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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ovoinhibitor is a serine protease-inhibiting protein that was originally purified from egg whites. It is secreted by the oviduct under the control of estrogen and progesterone and it specifically inhibits serine proteinases such as trypsin and chymotrypsin. During recent attempts to raise monoclonal antibodies (Mabs) against chicken bursa of Fabricius proteins, one Mab was produced that specifically recognized chicken ovoinhibitor. This was the first demonstration of ovoinhibitor in an avian immune organ. We presently report on the expression of an ovoinhibitor-like molecule by the pituitary of the chicken as revealed by immunocytochemistry and RT-PCR. Immunofluorescent dual staining experiments using the mouse anti-ovoinhibitor Mab in conjunction with polyclonal antibodies against various hypophysial hormones revealed partial co-localization of an ovoinhibitor-like molecule with growth hormone (GH), luteinizing hormone (LH), and pro-opiomelanocortin (POMC), in a subset of the respective hormone producing cells. By contrast, no co-localization with prolactin (PRL) could be reliably demonstrated. RT-PCR of hypophysial mRNA using ovoinhibitor gene-specific primers yielded an amplicon that was 20% shorter than predicted on the basis of the published ovoinhibitor sequence. Sequencing revealed that of the represented exons only the central portion was expressed in the pituitary and that both 5' and 3' ends of each exon had been truncated. While expression of ovalbumin-like serine protease inhibitors (serpins) has been previously reported in the rat pituitary, to our knowledge, this is the first report of a Kazal-type serine protease inhibitor in the vertebrate neuroendocrine system.
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PMID:The chicken pituitary expresses an ovoinhibitor-like protein in subpopulations of some, but not all, hormone-producing cell types. 1465 38

The objective of this study was to examine the degradation of short peptides corresponding to modified fragments of human growth hormone-releasing hormone by trypsin. Six analogues of pentapeptide 9-13 of human growth hormone-releasing hormone containing homoarginine, ornithine, glutamic acid, glycine, leucine or phenylalanine residue in position 11, two analogues of hexapeptide 8-13 of human growth hormone-releasing hormone and two analogues of heptapeptide 7-13 of human growth hormone-releasing hormone containing homoarginine or glycine residue in position 11 were obtained. The peptides were subjected to digestion by trypsin and the course of reaction was monitored using HPLC. It was found that the rate of hydrolysis of the Lys(12)-Val(13) peptide bond depends on the amino-acid residue preceding Lys(12). The extension of the peptide chain towards the N-terminus by introduction of consecutive amino-acid residues corresponding to the human growth hormone-releasing hormone sequence accelerates the hydrolysis process. These results may be of assistance in designing new analogues of human growth hormone-releasing hormone, more resistant to the activity of proteolytic enzymes.
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PMID:Tryptic digestion of peptides corresponding to modified fragments of human growth hormone-releasing hormone. 1509 24

We report the morphological characteristics of 30 cases of sclerosing hemangioma (SH) of the lung and explore the histological origin of the major cells in these tumors. In addition to routine light and electron microscopy, immunohistochemistry was performed by using 12 monoclonal primary and 5 polyclonal primary antibodies. These included surfactant protein B (SP-B), thyroid transcription factor-1 (TTF-1), mast cell trypsin, CD68, epithelial antigen markers (high molecular weight cytokeratin, low molecular weight cytokeratin [CK-L], epithelial membrane antigen [EMA], cancer embryonic antigen), mesothelial antigen, neuroendocrine markers (neuron-specific enolase [NSE], chromogranin A, synaptophysin, calcitonin, adrenocorticotropic hormone, human growth hormone [hHG]), vimentin, and CD34. Surface cuboidal cells have short microvilli and have lamellar bodies in their cytoplasm. They can sometimes merge into multinuclear giant cells. Immunohistochemical results showed that these cells are strongly positive for SP-B, TTF-1, CK-L, EMA, and cancer embryonic antigen, whereas polygonal cells, previously also described as round or pale cells, were strongly positive for vimentin and TTF-1, and positive or weakly positive for 2 to 3 kinds of neuroendocrine markers. Sparse neuroendocrine granules and abundant microfilaments were observed in their cytoplasm. Some cell clusters in the solid regions were positive for SP-B and EMA. Mast cells existed sparsely in almost every field. Both cuboidal and polygonal cells were negative to CD34 and mesothelial antigen staining. We conclude that cuboidal cells of SH originate from reactive proliferating type II pneumocytes, which can fuse into multinuclear giant cells. Polygonal cells, as true tumor cells, likely originate from multipotential primitive respiratory epithelium and possess the capability for multipotential differentiation. The antibodies of SP-B, TTF-1, vimentin, and CK-L are very helpful to diagnosis and differential diagnosis of SH.
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PMID:Immunohistochemical and ultrastructural markers suggest different origins for cuboidal and polygonal cells in pulmonary sclerosing hemangioma. 1511 33

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.
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PMID:Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry. 1560 7

