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)

Phytohemagglutinin M (PHAM) has been purified from the commercial mixture of proteins produced by Phaseolus vulgaris, using a Sepharose-thyroglobulin column. The protein gave one band on gel electrophoresis and two bands on SDS-gel electrophoresis (mol. wt. 33 700 and 32 100, respectively). Molecular weight determination by ultracentrifugation gave a value of 61 200 +/- 700. The protein had a minimum sugar content of 16%. Binding studies of PHAM to purified rat spleen lymphocytes have been performed at 0, 25, and 37 degrees C. It was shown that the cells bound about the same amount of lectin at 0 and 37 degrees C, but less protein was bound at 25 degrees C. The binding phenomenon showed saturability at all temperatures. Data were analyzed by Scatchard plots and two kinds of binding sites were found. High-affinity sites and low-affinity sites have been characterized in terms of association constants and (apparent) number. It was also shown that cells treated with trypsin or sodium azide bound less lectin. Bound concanavalin A did not appear to affect the amount of bound PHAM, but its influence was reflected in the value of the association constant for the binding of PHAM. Unlabelled PHAM was shown to displace radioactive PHAM from the cells, but could not remove bound concanavalin A. The significance of these results is discussed in terms of the fluid plasma membrane model and cellular metabolism.
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PMID:The binding of phytohemagglutinin M to rat spleen lymphocytes. Quantitative studies. 95 51

Diazotized (125I)-diiodosulfanilic acid (DD125ISA) binds specifically to the exposed proteins on the surface of the rabbit platelet plasma membrane. This was demonstrated by the following observations with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole platelets and the isolated plasma membrane fraction: (1) the specific activity of isolated membrane protein was sevenfold that of whole platelet protein, (2) no proteins of intact platelets were labeled which were not represented in the isolated plasma membrane, (3) DD125ISA-labeled proteins were altered by trypsin treatment of intact, labeled platelets, and (4) the pattern of labeling produced by reaction of isolated membranes with DD125ISA differed from that produced by the labeling of intact platelets. Reaction of DD125ISA with intact platelets produced labeling of only the three membrane glycoproteins (molecular weights: 180,000, 125,000, and 92,000 daltons) with greatest labeling of the largest glycoprotein and least labeling of the smallest glycoprotein. When rabbit platelets were labeled simultaneously with DD125ISA and 51Cr, the two isotopes were similarly distributed in various density populations of platelets. Some DD125ISA was solubilized from labeled and washed platelets by sonication, but all platelet DD125ISA was recovered in the plasma membrane fraction after 30 minutes' circulation in vivo. In vivo 51Cr recovery and survival were not altered by simultaneous labeling of platelets with DD125ISA. The disappearance of DD125ISA from circulating platelets (T 1/2 = 17 hours) was more rapid than 51Cr (T 1/2 = 30 hours) and appeared exponential in contrast to the linear 51Cr disappearance. On the other hand, DD125ISA did not disappear from platelets faster than 51Cr when doubly labeled platelets were harvested after 3 hours' circulation and incubated in autologous plasma (T 1/2 of DD125ISA elution = 43 hours, 51Cr = 33 hours). SDS-PAGE analysis of DD125ISA-labeled platelets after 14 to 20 hours' circulation in vivo demonstrated the same pattern of DD125ISA distribution on membrane glycoproteins as on the platelets prior to infusion. We interpret this symmetrical loss of the membrane label to indicate symmetrical loss of membrane proteins, suggesting that the platelet may lose pieces of membrane and not specific surface proteins during circulation. This could occur during reversible adhesion encounters during the process of hemostasis and cause the smaller size and decreased effectiveness of older platelets.
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PMID:Studies on platelet plasma membranes. II. Characterization of surface proteins of rabbit platelets in vitro and during circulation in vivo using diazotized (125i)-diiodosulfanilic acid as a label. 95 84

