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)

The protease-catalyzed conversion of peptides and proteins produced by recombinant DNA technology is a promising method for large-scale production of peptides including those with non-proteinogenic structural elements. As a model system we have investigated the proteinase-catalyzed modification of a chemically synthesized substance P-precursor. In the precursor peptide the residues of substance P(1-8) were flanked by tripeptide linkers on both sides. In the first step the C-terminal tripeptide amide was replaced by the authentic C-terminal tripeptide amide of substance P via alpha-chymotrypsin-catalyzed transpeptidation. The enzyme simultaneously attacks two peptide bonds in the precursor molecule leading to the formation of several side-products. The desired peptide was obtained with 25% yield. In the second step the other tripeptide linker was selectively and almost quantitatively removed from the N-terminus of the precursor via trypsin-catalyzed hydrolysis. This study demonstrates that substance P can be obtained from an engineered protein by proteinase-catalyzed processing.
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PMID:Proteinase-catalyzed conversion of a substance P-precursor peptide. 138 46

Thyrotrophin-releasing hormone (TRH) and related peptides occur in high concentrations in human semen. TRH derives from a 242-amino acid precursor protein, prepro-TRH, with six repetitive sequences of -Lys-Arg-Gln-His-Pro-Gly-Lys/Arg)-Arg- connected by hydrophobic linking sequences. Antibodies to TRH-Gly (pGlu-His-Pro-Gly), a final precursor for TRH formation, were used to detect this tetrapeptide as well as other prepro-TRH fragments which cross-react with these antibodies. The total TRH-Gly immunoreactivity decreased significantly after vasectomy. The TRH-Gly immunoreactivity in semen increased significantly during in-vitro incubation at 0 or 37 degrees C, to a peak value at 5 h, followed by an exponential decline, with t 1/2 equal to 11 h at 37 degrees C. At 60 degrees C, however, the TRH-Gly immunoreactivity rose continuously, attaining, after 20 h, a level 2.2 times that at the start of the incubation (P less than 0.001). Reversed-phase high pressure liquid chromatography (HPLC) revealed both hydrophobic and hydrophilic TRH-Gly immunoreactive peptides in semen with both classes of peptides increasing significantly with heating to 60 degrees C. Cation exchange chromatography of pooled human semen incubated at 60 degrees C revealed a 4.3-fold increase in a TRH-Gly immunoreactive peak which co-eluted with synthetic TRH-Gly, and a 30% increase in another TRH-Gly immunoreactive peak identified as Glu-His-Pro-Gly. A minor, TRH-Gly immunoreactive peak increased 50-fold (P less than 0.001) during 20 h at 60 degrees C. This material co-eluted with Arg-Gln-His-Pro-Gly which is formed by enzymic cleavage of the paired basic residues flanking this sequence in prepro-TRH. When synthetic Arg-Gln-His-Pro-Gly was incubated with fresh semen at 60 degrees C a rapid conversion of most of this peptide to Glu-His-Pro-Gly, Gln-His-Pro-Gly and TRH-Gly occurred within 30 min. These data are consistent with thermal inactivation of the amidation and degrading enzymes at 60 degrees C while the trypsin-like enzymes which cleave the precursor peptide at the paired basic residues remain relatively unaffected. Because other investigators have found the C-terminal amidating enzymes to be associated with secretory vesicles and to be co-secreted with the vesicular contents, we suggest that secretory epithelia of the male reproductive system secrete TRH and TRH-related precursor peptides along with the alpha-amidating enzymes which continue processing of prepro-TRH in the post-ejaculatory seminal fluid.
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PMID:In-vitro production of precursor peptides for thyrotropin-releasing hormone by human semen. 211 83

Angiotensin converting enzyme (ACE) plays an important role in the regulation of renal blood pressure by the hydrolysis of the inactive precursor peptide angiotensin I to the potent vasopressor angiotensin II. Renal ACE is a surface membrane protein of both endothelium and tubular epithelium. Enzymatically active ACE was isolated from renal homogenates by chromatography using an affinity column constructed by linking an ACE inhibitor, lisinopril, to Affi-Gel 15. Analysis of eluates from this column showed that ACE activity was increased greater than 500-fold. SDS-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 144 kD (mouse) and 149 kD (bovine). N-terminal amino acid sequence analysis revealed: (formula; see text) Though bovine ACE has one additional N-terminal amino acid, these two partial sequences are highly homologous (16 of 20 positions are identical). Mouse ACE was digested with trypsin and the peptides were isolated by reverse phase HPLC. Analysis of the amino acid sequences showed that these tryptic peptides were unique to ACE. Thus, we were able to isolate ACE from bovine and mouse kidneys and show that they had substantial structural homology. They were also quite similar to that from rabbit lung.
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PMID:Partial protein sequence of mouse and bovine kidney angiotensin converting enzyme. 283 38

