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

A manual high-sensitivity sequencing method is described, in which 4-NN-dimethylaminoazobenzene 4'-isothiocyanate is used for the stepwise degradation of amino acid residues from the peptides. The 4-NN-dimethylaminoazobenzene 4'-thiazolinones of amino acids that were released, after conversion into their thiohydantoin derivatives, were identified by t.l.c. on polyamide sheets. This new method is simple and sensitive, and requires only 2-10nmol of peptides or proteins for extended sequence analysis. The method was tested on the sequence analysis of a hexapeptide (Leu-Trp-Met-Arg-Phe-Ala), bradykinin, glucagon and native lysozyme. Results show that the proposed procedure is a sensitive method for the sequence determination of short peptides as well as for the partial sequence determination of intact proteins.
 ...
PMID:High-sensitivity sequence analysis of peptides and proteins by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. 40

A chemotactic peptide CHO.Met.Leu.Phe.OH has been synthesized classically using the mixed anhydride procedure. The formyl group was introduced by coupling formic acid in the presence of dicyclohexylcarbodiimide to the partially protected triptide. The final product was obtained by treatment of the intermediate CHO.Met.Leu.Phe.OBzl with hydrogen fluoride. The ED50 of the peptide in the Boyden chamber assay was 7 x 10(-11) M; in the lysozyme release assay 2.4 x 10(-10) M and in the beta-glucuronidase release assay 2.6 x 10(-10) M. In a radioreceptor assay the ID50 of the peptide was 3.3 x 10(-10) M.
 ...
PMID:Synthesis of Nalpha-formyl-Met-Leu-Phe-OH: an inducer of chemotaxis in peritoneal polymorphonuclear neutrophils. 42 7

1. Previous reports from this laboratory have shown that both Lys-33 and Lys-116 are parts of an antigenic site in native lysozyme. Similar studies of tyrosine derivatives indicated that one or both of Tyr-20 and Tyr-23 are located in or very close to an antigenic site in lysozyme. The site, which was located around the disulphide bridge 30-115, was recently shown unequivocally to include the residues Tyr-20, Arg-21, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. This was confirmed by the ;surface-simulation' synthetic approach that we have recently developed, in which the foregoing eight surface residues were directly linked via peptide bonds, with intervening spacers where appropriate, into a single peptide. The peptide does not exist in native lysozyme, but simulates a surface region of it. 2. In the present work several surface-simulation peptides were synthesized representing various parts of the region, to determine the minimum structural feature that retains full antigenic reactivity and to investigate if the spatially constructed antigenic site has a preferred direction. 3. The peptide Lys-Asn-Arg-Gly-Phe-Lys exhibited a remarkable inhibitory activity towards the immune reaction of lysozyme and accounted entirely for the maximum expected reactivity of the site in the native protein (i.e. about one-third of the total lysozyme reactivity). An immunoadsorbent of the peptide bound about one-third of the total antibody to lysozyme. 4. The residues Tyr-20 and Arg-21 are not part of the site. The previously reported immunochemical effect observed on nitration of Tyr-20 was due to a deleterious ionic effect exerted by the modified tyrosine residue on the adjacent Lys-96, which is in an entirely different antigenic site of lysozyme. Thus the modification of Tyr-20 impairs the reactivity of an adjacent antigenic site, even though the residue itself is not part of a site. The conformational and immunochemical implications of this finding are discussed. 5. The antigenic site therefore comprises the five spatially adjacent residues Lys-116, Asn-113, Arg-114, Phe-34, Lys-33. The antigenic site exhibited a preferred direction (Lys-116 to Lys-33), since the reverse surface-simulation synthetic sequence was immunochemically inefficient. The site describes a line which circumscribes part [2.1nm in C((alpha))-C((alpha)) distance from Lys-116 to Lys-33] of the surface of the molecule.
 ...
PMID:Enzymic and immunochemical properties of lysozyme. Accurate definition of the antigenic site around the disulphide bridge 30-115 (site 3) by 'surface-simulation' synthesis. 60 22

