Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The C5a molecule is one of two spasmogenic fragments (i.e. C3a and C5a) released from serum components C3 and C5 during complement activation. These fragments are called anaphylatoxins because their ability to stimulate mast cell histamine release, smooth muscle contraction, and increased vascular permeability may lead to a fatal reaction resembling anaphylactic shock in experimental animals. In addition, the C5a molecule, which is a glycoprotein, is perhaps the most potent of all humoral chemoattractants for polymorphonuclear leukocytes. Most of the structural analyses in this study were performed on the desArg 74 form of human C5a (C5adesArg). C5adesArg represents a natural form of C5a that is recovered from activated serum when no inhibitors are added to block the action of serum carboxypeptidase. The complete primary structure of the human C5a polypeptide portion is reported here. A partial characterization of intact human C5a has been previously reported (Fernandez, H. N., and Hugli, T. E. (1976) J. Immunol. 117, 1688--1694). The polypeptide portion of C5a contains 74 amino acids, accounting for a molecular weight of 8,200 while the carbohydrate portion accounts for approximately 3,000. The carbohydrate portion of C5a exists as a single complex oligosaccharide unit attached to an asparagine at position 64. An unusual feature of the C5a molecule is its large content of half-cystine, which accounts for more than 9% of its total residues. Two repeating Cys sequences occur in the linear structure and 6 of the 7 half-cystines in C5a are located at nearly identical positions to those in the human C3a molecule. In fact, sequence similarities between C3a and C5a indicate their common genetic ancestry. The role of C5a and C5adesArg as chemotactic factors prompted comparisons of their structural features with those of the chemotactically active formyl-Met peptides (Schiffman E., Corcoran, B. A., and Wahl, S. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1059--1062). Removal of the COOH-terminal arginyl residue from C5a reduces chemotactic activity; therefore, the terminal portion of this molecule appears to play an active role in stimulating leukocyte migration. Hence the COOH-terminal sequence of C5a was examined for structural similarities to that of the formyl-Met peptides. Since methionine assumes a special functional importance in the formyl-Met peptides, attention is focused on the single methionyl residue in C5a. This methionyl residue, located near the COOH terminus of the molecule, may play an active role in the functional expression of C5a as a chemotactic factor. Although human and pig C3a show a close structural and functional relationship to C5a they lack the ability to excite leukotaxis, and this difference may correlate with the absence of a methionyl residue near the COOH terminus of C3a.
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PMID:Primary structural analysis of the polypeptide portion of human C5a anaphylatoxin. Polypeptide sequence determination and assignment of the oligosaccharide attachment site in C5a. 69 Jan 34

[Asn A21]Insulin is formed as the main product during alkaline saponification of insulin hexamethyl ester. Purification was achieved by gel chromatography followed by ion-exchange chromatography on carboxymethyl cellulose at pH 4 or by preparative isoelectric focusing in a granulated gel over a narrow pH range. Two main products could be isolated. One of them showed the electrophoretic behaviour of insulin (A), whilst the other corresponded to insulin with a blocked carboxyl function (B). Incubation of this product B with carboxypeptidase A liberated only the C-terminal alanine of the B-chain, but not the asparagine of the C-terminus of the A-chain. Chymotryptic digestion of the isolated S-sulfonate A-chain derivative (C) followed by high-voltage electrophoresis confirmed that the carboxyl function of asparagine A21 was blocked. In order to determine the free carboxyl functions of the A-chain derivative C, it was coupled with glycine methyl ester yielding D. Amino acid analysis of the chymotryptic peptides of D showed that the carboxyl functions of glutamic acid A4 and A17 had been free prior to coupling. The amino acid analysis of the enzymatic hydrolysate (subtilisin, aminopeptidase M) of the A-chain derivative C showed an additional peak with an elution position identical to the model compound aminosuccinimide. The biological activity of the [Asm A21[insulin was found to be about 40% in the fat cell test and 13.2 units/mg measured by the mouse convulsion method.
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PMID:[A21-Asparaginimide] insulin. Saponification of insulin hexamethyl ester, I. 83 63

The synthesis by conventional methods of the following three peptides is described: MCD(8-11) Boc-His(Trt)-Val-Ile-Lys(Z) (III) MCD(5-7) Boc-Cys(SiPr)-Lys(Z)-Arg(Tos) (IV) and MCD(1-4) Boc-Ile-Lys(Z)-Cys(Trt)-Asn(Mbh) (V). These peptides are fragments of the mast cell degranulating peptide from bee venom. The purity of the fragments synthesized was examined by thin-layer chromatography, amino acid and elementary analysis. Including the fragments Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z) (I) and Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II), which were described earlier, the synthesis of the mast-cell-degranulating peptide on a polyethylene-asparagine support appears possible.
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PMID:[Basic peptides in bee venom, III. Synthesis of peptide fragments from the sequence of the mast-cell-degranulating peptide (author's transl)]. 85 9