A novel type of low-molecular-weight growth-inhibitory factor responsible for the density inhibition of tumorigenic V79 Chinese hamster cells has been purified, if not homogenously, by a series of reverse-phase and gel filtration high-performance liquid chromatography. The factor is an acid-stable, heat-labile substance distinct from antiproliferative nucleoside analogues or polyamines and has a molecular weight of approximately 2000. The biological activity of this inhibitor was enhanced nearly 5-fold by trypsin treatment, thereby suggesting that the inhibitor may be a precursor peptide which becomes an oligopeptide with intense biological activity by proteolysis, or that trypsin treatment allows resultant small molecules to efficiently transfer across the cytoplasmic membrane. This inhibitor reversibly inhibits the growth of a broad spectrum of cell types from neoplastic and nonneoplastic cells from various species. These data suggest that this inhibitor is primarily a growth-regulatory molecule common to mammalian cells and may play an important role in regulating growth of both normal and neoplastic cells.
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PMID:Characterization of a low-molecular-weight growth inhibitor formed by density-inhibited, tumorigenic V79 Chinese hamster cells. 337 15

Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence. Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4-->Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it. The Ala-4-->Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide. Several mutations, i.e. Pro-2-->Gly,Pro-2-->Val, Asp-7-->Ala,Lys-9-->Leu,Ser-10-->Ala/Ser-12-->Ala and Val-11-->Asp/Val-13-->Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues. When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e. Phe-18-->Leu,Leu-16-->Lys,Asp-15-->Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.
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PMID:Influence of amino acid substitutions in the nisin leader peptide on biosynthesis and secretion of nisin by Lactococcus lactis. 810 98

Cadherins are Ca(2+)-dependent cell adhesion molecules that mediate cell adhesion by homophilic binding. Structural and functional analysis of the extracellular part of cadherins that mediates this binding has often been hampered by the availability of sufficient amount of protein. Therefore, we have expressed the extracellular region of E-cadherin (uvomorulin) using the baculovirus expression vector system (BEVS). A recombinant baculovirus was generated that encodes the signal peptide, the precursor region and the extracellular part of the mature protein, under the control of the promotor for polyhedrin. Infection of insect cells with recombinant virus led to the expression of about 40 mg of the E-cadherin fragment per 2 x 10(9) infected cells. About half of the protein synthesized was secreted, either as mature protein or in its unprocessed form. The precursor peptide was removed by trypsin treatment in the presence of Ca2+ and recombinant protein was purified to homogeneity. Biochemical characterization of the recombinant protein revealed a high degree of similarity with the mouse wild-type protein. Recombinant protein exhibited the known resistance to trypsin in the presence of Ca2+ and was recognized by two different conformation-sensitive monoclonal anti-E-cadherin antibodies. Rabbit antibodies made against the recombinant protein recognized E-cadherin from different species. In spite of the high degree of structural resemblance recombinant E-cadherin was not able to inhibit E-cadherin mediated cell-cell adhesion.
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PMID:Characterization of recombinant E-cadherin (uvomorulin) expressed in insect cells. 814 91

Trypsin is the most abundant protease in Crustacea. This enzyme was purified from the digestive gland of Penaeus vannamei, revealing three major isoforms (molecular weights 31-32 kDa) and several minor components. Five cDNAs encoding five isoforms of trypsin were detected by two successive screenings of an amplified cDNA library from the digestive gland of P. vannamei. The longest isolated and sequenced cDNA encoded a preproenzyme of 255 amino acids containing a putative precursor peptide of 14 residues and a highly hydrophobic signal sequence of 14 amino acids. Amino acid sequence alignments revealed a high degree of identity between the trypsin from P. vannamei and that from crayfish (74%) and an equal level of sequence similarity to that from mammals and insects (approximately 40). Dot blot hybridization and subsequent analysis of the variation in trypsin-specific activities revealed that mRNA expression is at a maximum during early premoult (D1), declining sharply in late premoult (D2-D3). The specific activity of trypsin also followed this pattern, suggesting the regulation of trypsin biosynthesis is, at least in part, transcriptional. The characterization of trypsin cDNA from P. vannamei provides the first description of a putative zymogen sequence in a crustacean species, enabling us to elucidate the regulatory mechanism of trypsin synthesis in these important marine organisms.
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PMID:Molecular cloning and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): use in assessing gene expression during the moult cycle. 869