The isolation and characterization of a microsomal arylaminopeptidase from rat kidney is reported. By treatment of a microsomal arylaminopeptidase-phosphatase-complex with trypsin and subsequent gel filtration of the solubilized proteins on Sepharose 6B a electrophoretic homogeneous arylaminopeptidase was obtained (yield, 3%; enrichment, 900 times). The following properties of the purified enzyme were determined: 1. Molecular weight: 182000 (gel filtration on Sepharose 6B) to 192000 (SDS-polyacrylamide gel electrophoresis). 2. Subunit structure: In the presence of 6 M guanidine - HC1 + 1% BETA-mercaptoethanol the enzyme dissociates into subunits (MW 46700, ESTIMATED BY SDS gel electrophoresis method). 3. Isoelectric point: 4,71 (agarose gel electrophoresis method). 4. UV characteristics: E 280nm/E260NM=1.3. 5. Substrate specifity: optimal substrates L-alanyl derivatives (anilide, beta-naphthyl amide, p-nitroanilide, 4-(phenylazo)-phenylamide and hydrazide). Among these compounds the anilide derivative was hydrolyzed most rapidly. Furthermore, di- and tripeptides, especially L-methionyl-L-leucine, were also split. No hydrolysis was observed with hemoglobin (pH 4.5 and 7.5) and amino acid- or peptide-ester substrates. 6. Optimal pH: 7.5 +/- 0,1; optimal temperature: 45 to 50 degrees C. 7. The enzyme has no transamidation activity with L-alanyl amide both as aminoacyl donator and -acceptor. 8. Influence of effectors: Heavy metal ions (Ni2+, Cd2+, Cu2+, Zn2+), chelating agents (EDTA, o-phenanthroline) and puromycin inhibit the enzyme significantly. SH-group reagents are without any influence. 9. L-alanyl-L-alanyl-4 (phenylazo)-phenylamide, a dipeptide aryl aminopeptidase substrate, is hydrolyzed by the purified enzyme preparation according to a consecutive or step by step mechanism.
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PMID:[Isolation and characterization of a microsomal arylaminopeptidase from rat kidney]. 97 46

We have developed a new method for identifying proteins which span the plasma membrane ("trans-membrane" proteins) of mammalian cells grown in tissue culture. The method involves labeling proteins exposed on the cell surface with 125I by the lactoperoxidase technique and then preparing sealed, "inside-out" membrane vesicles (phagosomes) from the labeled cells using the polystyrene latex bead procedure. These inside-out vesicles are then treated briefly with trypsin and analyzed by SDS-polyacrylamide gel electrophoresis for the presence of 125I-labeled protein species which were degraded by proteolytic attack. Such proteins must be exposed on both the outer and inner membrane surfaces and, therefore, they must pass through the lipid barrier. This method is a general one in the sense that it is suitable for use with a wide variety of cell types, and here we show how it has been employed to prove that a particular high molecular weight polypeptide, called band 1, spans the plasma membrane of mouse L cells. Further studies of the band 1 polypeptide have demonstrated that it is preferentially exposed on the L cell surface during G1 phase of the cell cycle. Progression of cells from G1 to S is accompanied by a marked decrease in the availability of band 1 to iodination and it remains unavailable until cells re-enter G1. It is suggested that the band 1 polypeptide may be functionally involved in the regulation of cell proliferation.
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PMID:Identification of a high molecular weight trans-membrane protein in mouse L cells. 103 Aug

Platelet elastase has been differenciated from various protein fractions into a trypsin dependent form and a trypsin independent form. Trypsin independent elastase has been purified by affinity chromatography on cellulose elastin column as a pure protein raction of molecular weight: 26,000 ou SDS acrylamide gels. Trypsin dependent elastase has been purified by preparative acrylamide disc gel electrophoresis. This fraction, proteolysed (limited proteolysis) and activated by trypsin into active elastase, has been identified as the precursor (platelet proelastase) of platelet elastase. Its molecular weight is 28,000 before trypsin and 26,000 after trypsin.
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PMID:Human blood platelet elastase and proelastase. 110 Nov 57

Milk fat globule membrane was solubilized with sodium dodecyl sulfate and mercaptoethanol and the membrane proteins were separated by SDS-polyacrylamide gel electrophoresis. The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons. At least five glycopeptides were separated of which two stained intensely with periodic acid-Schiff reagent, but poorly with coomassie blue. Trypsin hydrolysis of whole cream and isolated milk fat globule membrane revealed major differences in the rates of protein hydrolysis. Many of the membrane proteins of whole cream resisted proteolysis compared with the same proteins in the isolated membrane. Two glycopeptides were resistant to trypsin digestion in either preparation. Treatment of whole cream with neuraminidase led to the release of at least 70% of the protein-bound sialic acid. Whole cream and isolated membrane samples were iodinated with 125I in the presence of lactoperoxidase and hydrogen peroxide. The membrane proteins were significantly more accessible to lactoperoxidase-125I i in isolated membrane compared with the proteins of whole cream. Polypeptides of molecular weight 43,500 and approximately 48,000 daltons were predominantly labelled in whole cream and could be eluted from the fat globules with magnesium chloride (1.5m). The results strongly suggest that the proteins of milk fat globule membrane are asymmetrically arranged in the membrane and that most of the protein-bound sialic acid is present on the external surface of milk fat globules.
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PMID:Studies on the structure of milk fat globule membrane. 119 40

