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)

Rat fat cells contain three species of spontaneously active inhibitor proteins of protein phosphatase 1, as resolved by SDS-PAGE, with apparent molecular masses of 40 kDa, and 28 kDa respectively. The 33-kDa, thermostable inhibitor was highly purified from bovine adipose tissue and shown to be very similar to inhibitor-2 of skeletal muscle. It was phosphorylated, on threonine only, by glycogen synthase kinase 3. It formed an inactivated complex with protein phosphatase 1, that was reactivated by incubation with ATP-Mg and glycogen synthase kinase 3. By gel filtration it had a Stokes radius of 3.4 nm. Peptide and phosphopeptide maps, generated by Staphylococcus aureus V8 proteinase, trypsin or thermolysin, of the inhibitor and of the skeletal muscle inhibitor-2 were similar. The 40-kDa inhibitor, which was denatured by boiling, represents a novel protein phosphatase inhibitor protein or an undegraded precursor of inhibitor-2. The total activity of inhibitor-2-like material (thermostable and macromolecular) in an adipocyte cytosol extract corresponded to an intracellular concentration of 0.3 microM inhibitor-2.
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PMID:Adipose tissue protein phosphatase inhibitor-2. 282 48

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.
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PMID:Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity. 304 19

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.
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PMID:Multiple phosphorylation sites of rat liver glycogen synthase. 309 Oct 84

We previously have isolated cDNA clones for several mRNAs that increase in abundance during the differentiation of 3T3 adipocytes but whose physiological role is unknown. We show here that a mRNA that is complementary to one of these clones and encodes a protein of 28 kDa is expressed abundantly in mouse fat pads but not in several other mouse tissues. Sequence analysis of the corresponding cDNA clone indicated that the encoded protein shows 30% overall amino acid homology to several serine proteases including trypsin, chymotrypsin, and elastase. Homology is much higher (64%) between the 28-kDa protein and regions that are strongly conserved among the members of the serine protease family. The derived protein also has key features characteristic of active serine proteases, including the histidine, aspartic acid, and serine residues, which comprise the charge relay system, and a potential cleavage site for activation of the zymogen. Primer extension analysis performed to obtain the sequence of the 5' end of mRNA that encodes the 28-kDa protein indicates that two forms of this mRNA exist and probably arise through alternative splicing. The two mRNAs encode signal sequences that differ by the deletion of one amino acid near the predicted cleavage site of the signal peptide. These results demonstrate that adipocyte differentiation is accompanied by the expression of mRNA encoding a serine protease homologue that can be synthesized with two different signal peptides.
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PMID:A developmentally regulated mRNA from 3T3 adipocytes encodes a novel serine protease homologue. 390 Oct 3

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

Limited proteolysis has been used to probe the domain structure of the type I DNA methyltransferase M.EcoR124I. Trypsin digestion of the methyltransferase generates two fragments derived from the HsdS subunit, a 28 kDa N-terminal domain and a 19 kDa C-terminal domain, leaving the HsdM subunit intact. Extensive digestion by chymotrypsin, however, removes 59 amino acid residues from the N terminus of the HsdM subunit to leave a 52 kDa C-terminal domain. Binding of the cofactor S-adenosyl methionine has no appreciable effect on the rate of cleavage, but binding of a 30 bp DNA duplex containing the cognate recognition sequence confers almost total protection. Following trypsin cleavage of the methyltransferase, a stable proteolytic product is produced which has been purified for biochemical characterisation. The trypsinised enzyme is shown to be a multimeric complex containing two intact HsdM subunits and both fragments of the HsdS subunit, consistent with the circular model proposed for the organisation of domains in the specificity subunit in type IC methyltransferases. Gel retardation studies show that the proteolysed enzyme still retains DNA binding activity, but its specificity for the DNA recognition sequence is dramatically reduced.
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PMID:Probing the domain structure of the type IC DNA methyltransferase M.EcoR124I by limited proteolysis. 760 69

Study of the isoenzymatic spectrum of nuclear DNase from rat liver demonstrated that the extracts obtained in the presence of the protease inhibitor, PMSF, contained four polypeptides with molecular masses of 120, 54, 31 and 28 kDa possessing the endo-DNase activity. Long-term storage at -20 degrees C and autodigestion of the nuclei revealed the presence of additional polypeptides with a lower molecular mass and possessing an endo-DNase activity. Treatment of rat liver nuclear extracts with trypsin caused the appearance of an active polypeptide (M(r) 145 kDa). This finding and the multiplicity of endo-DNases of different molecular masses suggest the existence of a precursor possessing a much higher molecular mass. Chromatographic separation of endo-DNases on Sephacryl S-300 revealed three fractions of 400 and more kDa possessing a Ca2+/Mg(2+)-dependent activity, however, only after trypsin treatment.
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PMID:[Characteristics of nuclear endo-DNAase from rat liver by molecular mass and cationic dependence]. 766 4

