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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)

The three-dimensional structures of apo- and holomutant human lysozymes (D86/92 lysozyme), in which a calcium binding site was designed and created for enhancing molecular stability by replacing both Gln86 and Ala92 with aspartic acids, were refined at 1.8-A resolution by x-ray crystallography. The overall structures and crystallographic thermal factors of all three proteins, the apo-, holo-D86/92, and the wild-type human lysozymes, were essentially identical; these results showed that the introduction of the calcium binding site did not affect either the overall structure or molecular rigidity of the proteins. However, structure analyses of the apo-D86/92 lysozyme revealed that the mutations affected the side chain conformation of residue 86 and hydrogen networks between the protein and the internal solvent molecules. In the structure of the holo-D86/92 lysozyme, seven oxygen ligands formed a slightly distorted pentagonal bipyramid around the calcium ion, indicating that the coordination around the calcium ion was quite similar to that in baboon alpha-lactalbumin. The pentagonal bipyramid coordination could be one of the most widely found and appropriate calcium binding schemes in proteins.
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PMID:Crystal structures of the apo- and holomutant human lysozymes with an introduced Ca2+ binding site. 193 16

A three-disulphide derivative of hen egg-white lysozyme was made by selective reduction and carboxymethylation of one of the four original disulphide bridges. N-Terminal sequencing and two-dimensional 1H-n.m.r. spectroscopy revealed that the disulphide bridge linking cysteine residues 6 and 127 had been modified and that the three remaining disulphide bonds were native-like in nature. Analysis of COSY and NOESY spectra indicated that the three-disulphide lysozyme (CM6.127-lysozyme retains the same secondary and tertiary structure as its four-disulphide counterpart; its stability to pH and temperature is, however, dramatically decreased. N.m.r. spectroscopy was used to characterize the thermal folding and unfolding transition of CM6.127-lysozyme. Not only is the transition still a highly co-operative event, but the enthalpy change associated with folding and unfolding resembles that of intact lysozyme when their differences in thermal stability are taken into consideration. The significance of these results in terms of the folding process of lysozyme is discussed. By contrast with authentic lysozyme, CM6.127-lysozyme was found to exist in an unfolded state at pH 2 at room temperature. N.m.r. spectroscopy and c.d. were used to characterize this state. Unlike their homologous relative, alpha-lactalbumin, which exists in a partially folded molten globule state under these conditions, only residual non-native-like structure persists in the acid-unfolded state of CM6.127-lysozyme. These results indicate that the difference in folding behaviour of lysozyme and alpha-lactalbumin cannot be accounted for simply by their differences in thermal stability.
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PMID:A three-disulphide derivative of hen lysozyme. Structure, dynamics and stability. 198 84

Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.
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PMID:Demonstration by NMR of folding domains in lysozyme. 200 Jan 38

The previously predicted structures of human alpha-lactalbumin by homology with hen egg white lysozyme by an automatic method, after the alignment stage, are compared to the X-ray determined structure of baboon alpha-lactalbumin. The root mean square by rotation method (RMSR) deviations for 122 C-alpha atoms between the two models and the X-ray structure are 2.0 A and 2.3 A. The RMSR deviations for all atoms, except for differences in human and baboon sequences, are 2.8 A and 3.1 A. If the flexible C-terminus (residues 112-122) are removed then these RMSR deviations are reduced to 2.4 A and 2.3 A respectively. These results are consistent with the fact that the RMSR deviation between the human and baboon X-ray structures increases from residue 112 onwards and is conformationally flexible.
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PMID:Comparison of the X-ray structure of baboon alpha-lactalbumin and the tertiary predicted computer models of human alpha-lactalbumin. 209 82

Spectroscopic methods have shown that Ca2+ chelators interact with Ca2(+)-binding proteins. These spectral alterations have been interpreted as evidence for the binding of chelator by the proteins. We show by direct examination of EDTA interaction with calmodulin and alpha-lactalbumin that these proteins repel EDTA rather than bind it. The repulsion is reduced by increased salt concentration but is unaffected by Ca2+ binding to the proteins. The acidic protein, alpha-lactalbumin, repells the negatively charged EDTA and inorganic phosphate whereas the basic protein, lysozyme, repells the positively charged spermine. Thus, spectroscopic changes induced by negatively charged Ca2+ chelators on negatively charged Ca2(+)-binding proteins are due to electrostatic repulsion, and not to binding. These observations underscore the possible pitfalls of using spectroscopic methods alone to analyze protein-ligand interactions.
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PMID:Electrostatic repulsion between molecules of like charge can be misinterpreted as binding. 212 11