Trypsin was purified from crayfish, Pacifastacus leniusculus, hepatopancreas, and the gene that encoded this enzyme was cloned from a hepatopancreas cDNA library. Crayfish trypsin is synthesized as a zymogen according to the sequence of the putative precursor peptide. The authenticity of the trypsinogen is supported by the deduced amino acid sequence and confirmed by the N-terminal amino acid sequence of the mature protein. The enzyme has features characteristic of a trypsin, such as a specific binding pocket. Sequence comparison shows that crayfish trypsin is similar to those of other species, with the exception that six cysteine residues present in vertebrates are missing. Some structural characteristics, such as the length of the signal peptide and a calcium binding site, are similar to bacterial trypsin.
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PMID:Trypsin from Pacifastacus leniusculus hepatopancreas: purification and cDNA cloning of the synthesized zymogen. 1035 37

A large 40-residue precursor peptide (propeptide 5) was synthesized by linking together four designed anticancer peptide analogs to the neuropeptides: vasoactive intestinal peptide, somatostatin, bombesin and substance P, using enzyme cleavable lysyl-lysine linkers. On incubation with the enzyme trypsin, propeptide 5 was cleaved in a sequence-specific manner at the lysyl-lysine residues in the linker to release the individual peptide fragments which were identified by LC-MS. Another precursor peptide (propeptide 5a), consisting of two of the peptide analogs linked through lysyl-lysine linker, was also preferentially cleaved at the Lys-Lys site on incubation with the enzyme trypsin. Propeptide 5 showed potent anticancer activity, both in vitro and in vivo, which was greater than that of the individual component peptides. The enhanced activity suggests that the propeptide is possibly cleaved in the biological system at the lysyl-lysine site to yield the individual peptide analogs, which together show a synergistic effect. On the basis of these experimental findings, it can be concluded that pairs of basic amino acids such as Lys-Lys can be used as facile linkers for delivering multiple biologically active peptides.
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PMID:Delivering multiple anticancer peptides as a single prodrug using lysyl-lysine as a facile linker. 1755 67

A common polymorphism at codon 72 of human TP53 gene determines a proline to arginine aminoacidic substitution within the proline-rich domain of p53 protein. The two resulting isoforms (p53P(72) and p53R(72)) are different from a biochemical and biological point of view and many reports suggest that they can modulate individual cancer susceptibility and overall survival. In the attempt to explain the observed biological differences, we characterized the two isoforms by mass spectrometry and circular dichroism (CD) to evaluate the possible alteration in the secondary structure of p53 introduced by this polymorphism. Recombinant human p53R(72) and p53P(72) were produced by using E. coli expression system then purified by chromatography (affinity chromatography and RP-HPLC), and the whole proteins identified by HPLC-ESI-IT and MALDI-TOF analysis. A bottom-up approach, using both MALDI-TOF and HPLC-ESI-QTOF analysis, was then adopted to obtain the sequence information on the two p53 isoforms. To this purpose, peptide maps were obtained by trypsin proteolysis on the two p53 isoforms. The two isoforms proteolytic digests were separated by LC and subsequent mass spectrometry analysis of both entire and fragmented peptides was performed. In particular, precursor peptide ions obtained by ESI were subjected to collision by the triple quadrupole and TOF separation, allowing us to determine the isoforms aminoacidic peptide sequence by peptide ladder sequencing. Because of the presence of arginine, a selective trypsin proteolytic cleavage at R(72), giving rise to two selective shorter peptides, occurred in p53R(72), but was missing in the case of p53P(72) trypsin digest, in which an uncleaved longer peptide was instead identified. Upon primary structure confirmation, the two p53 isoforms were studied by CD in order to investigate the experimental variables, which affect ordered secondary structure adoption. CD analysis indicated that the two isoforms are not structurally different, thus allowing us to exclude that the observed biological differences can be due to a different conformation of the two isoforms introduced by this polymorphism. Furthermore, these studies establish a mass spectrometry method to identify the two isoforms that can be useful for future interactome studies and cancer drug discovery.
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PMID:Structural characterization of p53 isoforms due to the polymorphism at codon 72 by mass spectrometry and circular dichroism. 2042 42


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