Of the five varicella-zoster virus (VZV) open reading frames (ORFs) known to encode proteins which influence viral transcriptional events, two (ORFs 10 and 62) encode proteins associated with the tegument of virus particles, where they may function during the immediate-early events of infection. In this study, antibodies which recognize the products of the three additional VZV ORFs, ORFs 4, 61, and 63, were made and used to characterize their association with virus particles. ORF 4 encoded a 52-kDa polypeptide, and antibodies to ORF 63 reacted with polypeptides of 47 and 28 kDa. Antibodies to ORF 61 recognized heterogeneous polypeptides of 62 to 66 kDa in cells infected with a vaccinia virus recombinant expressing ORF 61 and in VZV-infected melanoma cells but reacted very weakly with polypeptides of VZV-infected human foreskin fibroblasts, suggesting that cell-specific factors were involved in ORF 61 protein accumulation. Analysis of virus particles purified from melanoma cells indicated that a 52-kDa polypeptide from ORF 4 and the 47-kDa polypeptide from ORF 63, but not any from ORF 61, were associated with virus particles. The virion proteins were likely components of the tegument, as they were not solubilized by treatment of virus with mild detergents and were completely resistant to trypsin digestion unless prior envelope solubilization was performed. The products of ORFs 4 and 63 were not found in purified VZV nucleocapsids. These results suggest that forms of the ORF 4- and ORF 63-encoded transcriptional regulatory proteins are also structural and may also have roles in the immediate-early events of infection.
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PMID:The transcriptional regulatory proteins encoded by varicella-zoster virus open reading frames (ORFs) 4 and 63, but not ORF 61, are associated with purified virus particles. 776 88

A polypeptide of high molecular mass has been detected in mammalian brain by a monoclonal antibody, 5C3, raised against skeletal muscle ryanodine receptor. 5C3 does not crossreact with the cardiac ryanodine receptor, the isoform which is believed to be located in many regions of the brain. Endogenous proteases in brain formed a prominent immunogenic fragment of 116 kDa whereas five immunostaining polypeptides greater than 200 kDa were observed in skeletal muscle. Mild trypsin digestion of brain microsomes resulted in fragments of approximately 400 and approximately 280 kDa, of similar mass to two peptides formed from the skeletal muscle ryanodine receptor. However a peptide of 28 kDa, resistant to trypsin, was observed in muscle but not in brain. The brain polypeptide recognised by 5C3 is therefore not identical to the skeletal muscle ryanodine receptor.
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PMID:Monoclonal antibody to skeletal muscle ryanodine receptor detects a polypeptide in rat brain: comparison of immunogenic fragments after limited proteolysis. 801 88

The processing of culture medium of rabbit synovial fibroblasts led to the isolation of three stromelysin-1 (MMP-3) cleavage products: A 31-kDa protein, which represents a C-truncated latent stromelysin-1, an active stromelysin-1 of 21 kDa, that originates from the 31-kDa proform by activation. A third protein had a molecular mass of 25 kDa representing the C-terminal part of prostromelysin-1 and is missing in the C-truncated latent stromelysin-1. The activation process of human prostromelysin-1 in vitro is known to lead to an active stromelysin-1 with a relative molecular mass of 45 kDa by removing the N-terminal prodomain. This active stromelysin-1 is further processed to a lower molecular mass active form of 28 kDa. Our results obtained for the highly homologous rabbit stromelysin-1 indicate that another activation pathway is possible. In a first step prostromelysin-1 is hydrolysed between Met261-Glu generating a C-truncated latent stromelysin-1, which is activated by cleavage of the Thr83-Phe bond to the 21-kDa stromelysin-1. The latent C-truncated stromelysin-1 is slowly converted even at 4 degrees C into the active form. In the presence of 50 microM ZnCl2 this activation was prevented for at least three weeks. The activation rate is largely enhanced by aminophenylmercury acetate and especially by trypsin. The differences of the 21-kDa stromelysin-1 to a 28-kDa stromelysin-1 isolated from human rheumatoid synovial fluids described earlier are discussed.
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PMID:Isolation of latent 31-kDa C-truncated stromelysin and 21-kDa stromelysin from rabbit synovial fibroblasts: an alternative activation pathway for stromelysin. 806 May 32


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