Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prostate-specific antigen (PSA) is one of the three most abundant prostatic-secreted proteins in human semen. It is a serine proteinase that, in its primary structure, manifests extensive similarities with that of the Arg-restricted glandular kallikrein-like proteinases. When isolated from semen by the addition of chromatography on aprotinin-Sepharose to a previously described procedure, PSA displayed chymotrypsin-like activity and cleaved semenogelin and the semenogelin-related proteins in a rapid and characteristic pattern, but had no trypsin-like activity. About one third of the purified protein was found to be enzymatically inactive, due to cleavage carboxy-terminal of Lys145. Active PSA formed SDS-stable complexes with alpha 1-antichymotrypsin, alpha 2-macroglobulin-analogue pregnancy zone protein. PSA formed inhibitory complexes with alpha 1-antichymotrypsin at a molar ratio of 1:1, a reaction in which PSA cleaved the inhibitor in a position identical to that reported from the reaction between chymotrypsin and alpha 1-antichymotrypsin. The formation of stable complexes between PSA and alpha 1-antichymotrypsin occurred at a much slower rate than that between chymotrypsin and alpha 1-antichymotrypsin, and at a similar or slightly slower rate than that between PSA and alpha 2-macroglobulin. When added to normal blood plasma in vitro, active PSA formed stable complexes both with alpha 2-macroglobulin and alpha 1-antichymotrypsin. This complex formation may be a crucial determinant of the turnover of active PSA in intercellular fluid or blood plasma in vivo.
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PMID:Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. 170 14

N-terminally extended forms of 5-oxoprolylglutamylprolinamide (Glp-Glu-Pro-NH2), a thyrotropin-releasing-hormone(TRH)-like peptide associated with the male reproductive system, were isolated from human semen by gel exclusion on Sephadex G50, ion-exchange chromatography on SP-Sephade C25 and QAE-Sephadex A25, and by HPLC. The peptides were located by trypsin-catalysed release of their C-terminal fragments which were detected by RIA with a TRH-specific antibody. A series of overlapping peptides containing 16, 18, 22 and 25 residues was obtained in homogeneous form and their sequences were determined by automatic Edman degradation. The peptides all terminated in -Lys-Gln-Glu-Pro-NH2 and were found to correspond to sequences occurring between residues 350-374 of semenogelin, a protein present in human semen. In semenogelin, however, the Gln-Glu-Pro sequence is followed by tryptophan and not glycine which is normally essential for formation of the C-terminal amide group. Model experiments with the synthetic peptide Glp-Glu-Pro-Trp showed that under a range of experimental conditions the tetrapeptide did not undergo conversion to Glp-Glu-Pro-NH2. This would indicate that the tripeptide and its extended forms are generated from a precursor that is related to semenogelin but in which Trp375 is replaced by glycine.
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PMID:Isolation and identification of N-terminally extended forms of 5-oxoprolylglutamylprolinamide (Glp-Glu-Pro-NH2), a thyrotropin-releasing-hormone (TRH)-like peptide present in human semen. 844 63

Two peptides with similar structures to thyrotropin-releasing hormone (TRH), pGlu-Glu-Pro amide and pGlu-Phe-Pro amide, have been identified in human seminal fluid and it has been shown that one of these peptides, pGlu-Glu-Pro amide, has the ability to increase the capacitation of sperm cells, consistent with a role in fertility. In order to select a species in which there is a high degree of expression of the genes that code for 'TRH-like' peptides, we have determined the levels of these peptides in the prostate, pancreas and thyroid of a range of species including rat, rabbit, ox, marmoset, macaque and man. The peptides were extracted from the tissues and purified before determination by RIA with TRH antibody. In addition, trypsin digestion and TRH RIA was used to investigate the presence of N-extended forms. The highest concentrations of TRH-immunoreactive peptides were found in the tissues of the marmoset, Callithrix jacchus. Ion-exchange chromatography demonstrated that marmoset thyroid contained principally authentic TRH, the pancreas contained both TRH and TRH-like peptides while the prostate contained TRH-like peptides alone. Further purification by HPLC showed that the main TRH-immunoreactive peptide in marmoset prostate was pGlu-Glu-Pro amide and a second component was identified as pGlu-Phe-Pro amide. The results indicate that the biosynthesis of these peptides could be studied to advantage in the marmoset. The biosynthetic precursors of the TRH-like peptides have not been identified. To examine whether pGlu-Glu-Pro amide might originate from semenogelin, we determined the sequence of semenogelin in the marmoset. It exhibited a high degree of homology with human semenogelin-I, but in place of the Lys-Gln-Glu-Pro sequence that might give rise to pGlu-Glu-Pro amide, marmoset semenogelin possessed the sequence Ser-Gln-Asp-Gln which cannot serve as a precursor for a TRH-like peptide. Further evidence was obtained by Northern blot analysis of a range of marmoset tissues. The results showed that semenogelin is not present in marmoset prostate. It is concluded that pGlu-Glu-Pro amide originates from a precursor distinct from semenogelin, both in marmoset and in man.
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PMID:Molecular cloning in the marmoset shows that semenogelin is not the precursor of the TRH-like peptide pGlu-Glu-Pro amide. 974 47

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.
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PMID:Purification and characterization of active recombinant rat kallikrein rK9. 1141 Feb 95

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first "washing" step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.
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PMID:Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science. 2284 16