The complete amino-acid sequence of pig alpha-lactalbumin has been determined. It was obtained by microsequencing of the native protein and the peptides derived after tryptic or cyanogen bromide cleavage. The tryptic peptides were separated by a rapid microbore HPLC method. Pig alpha-lactalbumin is 122 amino acids long and differs from the bovine homologue by 26 exchanged residues. Of the two prolines present in bovine alpha-lactalbumin, one has been deleted in the pig structure. All previously sequenced alpha-lactalbumins have shown glutamic acid at position 49, which is known to be the active site in the homologous lysozyme c structure. This residue is replaced by phenylalanine in pig alpha-lactalbumin indicating that the pig protein is the first alpha-lactalbumin with complete loss of all lysozyme functional residues.
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PMID:The complete primary structure of alpha-lactalbumin isolated from pig (Sus scrofa) milk. 222 64

1. Both alpha-lactalbumin and beta-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to alpha-lactalbumin and beta-lactoglobulin, respectively. 2. alpha-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline alpha-lactalbumin was found. 3. Feline beta-lactoglobulin-like proteins produced complex electrophoretic patterns that appear to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. The identifications of the proteins as alpha-lactalbumin, beta-lactoglobulin and lysozyme were confirmed by determination of N-terminal amino acid sequences.
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PMID:Feline whey proteins: identification, isolation and initial characterization of alpha-lactalbumin, beta-lactoglobulin and lysozyme. 234 34

Six cases of adenoid cystic carcinoma (ACC) of the breast were reviewed. Immunohistochemical studies were carried out for actin, S-100 protein, EMA, keratin, CEA, vimentin, NSE, alpha-lactalbumin, and lysozyme. Fine needle aspiration biopsy smears of five patients were also reexamined. Patients were treated by tumorectomy, quadrantectomy, or modified radical mastectomy. Axillary dissection was carried out in five cases, with negative lymph nodes in all. Five patients are alive without evidence of disease from 1 year 10 months to 13 years 4 months following surgery. One patient died 7 1/4 years after mastectomy, without evidence of disease. Histologically, a diagnostic biphasic cellular pattern was seen in all cases. In addition, several unusual features were encountered in some cases: squamous metaplasia, stromal myxoid pseudocartilaginous foci, and well-formed neoplastic ducts. Actin and/or S-100 protein were variably positive in all cases. The reaction was usually present in occasional basaloid cells predominantly at the periphery of neoplastic structures. Keratin, EMA, and CEA immunostaining disclosed ductal type cells in all cases. Vimentin was positive in four cases, usually in many basaloid cells. Aspiration cytology was suspicious in two cases and yielded a definitive diagnosis of ACC in three cases. Cytologic diagnosis was based on cellular morphology and on the presence of characteristic globoid structures. Immunohistochemical results show that in ACC dual myoepithelial-ductal differentiation occurs but is relatively limited. Most of the tumor cells are not differentiated ("indifferent" cells) and often express strong vimentin positivity. Such cells are regarded as precursor cells for either differentiated element. Unusual metaplastic changes in breast ACC suggest a possible relation with pleomorphic adenoma-type tumors, and this might be of prognostic significance.
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PMID:Adenoid cystic carcinoma of the breast: a histologic, cytologic, and immunohistochemical study. 247 45

We have isolated two overlapping recombinant lambda-phage clones from a genomic lambda-EMBL3 library containing 25 kb of the human lysozyme gene region. Furthermore a full-lenght human lysozyme cDNA clone of 1.5 kb was isolated from a human placenta cDNA library. Nucleotide sequences of the entire structural gene and the cDNA clone were determined. The human lysozyme gene spans 5856 bp and its sequence organization with four exons and three introns is homologous to the chicken lysozyme gene and the human alpha-lactalbumin gene. Human and chicken lysozyme genes differ mainly in the size of their introns and 3' non-coding region. Four Alu repetitive elements were found in the human lysozyme gene, one in each intron and one on the fourth exon. Lysozyme transcripts of 1.6 kb and 0.6 kb in size were detected in human myeloid cell lines U-937, HL-60 and THP-1 and surprisingly in human hepatoma cell lines HepG2 and Hep3B. The lysozyme gene locus was assigned to human chromosome 12 by hybridization to a panel of DNAs from human-rodent somatic cell hybrids.
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PMID:The human lysozyme gene. Sequence organization and chromosomal localization. 254 58

In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation. Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1). Only one E2 appears to function in ubiquitin-dependent protein degradation. The others have been postulated to function in regulatory ubiquitin conjugation. We have purified and characterized a previously undescribed E2 from rabbit reticulocytes. E2(230K) is an apparent monomer with a molecular mass of 230 kDa. The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates. Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein. E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation. E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation. Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C). E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C). Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds. Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible. We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite.
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PMID:A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties. 255 69


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