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 have recently reported that lipase may play a role in the pathogenesis of acute pancreatitis by its ability to release fatty acids from triglycerides. The aim of this study was to further investigate the effect of lipase and its various digestive products on the integrity of isolated pancreatic rat acini. Pancreatic acini were prepared by collagenase digestion and their newly synthesized proteins labeled with 35S-methionine. Acini were later incubated in buffer to which various factors were added: Products of lipolytic digestion, such as various fatty acids and monoglycerides, fat tissue, nonactivated or trypsin activated homogenized pancreatic tissue, and a specific lipase inhibitor (THL, tetrahydrolipstatin). Cellular destruction was quantified by the degree of radiolabeled proteins released. Short chain fatty acids and monoglycerides (up to C-12) caused cellular destruction, whereas long chain fatty acids and their respective monoglycerides were not harmful. With regard to unsaturated fatty acids, long chain fatty acids (C-18 to C-22) were also able to destroy cells. The degree of cellular necrosis correlated with incubation time and fatty acid concentration. The cellular damage caused by incubation of acini with either inactive or trypsin activated pancreatic homogenates together with triglycerides could be completely inhibited by the specific lipase inhibitor THL. Bile alone caused no damage. When bile was combined with activated-pancreatic homogenates, about 25% of newly synthesized proteins were released by acini within 30 min. Incubation with a combination out of bile activated pancreatic homogenates and triglycerides resulted in the most pronounced damage. This acinar destruction could only be partly inhibited by THL. These studies suggest that both lipase and phospholipase-A2 may play an important role in the pathogenesis of acinar cell destruction.
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PMID:Isolated rat pancreatic acini as a model to study the potential role of lipase in the pathogenesis of acinar cell destruction. 128 21

A study has been made of the association properties of the two GM1 ganglioside molecular species GM1-C18 and GM1-C20 (containing C18 and C20 long chain bases, respectively) to rat cerebellar granule cells in culture. Both gangliosides recognized, to the same extent, and associated with them to give a form of association, the trypsin-labile form. This form was removed by treatment with trypsin enzyme. Both gangliosides associated stably with the cells to become components of the cell membranes. Although similar amounts of the two gangliosides entered the cells, being then metabolized, the time course of the association was different for the two gangliosides: after 15 h of ganglioside-cell incubation the amount of GM1-C18 inserted into the cell membrane was 2.43 times higher than that of GM1-C20.
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PMID:Rat cerebellar granule cells in culture associate and metabolize differently exogenous GM1 ganglioside molecular species containing a C18 or C20 long chain base. 150 63

Our recent researches toward enzymic condensation of long chain peptides are discussed. These involve two methods of peptide C-terminal activation and transpeptidation catalyzed by clostripain, trypsin and their derivatives.
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PMID:Studies on enzymic condensation of long chain peptides. 182 42

Upon differential centrifugation of guinea pig intestine mucosal cells homogenate, fatty acyl-CoA:NADPH oxidoreductase (long chain alcohol forming) was found to be enriched in the light mitochondrial (L) fraction (sedimenting between 66,000 x g min and 500,000 x g min) which contained mainly mitochondria, lysosomes, and peroxisomes. Peroxisomes (marker enzymes: catalase and dihydroxyacetone phosphate acyltransferase) present in the L fraction were separated from other organelles in a Nycodenz density gradient centrifugation employing a vertical rotor. By comparing the distribution of acyl-CoA reductase with different marker enzymes in the gradient, it was concluded that this reductase is primarily localized in the microperoxisomes (microbodies). The topography of the membrane-bound enzyme in the isolated organelles was studied by checking its lability toward trypsin in the absence and presence of the detergent Triton X-100. The results suggested that acyl-CoA reductase is localized on the outer surface (cytosolic side) of microperoxisomal membrane.
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PMID:Peroxisomal localization of acyl-coenzyme A reductase (long chain alcohol forming) in guinea pig intestine mucosal cells. 206 6

Sphingosine-1-phosphate lyase is responsible for the ultimate step in sphingolipid breakdown, converting phosphorylated long chain bases into ethanolamine phosphate and a fatty aldehyde. Using tritiated dihydrosphingosine-1-phosphate, prepared enzymatically from [4,5-3H]dihydrosphingosylphosphocholine, we have reinvestigated the subcellular distribution of this enzyme in rat liver. Upon cell fractionation by differential centrifugation, the enzyme showed a microsomal distribution. Further separation of the microsomal fraction by sucrose gradient centrifugation confirmed an association with the endoplasmic reticulum. By means of constrained nonlinear regression, no evidence for a significant association with mitochondrial membranes, as reported previously (Stoffel, W., LeKim, D., and Sticht, G. (1969) Hoppe Seyler's Z. Physiol. Chem. 350, 1233-1241), nor with other cell compartments was found. The lyase activity, which appeared to be sensitive to different detergents, but not to Triton X-100, was not latent. It could be solubilized with Triton X-100, but not by high ionic strength, indicating that it is an integral membrane protein whose catalytic site is most probably exposed to the cytosol. Treatment of intact microsomal vesicles with trypsin or thermolysin inactivated the lyase activity, confirming that its catalytic site(s) or other domains essential for activity face the cytosol.
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PMID:Subcellular localization and membrane topology of sphingosine-1-phosphate lyase in rat liver. 206 24