We utilized mass spectrometry (MS) and bioinformatics to investigate the proteome of the anterior pituitary gland (AP). Subcellular fractions of APs from 2-month-old male Golden Syrian hamsters were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography MS/MS and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 x g and cytosolic fractions we identified 76, 52 and 52 different proteins, respectively. A total of 145 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the alpha-subunit for the glycoprotein hormones, luteinizing hormone-beta and follicle-stimulating hormone-beta. Groups of other identified proteins included hormone processing, secretion granule associated, non-hormonal endoplasmic reticulum associated, calcium binding, protein kinase C associated histone and non-histone chromosomal material, other RNA-binding, splicing factors, heterogeneous nuclear ribonucleoproteins, helicases, lamins, microfilament associated, microtubule associated, adenosine triphosphate and guanosine diphosphate associated, keratins, lysosomal, ribosomal, enzymes in glycolysis and the tricarboxylic and pentose phosphate paths, glutathione associated, transmethylation, catabolic and unknown protein products as well as blood hemoglobins. Proteins previously not reported in the AP, such as fertility protein SP22, were identified. The proteins identified in the present study form a foundation for defining the proteome in normal adult male AP.
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PMID:Analysis of the golden Syrian hamster anterior pituitary gland proteome by ion trap mass spectrometry. 1569 19

The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.
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PMID:Use of self assembled magnetic beads for on-chip protein digestion. 1610 May 77

We investigated the proteome of the anterior pituitary gland (AP) in a species in which the genome has been sequenced. Subcellular fractions of APs from 2-month-old male mice were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography tandem mass spectrometry and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 g and cytosolic fractions, we identified 49, 36 and 68 different proteins, respectively. A total of 115 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the alpha-subunit for the glycoprotein hormones, and luteinizing hormone-beta. Groups of other identified proteins included hormone-processing, secretion granule-associated, non-hormonal endoplasmic reticulum-associated, calcium-binding, protein kinase C-associated, histones, non-histone chromosomal, other RNA-binding, heterogeneous nuclear ribonucleoproteins, splicing factors, helicases, lamins, ribosomal, microtubule-associated, microfilament-associated, adenosine triphosphate- and guanosine triphosphate-associated, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation, enzymes in glycolysis and the tricarboxylic and urea cycles and the pentose phosphate path, heat-shock, glutathione-associated, peroxidases, ubiquitin-associated, catabolic, protease inhibitors, other, and blood proteins. The 115 proteins reported in this study and the 145 proteins reported in a previous study on the AP of the adult male Golden Syrian hamster are compared and form a foundation for defining the proteome in normal adult male AP.
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PMID:Mouse anterior pituitary gland: analysis by ion trap mass spectrometry. 1610 33

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.
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PMID:Characterization of N-palmitoylated human growth hormone by in situ liquid-liquid extraction and MALDI tandem mass spectrometry. 1742

Equine growth hormone (eGH) has been available since 1998 as an approved drug (EquiGen-5, Bresagen) containing recombinant eGH (reGH). It is suspected of being illegally administered to racehorses in order to improve physical performance and to speed-up wound healing. Thus it may be considered a doping agent which would require a sensitive and reliable method of identification and confirmation in order to regulate its use in racehorses. reGH differs from the native eGH by an additional methionine at the N-terminal (met-eGH) and has never been unambiguously detected in any type of biological matrix at trace concentrations (1-10 microg/L). A plasma sample (4 mL) was treated with ammonium sulfate at the reGH isoelectric point and the pellet was purified by solid-phase extraction. Specific peptides were generated by trypsin digestion and analyzed by LC-MS/MS. The detection limit was 1 microg/L. The method was validated according to European Union regulation (DEC/2002/657/EC) and the Association of Official Racing Chemists (AORC) requirements. Furthermore, it was successfully applied to determining the plasma concentrations of reGH with time using linear ion trap mass analyzer. The presence of this prohibited hormone (reGH) was also successfully detected by triple quadrupole mass spectrometry up to 48 h postadministration of reGH to a horse. The present LC-MS/MS method is the first with adequate sensitivity and specificity for detection of reGH, rbGH, and endogenous eGH. Hence, an efficient analytical tool is proposed as a means to fulfilling the regulation of reGH abuse in the horse racing industry.
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PMID:Identification of recombinant equine growth hormone in horse plasma by LC-MS/MS: a confirmatory analysis in doping control. 1881 43

Human growth hormone (HGH) is a relatively small protein consisting of 191 amino acids and has an average mass of 22,125 amu. The forensic analysis of proteins such as HGH must meet the analytical sufficiency requirements for the laboratory and consists of a binary approach. A suspected sample is analyzed as the whole protein for retention time and mass determination using high performance liquid chromatography equipped with a photodiode array and liquid chromatography mass spectrometry. Further fragmentation of the protein using a proteolytic enzyme adds another dimension to the specificity of the analysis. Porcine trypsin digests proteins in a very predictable manner and yields peptide fragments of the original protein that can be used as a means for fingerprinting the larger biomolecule. In silico, or theoretical, digestion of HGH by trypsin yields 21 peptides ranging in size from 1 to 23 amino acids in length. The larger fragments containing higher numbers of amino acids give more specificity to identifying a protein based on a fragment produced by the digestion of trypsin. Herein, the analysis of HGH using a proteolytic approach is presented that meets the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) recommendations for the identification of unknown substances.
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PMID:Proteolytic-based method for the identification of human growth hormone. 1912 Aug 27


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