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)

Trypsin has been isolated and purified from the digestive glands of the slipper lobster, Thenus orientalis. It is a glycoprotein with a molecular mass of approximately 35 kDa as judged by both SDS-PAGE and gel filtration. The N-terminal amino acid sequence has strong homology to crustacean trypsins. This is confirmed by the cross-reaction of crustacean trypsins with antibodies to the T. orientalis enzyme. Despite a 40% identity with the bovine trypsin N-terminal sequence, there was no cross-reaction with the mammalian serine proteases. The optimum kcat and kcat/Km values for N-alpha-benzoylarginine-p-nitroanalide were 0.91 s-1 and 9.7 x 10(3) M-1 s-1, respectively, with this specificity constant being lower than those reported for other crustacean trypsins. Inhibition studies indicated the presence of serine and histidine at the active site and pKa of the catalytic histidine residue was found to be 5.7 in the free enzyme and 4.7 in the Michaelis complex.
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PMID:Isolation and characterization of a trypsin from the slipper lobster, Thenus orientalis (Lund). 750 56

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.
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PMID:Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A. 759 1

Membrane vesicles from rat brain have been subjected to trypsin treatment in the absence and presence of substrates of the (Na+ + K+)-coupled L-glutamate transporter GLT-1. The fragments of this transporter have been detected upon immunoblotting employing several antibodies raised against sequences from this transporter. At the amino terminus, initially a fragment of an apparent molecular mass of 30 kDa is generated. This fragment is subsequently cleaved to one of 16 kDa. The generation of these bands is greatly inhibited in the presence of lithium. Moreover, lithium abolishes the positive cooperative activation of the transporter by sodium. The generation of the 30- and 16-kDa fragments is accelerated in the presence of L-glutamate and other transportable analogues, provided sodium is present as well. The 30-kDa fragment also contains an epitope from the loop connecting the putative membrane-spanning alpha-helices 3 and 4. This epitope, in contrast with the amino-terminal one, is destroyed with time. The carboxyl-terminal epitope is predominantly located on a 43-kDa fragment which is slowly converted to one of 35 kDa. This conversion is not inhibited by lithium. It is, however, stimulated by L-glutamate and other transportable analogues, but only in sodium-containing media. Potassium also stimulates this conversion regardless of the presence of L-glutamate. The stimulation of generation of amino- and carboxyl-terminal fragments by L-glutamate is not mimicked by the nontransportable analogue dihydrokainate. However, the analogue blocks the stimulation exerted by L-glutamate. In addition to new experimental information on the transporters topology, our observations provide novel information on the function of the GLT-1 transporter. Although lithium by itself does not sustain transport, it may occupy one of the sodium sites and be transported. Furthermore, the transporter-glutamate complex appears to exist in at least two states. After the initial binding (suggested to be important for the decay of synaptic glutamate), it undergoes a conformational change which represents, or is tightly associated with, the transport step.
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PMID:Conformational changes monitored on the glutamate transporter GLT-1 indicate the existence of two neurotransmitter-bound states. 762 23

Analysis of amyloid fibril material associated with familial amyloidotic cardiomyopathy revealed that it contains a mixture of transthyretin-related polypeptides. The major protein band in SDS/polyacrylamide gel corresponding to a molecular mass of 14.5 kDa, consists of transthyretin fragments starting at positions 46, 49 and 59, the latter not previously identified, and one blocked fragment derived from the N-terminal part of transthyretin. In reverse-phase HPLC, the major fragment recovered was that starting at Thr49, indicating a trypsin-like cleavage (Lys at position 48). Two minor bands, corresponding to 17 kDa and 35 kDa, contained proteins with blocked N-termini, and migrated as monomeric and dimeric transthyretin, respectively. A 13-kDa protein band was found to contain transthyretin with a ragged N-terminus, mainly starting at positions 2 and 5. Three more bands, corresponding to 10, 25 and 29 kDa, consist of transthyretin molecules with blocked N-termini and most likely of aggregates of truncated molecules. A point mutation of amyloid transthyretin was identified at position 111 (Met instead of Leu in normal serum transthyretin) which confirms the mutation found for Danish siblings with familial amyloidotic cardiomyopathy. However, the presence of a non-variant amyloid transthyretin was also observed, indicating that the Danish kindred is heterozygous with respect to this point mutation. Isoelectric focusing of the amyloid fibril material resolved multiple protein bands ranging over pH 4.5-6.5, confirming heterogeneities. Methanol extraction of the cardiac amyloid fibril material prior to the purification steps reveals a methanol-soluble substance amounting to about 10% (by mass dry material) of the amyloid fibril material. A yellow substance in this fraction shows absorbance maxima (270, 280 and 430 nm) similar to those observed for transthyretin in normal serum. Gas chromatography/mass spectrometry of the methanol extract revealed the presence of saturated fatty acids (C14:0, C16:0 and C18:0 in the corresponding ratio 2:8:5) and polyunsaturated fatty acids (C16:1, C18:1, C18:2 and C20:4 in the corresponding ratio of 1:2:1:1) as further constituents of the amyloid fibril material.
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PMID:Purification and characterization of amyloid-related transthyretin associated with familial amyloidotic cardiomyopathy. 786 37

