protease during anaerobic growth of Bacillus licheniformis in chemostat and batch-culture. 180 2

We studied the inhibitory effects of chicken egg-white ovomacroglobulin (ovoM) on keratitis induced by 56,000-Da protease (56 KP) of Serratia marcescens and by elastase (PE) and alkaline protease (PAP) of Pseudomonas aeruginosa. The effects of ovoM on the serratial and pseudomonal keratitis in rabbits were also elucidated. In one model, four drops of 56 KP, PE, or PAP (1 mg/ml) were applied to wounded corneas of eight eyes. Thereafter, 80 microliters ovoM (10 mg/ml) was dropped into four eyes and 0.01 M phosphate-buffed 0.15 M saline (pH 7.4) into the other eyes as a control. The other in vivo test system involved intrastromal injection of S. marcescens or P. aeruginosa, by which each sample (10(5)-10(7) colony-forming units) mixed with ovoM was injected into one cornea and the other cornea received organisms without ovoM. OvoM completely inhibited the activity of these bacterial proteases in vitro and reduced corneal destruction in experimental keratitis in rabbits. In addition, greatly accelerated wound healing was observed.
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PMID:Inhibitory effects of ovomacroglobulin on bacterial keratitis in rabbits. 186 68

The Bacillus subtilis sacU locus consists of two genes, degS and degU, which positively regulate the synthesis of several extracellular enzymes including the neutral and alkaline proteases. Both the DegS and DegU proteins have been purified from overproducing Escherichia coli strains harboring degS or degU gene-carrying plasmids, and the following results were obtained. DegS was autophosphorylated in the presence of [gamma-32P]ATP, and transferred the phosphoryl group to DegU. The transfer reaction was rapid in contrast to the autophosphorylation reaction. The phosphoryl groups incorporated into DegS and DegU were released at their own specific rates, the latter being twice faster than the former. The linkage between DegS and the phosphoryl moiety was unstable at acidic pH, whereas reverse was the case for the linkage between DegU and its phosphoryl group, suggesting that His and Asp are involved in the formation of DegS-phosphate and DegU-phosphate, respectively. Deletion of degS resulted in the reduced expression of the exocellular alkaline protease gene, aprE. These results suggest that phosphorylation of DegS by its own kinase activity and subsequent transfer of the phosphoryl group to DegU play a role in the activation of the aprE gene.
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PMID:Isolation and phosphorylation of the Bacillus subtilis degS and degU gene products. 212 96

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

A method for the determination of lorazepam in rat brain is described using liquid/liquid and solid-phase extraction, followed by high performance liquid chromatography. After addition of chlordiazepoxide as the internal standard, 100 mg brain tissue was homogenized and incubated with alkaline protease. Lorazepam and chlordiazepoxide were extracted three times with toluene. After treatment through a C18-Bond Elut column, lorazepam and chlordiazepoxide were analyzed isocratically on a reversed-phase column with a mobile phase consisting of methanol +0.025 M sodium phosphate buffer (66:34, v/v). The eluted drugs were monitored by their absorption at 240 nm. The sensitivity limit of this method was 10 ng of lorazepam per 100 mg of brain tissue sample. The standard curve was linear over the range of 20 to 200 ng lorazepam. The coefficient of variation for day-to-day precision established by 21 replicate analyses was 4.5 to 13.6%.
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PMID:Analysis of lorazepam in rat brain using liquid/liquid and solid-phase extraction in combination with high performance liquid chromatography. 220 80

The water-soluble ammonium salt of 3'-azido-5'-(O-ethoxycarbonylphosphinyl)-3'-deoxythymidine (ECP-AZT), the prototype of a novel class of compounds incorporating two active antiretroviral agents, in this case 3'-azido-3'-deoxythymidine (AZT) and phosphonoformic acid (PFA), within the same structure, was synthesized and tested as an inhibitor of the replication of human immunodeficiency virus type 1 (HIV-1) in Jurkat cells, a CD4+ human T-lymphocyte cell line. The corresponding 5'-(O-methoxycarbonylphosphinyl) derivative (MCP-AZT) was also prepared. The rationale for the synthesis of ECP-AZT and MCP-AZT was that they may be cleaved intracellularly to AZT and PFA via hydrolysis of the phosphate ester bond or to AZT 5'-monophosphate by oxidative cleavage of the carbon-phosphorus bond. ECP-AZT was found to block viral replication at a 50% inhibitory concentration (IC50) of ca. 10(-6) M as measured by reverse transcriptase (RT) activity in supernatants from cultures of infected cells. Little or no inhibition of cell growth was observed at this concentration, and there was less than 20% inhibition of cell growth at 10(-4) M. AZT itself was a more potent inhibitor of HIV-1 replication than ECP-AZT, but was also more cytotoxic. The antiviral selectivity of ECP-AZT, defined as the ratio IC50 (virus inhibition)/IC50(cell growth inhibition), was in the range considered to be therapeutic for anti-AIDS nucleosides.
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PMID:Inhibition of human immunodeficiency virus type 1 replication by phosphonoformate esters of 3'-azido-3'-deoxythymidine. 222 76

Two forms of a high-molecular-weight proteinase were isolated from rat liver. The purification procedure involved homogenization of the tissue, chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC: TSK 3000 SWG) and hydroxyapatite chromatography. The breakthrough fraction from the hydroxyapatite column contained the sodium dodecyl sulphate (SDS)- and linoleic acid-activated proteinase, ingensin A, but the other form, ingensin B, which was also activated by SDS and linoleic acid, was bound to the hydroxyapatite and eluted at 200 mM phosphate. A distinct feature of ingensin A was its activation by a brief sonication procedure. The optimum pH of the two forms was 7.5-9.5, and both of them were activated by monovalent cations. Although both enzymes show similar molecular weights of 700,000 on gel filtration, ingensins A and B were separated into a major subunit of 120,000 and subunits of 25,000-35,000, respectively, under the denaturing conditions.
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PMID:Purification of the two forms of the high-molecular-weight neutral proteinase ingensin from rat liver. 301 26


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