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)

We have developed and tested a multifrequency phase/modulation fluorometer based on the Hamamatsu Model R2024U gatable microchannel plate photomultiplier (MCP-PMT), using internal MCP-PMT cross-correlation. This internal mixing is accomplished by biasing and modulating the gating mesh which is located 0.2 mm behind the photocathode. Near the photocathode center, no high-frequency photocurrent modulation was achieved. Within a circular area near the photocathode edge, however, the R2024U allows accurate phase shift and demodulation measurements up to at least 4.5 GHz, the frequency limit of our PMT-modulation amplifier. By mixing immediately after the photocathode, there is no decrease in the time resolution due to transit time spread, and the MCP has to process only low-frequency signals. This means no low-level high-frequency signal voltages have to be handled in this fluorometer, and the problems of RF shielding become much less critical. Also, the effective output impedance of the PMT has been increased, resulting in a 43-dB increase in the PMT output signal power. In principle, more MCPs could be built into the PMT, allowing an improved fluorescence detection limit. We have used the method of reference fluorophores in order to compensate for pronounced PMT color effects, a wavelength-dependent modulation, and a wavelength-dependent time shift. No color correction is required in the case of time-dependent depolarization. The performance of the instrument was verified by measurements of the intensity decay of perylene, which showed a single-exponential decay, and by measurements of the decay of tryptophan in water, which showed a double-exponential decay, as expected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A 4-GHz frequency-domain fluorometer with internal microchannel plate photomultiplier cross-correlation. 204 14

Synthesis and release of NAD(P)ase by Neurospora crassa wild type was studied in experiments in which mycelia grown in Vogel minimal medium were transferred to media containing protein as the only carbon source. Several results are presented suggesting that the NAD(P)ase may be induced by the presence of protein in the culture medium. Low concentrations of sucrose or glucose (0.1%), Casamino acids or some amino acids such as methionine, cysteine, phenylalanine and tryptophan strongly repressed the enzyme synthesis. Under induction conditions NAD(P)ase and alkaline protease appeared together in the culture medium. It would appear that NAD(P)ase and alkaline protease are coordinately regulated by a common control mechanism related to carbon catabolism.
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PMID:Carbon source regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa: induction and repression of enzyme synthesis. 623 74

An endogenous activator of 20S proteasome was purified from human platelets and its effect on three peptidase activities of proteasome was studied. This activator had a molecular weight of 170 kDa, and was composed of 32 kDa polypeptides as determined by SDS-PAGE. It was highly labile upon heat treatment (56 degrees C, 20 s) and proteinase (pronase CB) digestion. Suc-LLVY-MCA degrading activity of the platelet proteasome showed positive cooperativity between two or more catalytic sites because the coefficient was 1.54 when analyzed by use of the Hill plot. The endogenous activator increased Vmax and caused a loss of cooperativity. The plot of reaction velocity as a function of activator concentration yielded a saturation curve, implying the binding of the activator to proteasome. Boc-LTR-MCA degrading activity followed Michaelis-Menten kinetics. The activator enhanced the activity by increasing Vmax and decreasing Km. In contrast, CBz-LLE-2NA degrading activity could not be analyzed according to any kinetic scheme reported so far. The activator stimulated this activity at lower substrate concentrations (below 200 microM), while it inhibited the activity at higher substrate concentrations (400-800 microM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.
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PMID:Purification and characterization of endogenous protein activator of human platelet proteasome. 828 19

During 1994 and 1995, the structures of the serum amyloid P component, the bacterial chaperonin GroEL, the 20S proteasome, the bacterial light-harvesting complexes and the tryptophan operon RNA-binding attenuation protein have been determined. These structures all form circular assemblies in which the individual subunits are related by rotational symmetry. In most cases the circular organization generates a new biophysical property and a specific biological function which have presumably been selected by evolution.
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PMID:Circular assemblies. 872 45

The treatment of chronic suppurative otitis media caused by Pseudomonas aeruginosa remains a challenging problem. The virulence of Pseudomonas is related to its secretion of two matrix metalloproteinases, alkaline protease and elastase. This experiment examines the effects of a synthetic inhibitor of matrix metalloproteinases GM 6001, or N-(2(R)-2(hydroxyamido carbonylmethyl)-4-methylpentanoyl)-L-tryptophane methylamide), in a chinchilla Pseudomonas otitis media model. Thirty chinchillas underwent bilateral subtotal tympanic membrane perforations. Twenty-four chinchillas underwent bilateral middle ear inoculation with P. aeruginosa. Chinchillas were divided into four groups of six animals after the establishment of otitis media. Animals in one group were controls; the other three groups received either gentamicin, GM 6001, or gentamicin plus GM 6001 into the external auditory canal three times daily for 4 weeks. Clearance of Pseudomonas infection occurred in three ears of three animals, all in gentamicin groups, with or without GM 6001. Otorrhea (p = 0.0014) and external canal erythema (p = 0.025) were mild in the two gentamicin groups and moderate in the GM 6001 group when compared with bacterial controls. Animals in the GM 6001 group had the highest survival rate, less severe facial paralysis, and less vestibular toxicity than the gentamicin, gentamicin plus GM 6001, or control groups, although these differences were not statistically significant. This pilot study showed encouraging results for a role of ototopical protease inhibitors in the treatment of Pseudomonas otitis media.
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PMID:Inhibition of proteases in Pseudomonas otitis media in chinchillas. 886 89

PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome. PA28 is composed of two homologous subunits, alpha and beta, arranged in alternating positions in a ring-shaped oligomer with a likely stoichiometry of (alphabeta)3. Our previous work demonstrated that the carboxyl terminus of the alpha subunit was necessary for PA28 to bind to and activate the proteasome. The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the alpha and beta subunits in proteasome activation. Each subunit and various mutants of the alpha subunit were expressed in Escherichia coli and purified. PA28alpha stimulated the proteasome, but had a much greater Kact than native heteromeric PA28. In contrast, PA28beta was unable to stimulate the proteasome. Mutants of the alpha subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome. However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents. Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28alpha. Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28alpha, had no effect on proteasome stimulatory activity. Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits. PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted from inactive alpha subunits and wild type beta subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active alpha mutants and wild type beta subunits had the same activity as native heteromeric PA28. These results indicate that the beta subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.
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PMID:Relative functions of the alpha and beta subunits of the proteasome activator, PA28. 934 51

Fluorescence emission properties of the alkaline protease Esperase have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gln 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca(2+)-binding sites.
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PMID:Steady-state and time-resolved fluorescence of Esperase: comparison with the X-ray structure in the region of the two tryptophans. 969 45

Four novel proteasome inhibitors, TMC-95A-D (1-4) have been isolated from the fermentation broth of Apiospora montagnei Sacc. TC 1093, isolated from a soil sample. All of the molecular formulas of 1-4 were established as C(33)H(38)N(6)O(10) by high-resolution FAB-MS. Their planar structures were determined on the basis of extensive analyses of 1D and 2D NMR, and degradation studies. Compounds 1-4 have the same planar structures to each other, and are unique highly modified cyclic peptides containing L-tyrosine, L-aspargine, highly oxidized L-tryptophan, (Z)-1-propenylamine, and 3-methyl-2-oxopentanoic acid units. The absolute configuration at C-11 and C-36 of 1-4 was determined based on chiral TLC and HPLC analyses of their chemical degradation products. The ROESY analysis along with (1)H-(1)H coupling constants clarified the absolute stereochemistry at C-6, -7, -8, and -14 of the cyclic moieties. These studies revealed the relationships of 1-4 to be diastereomers at C-7 and C-36.
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PMID:Structures of TMC-95A-D: novel proteasome inhibitors from Apiospora montagnei sacc. TC 1093. 1081 45

The structure--function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle.
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PMID:Structure--function relationships in bovine thymus 20S proteasome: a fluorimetric study. 1131 22

Patients with cancer cachexia experience a profound wasting of adipose tissue and lean body mass. Anorexia, although often present, is insufficient to account for tissue wasting because 1) cachexia involves massive depletion of skeletal muscle that does not occur during anorexia, 2) nutritional supplementation cannot replenish the loss of lean body mass, 3) cachexia can occur without anorexia, and 4) food intake might be normal for the lower weight of the cancer patient. Anorexia can arise from 1) decreased taste and smell of food, 2) early satiety, 3) dysfunctional hypothalamic membrane adenylate cyclase, 4) increased brain tryptophan, and 5) cytokine production. Appetite stimulants such as cyproheptadine, medroxyprogesterone acetate, and megestrol acetate do not significantly improve lean body mass. Tumor products might be more important in the development of cachexia. Cachectic patients excrete in their urine a lipid-mobilizing factor that directly stimulates lipolysis in a cyclic AMP-dependent manner and increases energy expenditure. Loss of skeletal muscle in cachexia is caused by upregulation of the ubiquitin-proteasome catabolic pathway. Cachexia-inducing tumors elaborate a sulfated glycoprotein, which directly initiates protein catabolism in skeletal muscle. The action of this proteolysis-inducing factor is attenuated by the polyunsaturated fatty acid eicosapentaenoic acid, which is also effective in preventing loss of skeletal muscle in cancer patients. Antagonists of tumor catabolic factors will provide important new agents in the treatment of cancer cachexia.
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PMID:Cancer anorexia and cachexia. 1137 46


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