The phytohemagglutinin mitogenic proteins derived from Phaseolus vulgaris comprise a class of five glycoproteins that are isomeric tetramers composed of varying proportions of two different subunits (L and R). Within the native tetramer, the L subunit is a potent leukoagglutinin and mitogen that lacks hemagglutinating properties, whereas the R subunit is a potent hemagglutinin with little or no mitogenic activity. The subunits have been isolated in homogeneous form by isoelectric focusing in 8 M urea. Previous work has shown that they have equal molecular weights and differ in amino-acid sequence from residues 1-7, but are identical in positions 8-24 [(1973) J. Exp. Med. 138, 939-951]. We now report amino-acid composition studies which reveal striking similarities between the subunits. Both lack methionine and cysteine. The twelfth residue in each subunit is a glycosylated asparagine, with the identical carbohydrate composition in each. The last three residues of the subunits, as determined by carboxypeptidase A digestion, are identical. Tryptic peptide mapping of the succinylated phytohemagglutinin subunits reveals a high degree of similarity. We conclude that the substantial difference in biological properties among the tetrameric phytohemagglutinin mitogens is a result of relatively restricted differences in the primary structure of their constituent subunits.
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PMID:Extensive homology between the subunits of the phytohemagglutinin mitogenic proteins derived from Phaseolus vulgaris. 105 14

The combined use in peptide synthesis of the Fmoc-group with methyl, benzyl or p-nitro benzyl esters is not practical because of the elimination of the Fmoc-group under basic conditions and by catalytic hydrogenation. Nevertheless the solution synthesis of peptides requires those combinations in some cases. For this purpose we have investigated enzymatic hydrolysis of some tri and tetrapeptide esters. The hydrolysis were carried out under pH-control. We measured deprotection of the carboxyl group by thermitase, porcine liver esterase, carboxypeptidase A and alpha-chymotrypsin. The main problems are to suppress proteolytic degradation of the peptide bond and to bring the protected peptides into solution. To solve both problems we used dimethylformamide and dimethylsulfoxide as cosolvents. The ratios between esterolytic and proteolytic activity were estimated under various cosolvent concentrations. Advantages of this method are to avoid side reactions of alkaline instable side chains (e.g. asparagine, glutamine), cleavage of base labile protecting groups and racemization by alkaline saponification. The enzymatic deprotection was followed by HPLC, HPTLC and titration. On a preparative scale this method gives good yields and sufficiently pure products.
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PMID:Hydrolysis of peptide esters by different enzymes. 144 67

The structural difference between two forms (basic and acidic) of guinea-pig beta 2-microglobulin (beta 2m) has been established. Both forms are present in urine from inbred guinea-pig strains. The beta 2m forms were each digested with carboxypeptidase Y and carboxypeptidase A contaminated with carboxypeptidase B. Released amino acids were separated from remaining protein, dansylated and analysed by 2-dimensional TLC on polyamide layer sheets. From the results it was concluded that the basic beta 2m form has lysine and the acidic beta 2m form has asparagine as their respective C-terminal amino acids. The acidic form is also 1 amino acid (lysine) shorter than the basic form, which is supported by electrophoretic studies on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the 2 forms of beta 2m in urine from inbred guinea-pig strains 2 and 13, shown by gel filtration and ion exchange chromatography, makes it unlikely that the 2 forms are a result of genetic polymorphism.
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PMID:Structural difference between the two forms of guinea-pig beta 2-microglobulin and their occurrence in inbred guinea-pig strains. 393 25

Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23-9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13mug/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b.
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PMID:The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of the venom of a sea snake Katicauda semifasciata. 466 80

The crystal structure of bovine carboxypeptidase A (Cox) has been refined at 1.54 A resolution using the restrained least-squares algorithm of Hendrickson & Konnert (1981). The crystallographic R factor (formula; see text) for structure factors calculated from the final model is 0.190. Bond lengths and bond angles in the carboxypeptidase A model have root-mean-square deviations from ideal values of 0.025 A and 3.6 degrees, respectively. Four examples of a reverse turn like structure (the "Asx" turn) requiring an aspartic acid or asparagine residue are observed in this structure. The Asx turn has the same number of atoms as a reverse turn, but only one peptide bond, and the hydrogen bond that closes the turn is between the Asx side-chain CO group and a main-chain NH group. The distributions of CO-N and NH-O hydrogen bond angles in the alpha-helices and beta-sheet structures of carboxypeptidase A are centered about 156 degrees. A total of 192 water molecules per molecule of enzyme are included in the final model. Unlike the hydrogen bonding geometry observed in the secondary structure of the enzyme, the CO-O(wat) hydrogen bond angle is distributed about 131 degrees, indicating the role of the lone pair electrons of the carbonyl oxygen in the hydrogen bond interaction. Twenty four solvent molecules are observed buried within the protein. Several of these waters are organized into hydrogen-bonded chains containing up to five waters. The average temperature factor for atoms in carboxypeptidase A is 8 A2, and varies from 5 A2 in the center of the protein, to over 30 A2 at the surface.
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PMID:Refined crystal structure of carboxypeptidase A at 1.54 A resolution. 688 46

The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.
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PMID:Human cathepsin G lacking functional glycosylation site is proteolytically processed and targeted for storage in granules after transfection to the rat basophilic/mast cell line RBL or the murine myeloid cell line 32D. 749 46

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.
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PMID:Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences. 773 67


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