Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the "soluble" enzyme subtypes and their "particulate" counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle.
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PMID:Purification of the major protein-tyrosine-phosphatases of human placenta. 283 86

The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.
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PMID:Mitochondrial ATP synthase. Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides. 290 92

Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.
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PMID:Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes. 299 75

Reorganization and activation energies for charge transfer reactions occurring inside a dielectric sphere have been calculated by solving the problem of polar medium reorganization within and outside a dielectric sphere placed in another infinite dielectric. The dielectric sphere is assumed to simulate a protein globule, i.e. an enzyme molecule. It has been shown that for some reaction types the activation energy tends to decrease as the globule radius increases and that for each of the reaction types considered there is an optimal globule radius an increase of which does not bring about any tangible activation energy reduction. The calculated optimal radii for different processes are in good agreement with the increasing molecular sizes in the series: ribonuclease less than or equal to lysozyme less than serine proteinases approximately equal to cysteine proteinases less than NAD-dependent dehydrogenases. The calculated radii are usually about 1.5 to 1.7 times (and molecular masses about 4-5 times) smaller than the experimental ones. The reasons for this discrepancy are discussed and it has been suggested that the approximate nature of the treatment of a protein globule as a structureless dielectric is the main reason. It is shown that charge transfer at an acute angle to the globule surface is the optimum process. For endoergonic reaction stages it is the net charge transfer towards the periphery and for exoergonic ones that in the reverse direction which are advantageous. These conclusions are consistent with the data about the structure of the above-mentioned enzymes.
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PMID:Medium reorganization energy and enzymatic reaction activation energy. 315 27

We have reported previously that phenylarsine oxide (PAO) blocks insulin-stimulated glucose transport in 3T3-L1 adipocytes (Frost, S. C., and Lane, M. D. (1985) J. Biol. Chem. 260, 2646-2652). As shown in the present study, the locus of inhibition is post-receptor. Insulin stimulated the extent of receptor autophosphorylation in solution and in the intact cell by approximately 4-fold. PAO had no effect on this activity. Using reduced and carboxamidomethylated lysozyme as a substrate for the tyrosine-specific receptor, insulin stimulated the rate of receptor kinase-catalyzed substrate phosphorylation by 2-fold; PAO had no effect on this stimulation. However, the insulin-stimulated, serine-specific phosphorylation of two endogenous phosphoproteins (pp24 and pp240) in the intact cell was blocked by 25 microM PAO. These complementary in situ and in vitro studies demonstrate that the inhibition by PAO must be distal to the insulin receptor's protein tyrosine kinase activity.
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PMID:Effect of phenylarsine oxide on insulin-dependent protein phosphorylation and glucose transport in 3T3-L1 adipocytes. 329 62

Wild-type T4 lysozyme contains unpaired cysteine residues at positions 54 and 97. To investigate the role these residues play in the thermal inactivation of the wild-type, we constructed a double mutant with these cysteines replaced with valine and serine. This molecule, T4 lysozyme (C54V/C97S), is more stable than the wild-type to inactivation at 70 degrees C at pH 6.5 and 8.0. Guanidine hydrochloride reactivation experiments and SDS-PAGE on the inactivated products show that the wild-type is susceptible to varying degrees of oxidative damage, depending on buffer conditions, while the cysteine-minus mutant inactivates only by other pathways. The products of thermal, oxidative inactivation of the wild-type are disulfide-linked oligomers. The dependence of inactivation rate on temperature suggests that the formation of these aggregates depends on prior thermal unfolding of the T4 lysozyme molecule.
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PMID:The role of cysteine oxidation in the thermal inactivation of T4 lysozyme. 350 92

The protease activities responsible for the cotranslational processing of the Semliki Forest virus structural polyprotein were investigated by using an in vitro transcription-translation system. Three cleavages released the individual chains from the nascent polyprotein in the order capsid, p62, 6K (a nonstructural peptide), and E1. We showed directly that the protease activity responsible for the release of the capsid protein resides in the capsid itself: by progressive truncation of the cDNA used for the SP6 transcription, we showed that a precursor containing as few as 38 residues of the p62 protein left at the C terminus of the capsid was still very efficiently cleaved in vitro. We further tested the possibility that serine-219 of the capsid is involved in autoproteolysis by site-directed in vitro mutagenesis. A change in the sequence Gly-Asp-Ser(219)-Gly, a tetrapeptide conserved among several animal serine proteases, to Gly-Asp-Arg-Ser-Thr was shown to completely abolish in vitro cleavage. This supports the notion that the capsid is a serine protease. The role of the capsid protease in the processing of the 6K junctions was then investigated by translations of a hybrid polyprotein in which the capsid and most of the p62 sequences are replaced by those of the secretory protein lysozyme. The cleavages and concomitant appearance of the 6K peptide occurred efficiently and were shown to require the presence of membranes. This demonstrates that the capsid protease is not required for those cleavages and suggests that a membrane-associated host protease is responsible for the cleavage.
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PMID:Processing of the Semliki Forest virus structural polyprotein: role of the capsid protease. 355 12

The preferential interactions of lysozyme with solvent components and the effects of solvent additives on its stability were examined for several neutral osmolytes: L-proline, L-serine, gamma-aminobutyric acid, sarcosine, taurine, alpha-alanine, beta-alanine, glycine, betaine, and trimethylamine N-oxide. It was shown that all these substances stabilize the protein structure against thermal denaturation and (except for trimethylamine N-oxide for which interaction measurements could not be made) are strongly excluded from the protein domain, rendering unlikely their direct binding to proteins. On the other hand, valine, not known as an osmolyte, had no stabilizing effect, although it induced a large protein-preferential hydration. A possible explanation is given for the use of these substances as osmotic-pressure-regulating agents in organisms living under high osmotic pressure.
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PMID:The stabilization of proteins by osmolytes. 397 11

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.
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PMID:Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin. 399 69

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.
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PMID:Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae. 400 47


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