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)

AJ-FS9 is one of a new series of monoclonal antibodies (mAbs) raised by immunizing mice with isolated human sperm tail fibrous sheath (FS). Using indirect immunofluorescence (IIF) of human spermatozoa dried onto slides, the AJ-FS9 mAb reacted with the principal piece of occasional spermatozoa. Following their enzymatic treatment with trypsin, dispase or collagenase, but not sulphatase, all the spermatozoa were stained at their principal piece. The ultrastructural localization of the antigens to the FS was established by immunogold electron microscopy, which showed the distribution of gold particles on the FS outer surface of spermatozoa sequentially treated with 1% Triton and dispase; spermatozoa demembranated by Triton alone showed no reaction. For biochemical characterization, spermatozoa were lysed with 1% Triton, and the sperm pellet was run through a reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotted and immunostained with AJ-FS9. The results showed the reaction of the antibody with three protein bands with molecular masses ranging between 46 and 56 kDa. IIF screening of human testicular cryostat sections with AJ-FS9 mAb showed its reactivity with occasional sperm tails; but following their dispase treatment, all spermatozoa were stained. The restricted staining of the assembled FS of maturing sperm tails indicated the late appearance of the antigens during spermatogenesis. The antibody did not react with sperm cell precursors or other cell types within/without the seminiferous tubules. Untreated and dispase-treated frozen sections of skin, oesophagus, tongue, liver, kidney, stomach, ileum or their blood vessels showed no reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:AJ-FS9 monoclonal antibody detects masked antigens within the human sperm tail fibrous sheath. 784 12

Administration of either thyroid hormone or epidermal growth factor (EGF) ameliorates injury in a variety of experimental acute renal failure (ARF) models. Since thyroid hormone augments EGF release and EGF receptor expression, a hypothesis suggesting that the mechanism of thyroid action is via EGF appears attractive. The present study is an attempt to evaluate the role of EGF in thyroid mediated protection from ARF induced in rats by dichromate. Renal parenchymal levels of acid extractable endogenous EGF were measured by RIA in dichromate exposed, otherwise untreated animals and in those receiving dichromate followed by thyroid. In the untreated case serum creatinine peaked at 2.5 mg % on the third day following dichromate exposure. Endogenous levels of EGF closely paralleled serum creatinine with a six-fold increase observed at peak injury. The source of EGF increase appeared to be a membrane bound precursor as soluble levels of EGF rose in injured kidneys at the expense of Triton-X-100 extractable, immunoreactive material that upon treatment with trypsin yielded additional EGF. T3 administered one hour following dichromate resulted in significant functional protection (peak injury serum creatinines 2.63 +/- 0.76 control vs. 0.98 +/- 0.14 with T3) as well as an approximate doubling in renal EGF levels at 24, 48 and 72 hours (4.7 +/- 0.3 vs. 9.7 +/- 0.8 at 24 hr, 33.5 +/- 6.5 vs. 63.2 +/- 20.0 at 48 hr, and 23.1 +/- 10.0 vs. 44.1 +/- 8.7 ng/g wet weight at 72 hr). There was no beneficial effect of exogenous EGF on renal function either when given in conjunction with T3 or when used alone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of renal EGF in dichromate-induced acute renal failure treated with thyroid hormone. 793 9

The actin-bundling protein fimbrin is homologous to 1-plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069-1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton-X-100 (soluble fraction), Tween 40-deoxy-cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone-extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC-extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic 32PO4 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins.
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PMID:Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages. 822