The strain 273 B, the type strain of a H serotype of Bacillus thuringiensis not yet characterized: B. thuringiensis subsp. cameroun, serotype H32, was isolated from soil samples collected in Cameroon. This strain produces cuboidal parasporal bodies composed of two major proteins of 53 kDa and 35 kDa. N-terminal sequences of the major proteins share no homology with published sequences. Only the 35 kDa protein is susceptible to digestion by trypsin. A complex array of 9 plasmids was revealed.
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PMID:Characterization of the type strain of Bacillus thuringiensis subsp. cameroun serotype H32. 795 77

Cluster 13 was defined by 2 independently derived murine monoclonal antibodies (MAbs), RS7 (IgG1) and MR54 (IgG2a), which were raised against human squamous-cell carcinoma of the lung and a human ovarian-carcinoma cell line, respectively. Immunologic and biochemical evidence demonstrated that RS7 and MR54, as well as 2 additional MAbs, MR6 (IgG2a) and MR23 (IgG1), generated in the same fusion as MR54, recognize the same antigen, a 46- to 48-kDa glycoprotein. Evaluation of the expression of antigen on the surface of tumor cell lines, Western blotting analyses, competitive binding studies, and double-determinant ELISA assays, support this conclusion. Two distinct epitopes are defined by these MAbs. In order to further characterize this antigen, amino-acid-sequence analyses were performed on peptides derived from antigen purified by affinity chromatography with MAb RS7. The sequence data obtained from 2 peptides, which were independently generated by CNBr cleavage and trypsin digestion respectively indicated identity to GA733-1. The GA733-1 genomic DNA sequence predicted a type-1 membrane protein of 35 kDa, with 4 potential N-linked glycosylation sites. The GA733-1 protein product has not been identified previously, and MAbs to this tumor-associated antigen were not previously known.
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PMID:Characterization of cluster 13: the epithelial/carcinoma antigen recognized by MAb RS7. 819 3

Streptococcus pyogenes adheres to human epithelial cells in vitro and in vivo. To identify adhesins, cell wall components were extracted from S. pyogenes M6 with alkali or by treatment with mutanolysin and lysozyme. HEp-2 cells were incubated with extracts of S. pyogenes M6 and then analyzed by Western blot (immunoblot) assays, using antibodies to S. pyogenes. Only one streptococcal component (62 kDa) was bound to HEp-2 cells and was identified serologically as M6 protein. Experiments with pepsin-cleaved fragments of M protein indicated that the binding site was located at the N-terminal half of the molecule. M protein was bound selectively to two trypsin-sensitive surface components, 97 and 205 kDa, of HEp-2 cells on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. Tritium-labeled lipoteichoic acid bound to different HEp-2 cell components, 34 and 35 kDa, in a parallel experiment, indicating that lipoteichoic acid was not complexed with M protein and does not mediate M-protein binding. The four HEp-2 components were unrelated to fibronectin since they did not react with specific antibodies. An M-protein-deficient (M-) strain of streptococcus (JRS75), grown in chemically defined medium, showed 73% less adhesion activity to HEp-2 monolayers than an M+ strain (JRS4). Streptococcal adhesion was insensitive to competitive inhibition by selected monosaccharides. These results indicate that M protein binds directly to certain HEp-2 cell membrane components and mediates streptococcal adhesion.
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PMID:M protein mediates streptococcal adhesion to HEp-2 cells. 830 Feb 5

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.
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PMID:Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis. 874 66

Active Ca2+ transport was measured in microsomal vesicles prepared from bovine retinae and was compared with that in disk membranes of the photoreceptor cells of the same retina. The 45Ca uptake was dependent on the presence of Mg(2+)-ATP and was inhibited by vanadate or when GTP substituted for ATP. The dependence of calcium uptake on the external free Ca2+ concentration gave a KM = 13 microM or a KM = 0.1 microM for disks and microsomal vesicles, respectively. A phosphorylated intermediate (E-P) of Ca(2+)-ATPase of about 100 kDa was isolated in microsomal vesicles. The E-P formation was strongly inhibited by thapsigargin and partially by 2,5-di-(-butyl)benzohydroquinone. Digestion of disks or microsomes with calpain had no effect on the phosphorylated intermediate, while digestion with trypsin produced two fragments of approximately 55 kDa and 35 kDa. These results suggest that bovine retinal microsomes contain a calcium pump belonging to the SERCA family.
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PMID:Endoplasmic reticulum Ca(2+)-ATPase in microsomal vesicles isolated from bovine retinae. 874 9

Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by autophosphorylation. Activation by phospholipids is inhibited by Ca2+-calmodulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049-27055), we identified the catalytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase. In this paper, we report the cloning and sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specificity similar to that of native kinase and resistance to trypsin similar to that of fully phosphorylated MIHC kinase. MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of protein kinase A and similarly low sequence identity to the catalytic domains of protein kinase C- and calmodulin-dependent kinases, but 50% sequence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regulated by small GTP-binding proteins. The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserved Tyr and Ser/Thr residues in the region corresponding to the P+1 loop of protein kinase A. A synthetic peptide corresponding to this region of MIHC kinase is phosphorylated by both the expressed catalytic domain and native MIHC kinase.
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PMID:The catalytic domain of acanthamoeba myosin I heavy chain kinase. II. Expression of active catalytic domain and sequence homology to p21-activated kinase (PAK). 890 Jan 96


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