The effects of serum and coatings of substrate-attached material (SAM, which remains tightly adherent to the substrate after EGTA-mediated removal of cells) on the kinetics of attachment of DNA-radiolabeled BALB/c 3T3. SV40-transformed 3T3, and concanavalin A-selected revertant cells to glass coverlips were studied. The presence of serum in the medium of attaching cells had a marked effect on (1) the initial time lag before stable attachment of cells, (2) the maximum level of attached cells, (3) the stability of attachment, and (4) pseudopodial spread of the cell over the substrate. These serum effects could be mimicked by measuring attachment in medium without serum and with use of serum-preadsorbed or 3T3 SAM-coated coverslips. Enzymatic treatment of serumpreadsorbed substrates indicated that the factor(s) in serum which affects attachment is very trypsin-sensitive. Serum preadsorption of substrates stimulated attachment of SVT2 cells in medium with serum in a manner very similar to the effects of 3T3 SAM coating, while attachment of 3T3 SAM coating, while attachment of 3T3 or revertant cells was unaffected. Slab gel electrophoretic analysis (PAGE-SDS gels) identified eight major serum proteins by Coomassie blue staining (a) which bind to the substrate in the absence of cells and (b) which persist on the substrate after growth to confluence of 3T3 or SVT2 cells; this suggests that major breakdown or serum-adsorbed components does not occur during growth of normal or transformed cells. Seven radioactive SAM proteins were detected by autoradiography in 3T3 or SVT2 SAM electropherograms -- two of which are high molecular weight components which correspond to the glucosamine-radiolabeled hyaluronate proteoglycans observed previously; the remaining five are newly-identified proteins in SAM (one of these proteins appears to be actin). 3T3 and SVT2 cells have unique proportions of these seven components. The data are consistent with the idea that normal or virus-transformed cells do not attach directly to the culture substrate, but to specific classes of substrate-adsorbed serum proteins via deposition of specific classes of cell surface proteins and polysaccharides.
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PMID:Substrate-attached serum and cell proteins in adhesion of mouse fibroblasts. 126 8

The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.
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PMID:Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis. 128 66

Serine proteinase inhibitory proteins (SPIs) were extracted from human disc tissues using 2 M GuHCl and subjected to CsCl density gradient ultracentrifugation. The SPIs recovered in the low buoyant density fractions (rho < or = 1.35 g/ml) were purified by a combination of gel-permeation, ion-exchange, trypsin affinity, and reverse-phase high performance chromatographies. Characterisation of the major disc SPI by polyacrylamide gel electrophoresis, isoelectric focussing, enzyme inhibition and pH stability studies indicated that this small molecular weight (12-14 kDa), highly basic (pI > 9.5), acid-stable but alkaline-labile protein possessed potent inhibitory activity against bovine pancreatic trypsin and chymotrypsin, and human leukocyte elastase and cathepsin G. Two-major and two-minor low molecular weight cationic SPI species were identified by reverse-phase HPLC. The predominant species was identical to a human articular cartilage SPI sharing amino terminal sequence homology with the mucus proteinase inhibitors (MPIs). It also cross-reacted with an antiserum to the MPIs and behaved identically to secretory leucocyte proteinase inhibitor (SLPI) when examined by reverse phase HPLC, and SDS PAGE. A higher molecular weight (54 kDa), anionic (pI approximately 4.6) SPI was also purified and identified as alpha 1-proteinase inhibitor (alpha 1-PI). Quantification of alpha 1-PI and the small molecular weight cationic disc inhibitors indicated that the latter were depleted in morphologically degenerate disc tissues while levels of alpha 1-PI were somewhat higher although a large proportion of the alpha 1-PI was inactive. A depletion of total SPI levels was evident overall in degenerate discs suggesting a functional role for these inhibitory proteins in the maintenance of IVD matrix homeostasis.
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PMID:The serine proteinase inhibitory proteins of the human intervertebral disc: their isolation, characterization and variation with ageing and degeneration. 128 14

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.
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PMID:Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease. 128 50


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