The oversynthesis of the secreted alkaline phosphatase (PhoA) in E.coli K12802 cells due to transformation with the PhoA+ plasmid pHI-7 leads to a change in its biogenesis--alternative localization and accumulation of the enzyme intermediate forms corresponding to different stages of the its post-translational modification. Instead of the soluble PhoA available in the parent strain mostly as a completely processed mature metazyme III localized in the periplasm, five enzyme forms were discovered in the PhoA overproducer: a cytoplasmic PhoA precursor (prePhoA) as insoluble aggregates; three soluble metazymes of a mature active form localized in the periplasm as in well as in culture medium; and a soluble high-molecular form in the periplasm. PrePhoA was isolated and purified by removal of soluble cell fractions using differential centrifugation, solubilization of membrane proteins with Triton X100, dissolution of the aggregates in the buffer with 8M urea and FPLC on MonoQ. Extracellular PhoA was purified by ultrafiltration, thermal treatment, and gel chromatography on Sepharose CL-4B. It was shown that the isolated prePhoA can be transformed into a mature form in the presence of a leader peptidase in 0.8 urea and is completely cleaved with proteinase K. Three forms of the mature PhoA vary in resistance to proteinase K and trypsin. Metazyme I, the unprocessed mature PhoA, is the most resistant to proteolysis.
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PMID:[Features of the biogenesis of Escherichia coli alkaline phosphatase during its supersynthesis]. 836 88

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.
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PMID:Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer. 935 32

1. We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the K(ATP) channel in the pancreatic beta-cell from mouse. 2. All three detergents blocked K(ATP) channel activity with the following order of potency: Tween 80 (Ki< approximately 83 nM)>Triton X100 (Ki=350 nM)>Cremophor. In all cases the block was poorly reversible. 3. Single-channel studies suggested that at low doses, the detergents act as slow blockers of the K(ATP) channel. 4. Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg2+-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel. 5. The detergents could block the pore-forming subunit, Kir6.2deltaC26, which can be expressed independently of SUR1 (the regulatory subunit of the K(ATP) channel). These data suggest that the detergents act on Kir6.2 and not SUR1. 6. The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1). 7. Voltage-dependent K-currents in the beta-cell were reversibly blocked by the detergents with a far lower potency than that found for the K(ATP) channel. 8. Like other insulin secretagogues that act by blocking the K(ATP) channel, Cremophor elevated intracellular Ca2+ in single beta-cells to levels that would be expected to elicit insulin secretion. 9. Given the role of the K(ATP) channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the K(ATP) channel.
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PMID:Inhibition of the ATP-sensitive potassium channel from mouse pancreatic beta-cells by surfactants. 964 78

Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium.
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PMID:Control of ciliary orientation through cAMP-dependent phosphorylation of axonemal proteins in paramecium caudatum. 1074 59

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.
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PMID:The assembly pathway of outer membrane protein PhoE of Escherichia coli. 1084 98

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.
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PMID:Titin determines the Frank-Starling relation in early diastole. 1256 38

The sensitivity of Invertebrate iridescent virus 6 (IIV-6) to a selection of organic solvents, detergents, enzymes and heat treatment was assayed in Spodoptera frugiperda (Sf9) cells and by injection of inoculum into larvae of Galleria mellonella. In several cases, the degree of sensitivity of the virus depended on the method of assay; cell culture assays indicated greater losses of activity than insect bioassay. IIV-6 was sensitive to chloroform but sensitivity to ether was only detected by cell culture assay. Sensitivity (defined as a reduction of at least 1 log activity) was detected following treatment by 1 and 0.1% SDS, 1% Triton-X100, 70% ethanol, 70% methanol, 1% sodium deoxycholate, pH 11.1 and 3.0. No sensitivity was detected to 1% Tween 80, 1 M MgCl2, 100 mM EDTA, lipase, phospholipase A2, proteinase K, or trypsin at the concentrations tested. Viral activity was reduced by approximately 4 logs following heating to 70 degrees C for 60 min or 80 degrees C for 30 min. The above observations highlight the need for studies on the role of the virus lipid component in the process of particle entry into cells, and may explain why vertebrate and invertebrate iridoviruses have been reported to differ in their sensitivity to organic solvents and enzymes.
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PMID:Sensitivity of Invertebrate iridescent virus 6 to organic solvents, detergents, enzymes and temperature treatment. 1257 4


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