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)

We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica. A similar protein is present in rat cerebral cortex. This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC). It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20. Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity. The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with [3H]arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC). PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E. coli alkaline phosphatase. These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons.
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PMID:A phospholipase A2-stimulating protein regulated by protein kinase C in Aplysia neurons. 164 37

Pharmacological and biochemical characteristics of the partially purified gamma-aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and beta-galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.
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PMID:Pharmacological and biochemical characteristics of partially purified GABAB receptor. 166 62

Aprotinin, a protease inhibitor, and promazine, an inhibitor of phospholipase A2, were tested for possible inhibition of pancreatic acinar cell (PAC) decline induced by uncoupling of oxidative phosphorylation with 2,4-dinitrophenol (DNP) or by temporary anoxia/reoxygenation. In incubates of acinar cells isolated from rat pancreas the presence of aprotinin did not influence the survival of cells treated with these noxae. This finding excludes that extracellulary acting trypsin, possibly released from damaged cells, contributes to further cell death. While promazine at concentrations of 15 to 20 nmol.(10(6) cells)-1 was well tolerated by untreated PAC, higher concentrations caused a clear reduction of cell viability. At optimum concentration promazine was without influence on DNP-treated cells, but it had a beneficial effect on survival and morphology of anoxia-treated PAC (p less than or equal to 0.05). Therefore, it can be assumed that after anoxia/reoxygenation the membrane phospholipase A2 becomes stimulated and causes phospholipid depletion with final death of the cells. It is suggested that such a mechanism may contribute to the initial cell damage in the pathogenesis of acute pancreatitis, too.
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PMID:Influence of aprotinin and promazine on survival of isolated pancreatic acinar cells. 172 49

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.
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PMID:Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation. 172 52

The human hepatoma cell line, HepG2, secreted an activity that degrades platelet-activating factor (PAF) by the hydrolysis of the sn-2 acetyl group. This activity was Ca++ independent, inhibited by diisopropylfluorophosphate but not by p-bromophenacyl bromide, and resistant to treatment with trypsin or pronase. Separation of HepG2-conditioned medium by gel filtration disclosed that the activity was associated with lipoproteins. An antiserum against PAF acetylhydrolase immunoprecipitated this activity. It was not recognized by an antibody against lecithin:cholesterol acyltransferase (LCAT), which also is secreted by HepG2 cells. Therefore the phospholipase A2 activity of LCAT was excluded as a source of the observed activity. PAF added to the culture medium stimulated the secretion of the PAF-degrading activity by HepG2 cells, while lyso-PAF was inactive. Maximal stimulation was observed with 5 ng/ml PAF, which induced a fivefold increase. The presence of 5 ng/ml PAF, enhanced the secretion of [35S]methionine-labeled PAF acetylhydrolase and cycloheximide inhibited both the basal and PAF-stimulated secretion of the labeled enzyme. We conclude that HepG2 cells produce PAF acetylhydrolase. The liver may be a major source of plasma PAF acetylhydrolase, and PAF may induce the production of its inactivating enzyme by the liver.
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PMID:Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2. 184 78

Porcine phospholipaseA2 expressed in E. coli as a fusion protein was isolated, renatured and specifically cleaved by trypsin as described in (1). Active phospholipaseA2, was purified to homogeneity on a column of PBE-94 over a pH region 7.4-4.5. Using this method, several phospholipase A2 mutant enzymes have now been purified in a single step and all behaved identically during chromatofocusing. The method will therefore be extremely useful not only for those interested in understanding the structure-function relationships of phospholipaseA2 but also for preparing the enzyme in large quantities for industrial and pharmaceutical purposes.
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PMID:A novel method for the purification of porcine phospholipase A2 expressed in E. coli. 185 Feb 64

In order to assess the adaptability and/or applicability of the restrained molecular dynamics (RMD) simulation for building a possible tertiary structure of a protein from the X-ray crystal structure of a family reference protein, the tertiary structure prediction of Crotalus atrox venom phospholipase A2 (PLA2) was attempted based on the X-ray crystal structure of bovine pancreatic PLA2. For the formation of secondary and tertiary structures from the fully extended starting structure, the RMD simulation with interatomic distance restraints and torsion angle restraints, which were derived from homologous amino acid sequence regions in the reference protein, was carried out until the molecular system was fully equilibrated. The predicted tertiary structure of C. atrox venom PLA2 was compared with its X-ray crystal structure, and furthermore the utility of this method was discussed by reference to the similar tertiary structure prediction of beta-trypsin from the X-ray crystal structure of an elastase.
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PMID:Adaptability of restrained molecular dynamics for tertiary structure prediction: application to Crotalus atrox venom phospholipase A2. 188 69

Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.
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PMID:Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes. 194 68

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of type I collagen on platelet membrane phospholipase A2 activity was also observed in a cell-free system utilizing platelet membranes. The release of arachidonic acid was enhanced by types I and III but not by type V collagen. The activation of platelet phospholipase A2 by type I collagen was inhibited by soybean trypsin inhibitor and mimicked by trypsin. However, type I collagen addition was not associated with any detectable changes in platelet membrane proteins while trypsin altered many proteins. These results point to acid soluble phospholipase A2 activity of platelets as an enzyme activated by type I collagen.
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PMID:Stimulation of phospholipase A2 activity in human platelets by trypsin and collagen. 198 2

Distinct peptide maps of two rabbit lung Ca2(+)-dependent phospholipid-binding proteins (PLBPs), 36,000 and 33,000, were generated by cyanogen bromide (CNBr) cleavage, trypsin or Staphylococcus aureus V8 proteinase digestion. The amino acid sequence of a CNBr-cleaved peptide of the 36,000 PLBP was aligned to the amino terminus of human lipocortin I with more than 77% identity, but had no identity with the known amino terminal sequence of other known annexins. Partial amino acid sequence of a 33,000 PLBP peptide demonstrated a close (56%) relationship to endonexin II, human placental anticoagulant protein, and porcine intestine protein II, but shared only 32% identity with lipocortin I, 30% with lipocortin II. Antiserum generated against purified 36,000 PLBP reacted strongly with the 33,000 PLBP, but did not react with any other rabbit lung cytosolic proteins. Both PLBPs inhibited the phospholipase A2 reaction when dioleoyl phosphatidylcholine and phosphatidylglycerol vesicles or monolayers were used as substrates. In the vesicle assay, the phospholipase A2 reaction was inhibited at lower substrate phospholipid concentrations but not at nearly saturating substrate concentrations. In the monolayer assay, the phospholipid-binding proteins did not inhibit phospholipase A2 at a low phospholipid surface concentration of 3.8.10(-3) molecules/A2, but they did at higher surface concentrations between 1.1 x 10(-2) and 3.8 x 10(-2) molecules/A2. The inhibition of phospholipase A2 by rabbit lung phospholipid-binding proteins is most likely due to the prevention of penetration by phospholipase A2 into the interface, a requirement for the enzyme to act on the substrate.
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PMID:Lung calcium-dependent phospholipid-binding proteins: structure and function. 199 31


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