The amino acid sequence of lysozyme c from chachalaca egg white was determined. Like other bird lysozymes c, that of the chachalaca has 129 amino acid residues. It differs from other avian lysozymes c by 2 to 31 amino acid substitutions as well as by being devoid of phenylalanine. It contains substitutions at 9 positions which are invariant in the other 7 bird lysozymes of known sequence. Although the chachalaca is classified zoologically in the order Galliformes, which includes chickens and other pheasant-like birds, its lysozyme differs more from those of pheasant-like birds than do the lysozymes c of ducks. Phylogenetic analysis of the sequence comparisons confirms that the lineage leading to chachalaca lysozyme c separated from that leading to other galliform lysozymes c before the duck lysozyme c lineage did. This indicates a contrast between protein evolution and evolution at the organismal level. Immunological comparison of chachalaca lysozyme c with other lysozymes of known sequence provides further support for the proposal that immunological cross-reactivity is strongly dependent on degree of sequence resemblance among bird lysozymes.
 ...
PMID:Amino acid sequence and immunological properties of chalchalaca egg white lysozyme. 94 Jan 73

In previous reports from this laboratory it was shown that an antigenic reactive site resides around the sequences 6-13 and 126-128 linked by the disulfide 6-127. The present work provides a strong support for the location of the reactive site by an independent approach. It also determines accurately the boundaries of the reactive site. 1. The two methionine residues in lysozyme were carboxyethylated by reaction with beta-propiolactone. The electrophoretically homogeneous derivative had no other modified amino acids and showed no conformational changes, relative to native lysozyme, as determined by ORD and CD measurements. However, it exhibited a slight increase in disulfide reducibility relative to native lysozyme and its lytic activity was about half that of native lysozyme, probably as a result of the slight conformational change. On the other hand, the antigenic reactivity of the derivative was equal to that of native lysozyme with several goat and rabbit antisera to lysozyem. It was therefore concluded that methionines 12 and 105 were not parts of antigenic reactive sites in native lysozyme. 2. Eleven peptides, corresponding to various sequences on the two sides of the disulfide 6-127 (i.e. two groups of peptides) were synthesized, purified and characterized. One group (A) of peptides comprised sequences 3-14, 5-14, 6-14, 5-13, 5-12 and an analog of sequence 5-14 in which methionine 12 is replaced by glycine. The second group (B) of peptides comprised sequences 125-129, 125-128, 126-128, 127-128, and 125-127. From groups A and B, nine disulfide-containing peptides (see Fig. 2) were synthesized, purified, characterized and their immunochemical interactions with antisera to native lysozyme studied. Towards each of the antisera studied here, Phe-3, Gly-4, Arg-5, Arg-125 and Leu-129 were not essential parts of the reactive site. On the other hand, Arg-14, Lys-13, Gly-126 and with some antisera Arg-128 were each critical for the reactivity of the site. Peptides from group A alone or group B alone did not inhibit the reaction of lysozyme with its antisera, confirming our previous findings that the integrity of the disulfide bond is essential for bringing the two distant (in sequence) parts of the site together. Finally, replacement of Met-12 by glycine did not influence the immunochemical reactivity of the site, confirming the above conclusion that neither of the two methionine residues takes part in interaction of lysozyme with its antibodies. An accurate delineation of the antigenic reactive site is, therefore derived here and its shape in the three-dimensional structure of native lysozyme is described.
 ...
PMID:Enzymic and immunochemical properties of lysozyme. XIII. Accurate delineation of the reactive site around the disulfide 6-127 by immunochemical study of beta-propiolactone lysozyme derivative and of synthetic disulfide peptides. 94 82