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with [3H]acetic anhydride of very high specific radioactivity. The fluorenyl group removed by ammonia treatment was substituted by a nitrophenyl azide group. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 and cold natural GM1 (1:10 by mol) for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive: one, of about 30 kDa, interacted with the ganglioside molecules inserted into the outer membrane layer; three, in the region of 46 kDa, interacted with the portion of associated ganglioside able to be released by trypsin treatment. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A photoreactive derivative of radiolabeled GM1 ganglioside: preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture. 270 71

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.
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PMID:Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling. 309 94

The mechanism by which fatty acids enter cardiomyocytes is unclear. Therefore, the influx kinetics of [3H]oleate into isolated rat heart myocytes were examined. Cells were incubated at 37 degrees C with [3H]oleate bound to albumin in various molar ratios and the initial rate of uptake (V0) was determined as a function of the unbound oleate concentration in the medium. V0 was saturable with increasing oleate concentrations incubated (Km 78 nM; Vmax 1.9 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Furthermore, binding of [3H]oleate to isolated plasma membranes of cardiomyocytes was saturable, revealing a KD of 42 nM, and was inhibited by heat denaturation or trypsin pretreatment of the membranes. From these membranes a single 40-kD protein with high affinity for a variety of long chain fatty acids was isolated. With a monospecific antibody to this membrane protein, binding as well as cellular influx of [3H]oleate was selectively inhibited. These data indicate that at least a portion of myocardial fatty acid uptake is mediated by a specific membrane protein.
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PMID:Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process. 334 44

The effects of exogenously added phospholipase A2 (PLA2) and its hydrolytic products in isolated bullfrog sciatic nerve were investigated. Nerves were pretreated for 3 h with a dose of trypsin which did not affect conduction in order to enhance penetration of the added agents. Treatment of nerves with beta-glucosidase, neuraminidase or chymotrypsin had no effect on conduction. Whereas incubation of the nerves with normal Ringers for 2 h had no significant effect on conduction, incubation with PLA2 in Ringers caused decrements in the height of the compound action potential in a dose-related manner. In addition, incubation of the nerves with 10 mg/ml lysolecithin, arachidonic acid, or docosahexaenoic acid caused marked decrements in the height of the compound action potential. Electron microscopic analysis of nerves after each treatment which caused conduction block revealed varying levels of myelin damage. Although myelin was damaged at the paranodal and/or internodal region, depending on the agents used, the axonal membrane appeared to be intact at the ultrastructural level. It was concluded that the block in conduction resulting from PLA2 was due to the formation of lysolecithin and long chain polyunsaturated fatty acids.
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PMID:Mechanism of phospholipase A2-induced conduction block in bullfrog sciatic nerve. I. Electrophysiology and morphology. 348 69

The synthesis, translocation, processing, and assembly of rat liver short chain acyl-CoA, medium chain acyl-CoA, long chain acyl-CoA, and isovaleryl-CoA dehydrogenases were studied. These four acyl-CoA dehydrogenases are homotetrameric flavoproteins which are located in the mitochondrial matrix. They were synthesized in a cell-free rabbit reticulocyte lysate system, programmed by rat liver polysomal RNA, as precursor polypeptides which are 2-4 kDa larger than their corresponding mature subunits (Mr 41,000-45,000). When the radiolabeled precursors were incubated with intact rat liver mitochondria, they appeared to bind tightly to the mitochondrial outer membrane. At this stage they were completely susceptible to the action of exogenous trypsin. The precursors bound to mitochondria at 0 degrees C were translocated into the mitochondria and processed when the temperature was raised to 30 degrees C. No reaction occurred when the temperature was kept at 0 degrees C, however, suggesting that the binding of the precursors is temperature independent while the subsequent steps of the pathway are energy dependent. Indeed, the translocation reaction was inhibited by compounds such as dinitrophenol and rhodamine 6G which inhibit mitochondrial energy metabolism. The newly imported (mature) enzymes were inaccessible to the proteolytic action of added trypsin. The processing of the precursors to mature subunits was proteolytically carried out in the mitochondrial matrix, and the processed mature subunits mostly assembled to their respective tetrameric forms. Newly synthesized larger precursors of each of the four acyl-CoA dehydrogenases were recovered from intact, cultured Buffalo rat liver cells in the presence of dinitrophenol. When dinitrophenol was removed in a pulse-chase protocol, the accumulated precursors were rapidly (t1/2 3-5 min) converted to their corresponding mature subunits. On the other hand, when the chase was performed in the presence of the inhibitor, the labeled precursors disappeared with t1/2 of greater than 4 h for long chain acyl-CoA dehydrogenase and 1-2 h for the other three enzyme precursors.
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PMID:Biosynthesis of four rat liver mitochondrial acyl-CoA dehydrogenases: in vitro synthesis, import into mitochondria, and processing of their precursors in a cell-free system and in cultured cells. 381 56


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