Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.
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PMID:The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis. 284 68

Methods were developed for the purification of the surface, membrane-bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin. Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16-residue peptide with amino-terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.
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PMID:Purification of haemagglutinin and neuraminidase from influenza virus strain 3QB and isolation of a peptide from an antigenic region of haemagglutinin. 615 31

Intestinal brush borders were isolated from vitamin D-3-treated and vitamin D-deficient chicks, and protein topography in the paired preparations assessed by the enzymatic release of four marker hydrolases. Exposure of the brush borders to the protease bromelain resulted in soluble levels of alkaline phosphatase, leucine aminopeptidase, maltase, and sucrase activities from preparations of vitamin D-3-treated birds that were 42%, 75%, 64%, and 56%, respectively, of corresponding activities released in preparations from rachitic chicks. Analyses for recovery of enzyme activity revealed that bromelain treatment selectively inactivated 43% of the alkaline phosphatase activity of brush borders obtained from vitamin D-3-replete birds, and preferentially diminished recovered sucrase activity in preparations from vitamin D-deficient chicks. In additional experiments, brush borders isolated from rachitic birds were treated in vitro with the polyene antibiotic filipin or an equivalent volume of vehicle. Subsequent exposure of such preparations to bromelain resulted in little or no differences in levels of marker hydrolase specific activities released from filipin- or vehicle-treated brush borders. However, analyses of membrane-bound specific activities after treatment of brush border preparations with a range of filipin concentrations, revealed a biphasic inhibition of approx. 30% for both maltase and sucrase, relative to vehicle controls, and a smaller effect on alkaline phosphatase and leucine aminopeptidase.
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PMID:Intestinal brush border hydrolase topography. Effects of vitamin D-3 and filipin. 629 47

Adherence and invasion studies were conducted in monolayers of Caco-2 cells. Three-day-old monolayers were inoculated with Campylobacter jejuni 81-176 at a bacterium/cell ratio of 1,000:1. Saturation studies demonstrated time- and dose-dependent saturation curves for C. jejuni cell association and invasion into Caco-2 cells. Electron microscopy revealed intracellular C. jejuni located within membrane-bound vacuoles. Cell association and invasion were inhibited by 0.3 and 0.5 M concentrations of various sugars, including D-glucose, D-mannose, and D-fucose. However, there was no inhibition with the corresponding L-sugars, indicating physiological specificity. The inhibition of cell association with phloridzin was less pronounced. There was no inhibition of bacterial entry with monodansylcadaverine or g-strophanthin, indicating that it was unlikely that coated-pit formation is important in the invasion of C. jejuni into Caco-2 cells. Furthermore, there was no inhibition with cytochalasin D, vincristine, or vinblastine. Inhibition of cell association was demonstrated at 4 degrees C. Significantly decreased cell association and invasion were seen in potassium-depleted cells. Treatment of cells with bromelain also caused reduction in the number of C. jejuni binding to cells. A nonmotile aflagellate variant of C. jejuni also showed reduced invasion. The results of this study are consistent with energy-dependent invasion mechanisms. The results do not support an endocytic method of invasion for C. jejuni into Caco-2 cells.
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PMID:Cell association and invasion of Caco-2 cells by Campylobacter jejuni. 806 93

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.
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PMID:pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers. 982 2

In this study, we investigated the presystemic metabolism of trypsin and bromelain and the influence of these proteolytic enzymes on the mucus layer covering the gastrointestinal (GI) epithelia. In vitro studies demonstrated that 77.3% +/- 4.0% (mean +/- SD, n = 3) of trypsin is autodegraded within 2 hr, whereas autodegradation of bromelain was negligible. In contrast to the metabolization of bromelain by all pancreatic serine proteases, trypsin is only degraded to some extent by elastase. Both therapeutically used enzymes remained stable after incubation with an excised porcine mucosa, demonstrating that proteolysis caused by brush border membrane-bound enzymes is negligible. Trypsin and bromelain were highly mucolytic active, thereby reducing the diffusion barrier based on the mucus gel layer. Strategies to improve the galenic of dosage forms for trypsin and bromelain include the use of bioadhesive polymers such as hydroxyethylcellulose or slightly modified chitosan-EDTA, providing strongly improved stability of these enzymes toward proteolytic degradation in vitro. The given information represents a good starting point to improve the galenic of dosage forms for orally administered proteolytic enzymes.
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PMID:Peroral administration of enzymes: strategies to improve the galenic of dosage forms for trypsin and bromelain. 1069 48

In the past year there have been many advances in the area of small bowel physiology and pathology and therapy. In preparation for this review, over 1500 papers were assessed. The focus is on presenting clinically useful information for the practising gastroenterologist. Selected important clinical learning points include the following: (1) glucose absorption mediated by SGLT1 is controlled by mRNA abundance, as well as by posttranscriptional processes including protein trafficking; (2) inducers of cytochrome P-450 decrease glucose and fructose absorption and increase glucose consumption in the intestine; (3) the regulated release of nutrients from the stomach into the upper intestine ensures that the modest intestinal transport reserve capacity is not exceeded; (4) hepatocyte growth factor and short-chain fatty acids may enhance intestinal adaptation and prevent the atrophy seen when total parenteral nutrition is infused; (5) inhibitors of pancreatic lipase and phospholipase H2 may be useful clinically to reduce absorption as part of a treatment program for obesity and hyperlipidemia; (6) several membrane-bound and cytosolic proteins have been identified in the enterocyte as well as in the hepatocyte and may be the target for the future therapeutic manipulation of bile acid metabolism and control of hyperlipidemia; (7) suspect bile acid malabsorption in the patient with otherwise unexplained chronic diarrhea; (8) a proportion of lipid absorption is protein-mediated, and this opens the way to targeting these proteins and thereby therapeutically modifying lipid absorption; (9) a high protein diet may be useful to increase the intestinal absorption of drugs transported by the H+/dipeptide cotransporter; (10) a metal transporter DCT1 has been identified, and this may open the way to a better understanding of disorders of, for example, iron and zinc metabolism; (11) the nutrient transporters such as SGLT1 are responsible for a portion of the intestinal absorption of water; (12) the influence of nitric oxide on intestinal water absorption and secretion depends on its concentration; (13) a trial of bile acid-sequestering agent may prove useful in the treatment of the patient who experiences diarrhea while taking an enteral diet; (14) a proteolytic extract from pineapple stems may prove to be useful to treat diarrhea, although the mechanism of this effect remains to be established; and (15) the antisecretory effect of the new peptide, sorbin, needs to be tested in a clinical situation on patients with diarrhea. Other new and promising antidiarrheal agents include bromelain, an extract from pineapple stems, and igmesine, a final sigma ligand.
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PMID:Small bowel review: normal physiology part 1. 1176 47