We have previously shown that an antigenic site in native lysozyme resides around the disulphide bridge 30-115 and incorporates Lys-33 and Lys-116 and one or both of Tyr-20 and Tyr-23. These residues fall in an imaginary line circumscribing part of the surface of the molecule and passing through the spatially adjacent residues Tyr-20, Arg-21, Tyr-23, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. The identity of the site was confirmed by demonstrating that the synthetic peptide Tyr-Arg-Tyr-Gly-Lys-Asn-Arg-Gly-Phe-Lys (which does not exist in lysozyme but simulates a surface region of it), and an analogue in which glycine replaced Tyr-23, possessed remarkable immuno-chemical reactivity that accounted entirely for the expected reactivity of the site in native lysozyme. Tyr-23 is not part of the site, and its contribution was satisfied by a glycine spacer. The novel approach presents a powerful technique for the delineation of antigenic (and other binding) sites in native proteins and confirms that these need not always comprise residues in direct peptide linkage.
 ...
PMID:Delineation of the third antigenic site of lysozyme by application of a novel 'surface-simulation' synthetic approach directly linking the conformationally adjacent residues forming the site. 99 47

The first derivatives of difference absorbance spectra of several proteins were measured to examine the applicability of this technique as a tool to investigate state changes of phenylalanine residues in proteins. It was found by this technique that phenylalanine residues in insulin and those in lysozyme are exposed to more aqueous environment by denaturation with guanidine hydrochloride. Heat denaturation of collagen caused similar changes of some of its phenylalanine residues. It was thus demonstrated that difference-derivative absorbance spectrophotometry gives the information about state changes of phenylalanine residues in native proteins, which are hardly detected by common difference spectrophotometry.
 ...
PMID:Difference-derivative absorbance spectrophotometry as a technique to measure state changes of phenylalanine residues in proteins. 116 78

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.
 ...
PMID:The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents. 120 14

24 di-, tri-, and tetrapeptides have been synthesized as a start of a systematic study of the structural requirements for chemotactic activity and lysosomal enzyme-releasing ability in rabbit neutrophils. All but two of them are N-formyl methionyl peptides. Using the method of Zigmond and Hirsch (10), two representative peptides, F-Met-Leu-Phe and F-Met-Met-Met, were shown to stimulate directed, as well as, random locomotion; thus, they were truly chemotactic. The various peptides showed a wide spread in activity. F-Met-Leu-Phe, the most active peptide studied, had an ED50 for induced migration of 7 X 10(-11) M and for lysozyme and beta-glucuronidase release of 2.4 X 10(-10) M and 2.6 X 10(-10) M, respectively; the least active, Met-Leu-Glu was 26 million times less active in these respects. The relation of activity to structure is exceedingly specific, very small changes in structure making large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on its constituent amino acids but on the position of the amino acid in the peptide chain. Most striking in this last regards is the high activity conferred by phenylalanine when it is in the carboxyl terminal position of a tripeptide, whereas, as the second amino acid from the NH2 terminal end whether in a tripeptide or a dipeptide, it contributes no more to the activity than other amino acids with hydrophobic side chains such as leucine or methionine. The high activity and the specificity and nature of the structural requirements strongly suggest that the primary interaction of peptide and neutrophil leading to either chemotaxis or lysosomal enzyme release is a binding of the peptide with a stereospecific receptor on the neutrophil surface. Whether all chemotactic factors act through the same receptor is not known. An essentially exact correlation exists between the concentrations of the various synthetic peptides required to induce migration and their ability to induce release of lysozyme or beta-glucuronidase. This implies that these two neutrophil functions are triggered by teh same primary interaction; possibly, the binding of the peptides to the same putative receptor. A higher concentration of a given peptide is required to stimulate lysosomal enzyme release than a corresponding migratory response. A slightly but significantly higher concentration of peptide is required to induce beta-glucuronidase secretion than lysozyme release.
 ...
PMID:The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils. 126 85

An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml. Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:A rapid microtiter plate method for the detection of lysozyme release from human neutrophils. 131 27


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>