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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bilirubin UDP-glucuronyltransferase displays marked latency in native microsomes. To examine whether this latency correlates with structural integrity of the microsomal vesicles and reflects lumenal orientation of the enzyme's catalytic center, we analyzed the relationship between transferase activity and the degree of expression of mannose (Man)-6-phosphatase, which is a marker enzyme of the cisternal face of the ER membrane. Using detergent, sonication, or the pore-forming Staphylococcus aureus alpha-toxin to breach the microsomal membrane permeability barrier, we found that after each of these pretreatments a remarkably close direct relationship existed between latency changes for bilirubin UDP-glucuronyltransferase and Man-6-phosphatase. This finding suggested that the transferase may have the same transverse topology as the phosphohydrolase. We also compared the effects of membrane-impermeant proteinases on bilirubin UDP-glucuronyltransferase activity in native and disrupted microsomes. Whereas the unspecific proteinase nagarse markedly inactivated (to less than 30% of activities in controls) the transferase in disrupted microsomes, treatment with the proteinase had little effect on transferase activity in sealed microsomal vesicles. The results suggest that the active site of bilirubin UDP-glucuronyltransferase is on the lumenal face of the endoplasmic reticulum membrane. It was also found that activation of transferase activity by UDP N-acetylglucosamine, which is the presumed allosteric effector of UDP-glucuronyltransferase, was markedly altered by relatively small changes in structural integrity of the microsomes and totally abolished when latency of Man-6-P hydrolysis fell below approximately 80%. Collectively, these findings demonstrate that the microsomal membrane permeability barrier is a major determinant of expression of microsomal UDP-glucuronyltransferase activity and that quantitative assessment of integrity of the microsomes is essential for studying kinetic properties and regulation of microsomal UDP-glucuronyltransferase.
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PMID:Topology and regulation of bilirubin UDP-glucuronyltransferase in sealed native microsomes from rat liver. 313 Aug 1

The behaviour of Ca2+ ATPase activity in relation to Ca2+ transport process was studied under different experimental conditions in canine cardiac microsomal fraction predominantly containing sarcoplasmic reticulum. The total Ca2+ concentration required for half maximal activation (Ka) of microsomal Ca2+ ATPase and Ca2+ uptake did not differ significantly, unless 0.1 mmol/l EGTA was present in the incubation media. Pretreatment of cardiac microsomes with membrane disruptive agents like phospholipase A, trypsin as well as deoxycholate strongly increased (2-3 fold) Ca2+ ATPase activity but uptake rate of Ca2+ declined. Only in phospholipase C and beta-glucuronidase pretreatment, a parallel decrease of Ca2+ ATPase and uptake was observed. In presence of excess (free)Ca2+ (greater than 10 mumol/l) both Ca2+ ATPase as well as Ca2+ uptake were inhibited, however, Ca2+ binding process remained unaltered. Likewise, low pH completely altered the relation between Ca2+ binding and ATPase activity; whereas Ca2+ ATPase was inhibited, Ca2+ binding did not change. Our present data provide evidence for some cellular factors that may be involved in producing uncoupling of microsomal Ca2+ ATPase from Ca2+ accumulation process that was previously observed in various pathological situations.
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PMID:Behaviour of cardiac microsomal Ca2+ pump under conditions that may simulate pathological situations. 316 76

We present data indicating that aminoacylase I (EC 3.5.1.14) from porcine kidney and 'renal dipeptidase' (EC 3.4.13.11) are closely related. We show that, in situ, a considerable fraction of aminoacylase activity ist attached to membranes. Incubation of washed microsomal membranes with phospholipase C from B. cereus results in the rapid solubilization of aminoacylase I, suggesting that aminoacylase--as shown for renal dipeptidase before--bears a glycolipid 'membrane anchor'. In agreement with this assumption, purified aminoacylase was found to contain myo-inositol, a characteristic component of phosphatidylinositol-anchored membrane proteins. A reexamination of the molecular mass of purified aminoacylase yielded values (46,000 +/- 2,000 Da by SDS polyacrylamide electrophoresis, 98,000 +/- 5,000 Da by sedimentation equilibrium centrifugation) similar to those reported for renal dipeptidase. The enzymes coelute during most of the procedures applied in the purification of aminoacylase or renal dipeptidase, but can be separated by hydrophobic interaction chromatography. A survey of the literature revealed a series of additional features of aminoacylase I and renal dipeptidase (amino-acid composition, isoelectric points, metal dependence, and more) that are strikingly similar.
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PMID:Further characterization of porcine kidney aminoacylase I reveals close similarity to 'renal dipeptidase'. 322 87

Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 x 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 x 10(3). The size difference between the 125 x 10(3). The size difference between the 125 x 10(3) Mr skeletal muscle form and the 120 x 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 x 10(3) which is processed by microsomal membranes to yield a 122 x 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 x 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.
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PMID:Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle. 325 57

Rats treated with a single 0.5 ml/kg dose (ip) of CCl4 exhibited a threefold increase in liver microsomal phospholipase C (PLC) activity that was enhanced by phenobarbital and diminished by metyrapone pretreatment, respectively. Hepatocytes and hepatocellular fractions exposed to 0.5 mM CCl4 in vitro also exhibited a rapid rise in PLC activity that was reduced by metyrapone. Metyrapone also reduced the CCl4-related increase in the PLC-mediated reductions in cellular phosphatidylcholine content. The influence of CCl4 biotransformation on the activation of liver cell PLC was assessed in vitro. Covalent binding of 14CCl4 metabolites to isolated hepatocyte proteins and lipids was linear through 20 min of incubation and then quickly plateaued. The association of CCl4 metabolites with cellular phospholipids was inhibited by metyrapone and preceded the CCl4-dependent rise in PLC activity. CCl4-mediated increases in PLC activity were rapid and preceded reductions in cell viability. The translocation of cytosolic PLC to membranes such as the endoplasmic reticulum may explain the rapid, metabolite-dependent activation of PLC.PLC activation by haloalkanes was proportional to dose and incubation time in the order of CBrCl3 greater than CCl4 greater than CHCl3 greater than CFCl3 which corresponds to the observed hepatotoxic potential of these agents in vivo and in vitro. Haloalkane-dependent increases in PLC activity were inhibited by metyrapone. These results suggest that chemical metabolites activate PLC in vitro and in vivo. Therefore, the activation of a PLC that degrades membrane phospholipids may represent an important step in the pathogenic scheme of chemical-mediated liver cell necrosis.
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PMID:The role of CCl4 biotransformation in the activation of hepatocyte phospholipase C in vivo and in vitro. 342 Jun 13

Phospholipase C activity and diglyceride lipase activity were studied in the renal cortex and medulla of 10- and 40-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched normotensive Wistar-Kyoto rats (WKY). Enhanced phospholipase C activity was found in the cortical and medullary cytosol of kidney from SHRSP, and microsomal diglyceride lipase in SHRSP also increased. In SHRSP, phospholipase C and diglyceride lipase activities increased with age, but this increase was not evident in WKY. Phospholipase C had high substrate specificity for phosphatidylinositol in renal cytosol of both WKY and SHRSP. The increased activities were accompanied by prostaglandin E2 synthesis in renal medullary microsomes of 10-week-old SHRSP and were also present in the kidney of 40-week-old SHRSP. Total phospholipid and arachidonic acid contents in kidney were markedly high in the medulla of 10-week-old SHRSP, but these lipids were decreased in 40-week-old SHRSP. These results suggest that phospholipids and arachidonic acid in SHRSP may be genetically high and that the activated phospholipase C and diglyceride lipase hydrolyze phospholipids, providing arachidonic acid for prostaglandin synthesis, which results in a decrease of phospholipids and arachidonic acid in the kidney of 40-week-old SHRSP. These studies demonstrate that a phosphatidylinositol-specific phospholipase C-prostaglandin synthetic system may play an important role in the course of hypertension in SHRSP.
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PMID:Renal phospholipase C and diglyceride lipase activity in spontaneously hypertensive rats. 347 8

Accumulating evidence suggests that the increase in cytosolic Ca2+ induced by receptor agonists is mediated by inositol 1,4,5-trisphosphate, a product of phospholipase C-mediated breakdown of phosphatidylinositol 4,5-bisphosphate. The present study employs inositol tris[32P]phosphate to demonstrate a specific receptor binding site in a microsomal fraction of rat liver.
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PMID:Binding of inositol trisphosphate by a liver microsomal fraction. 348 56

The characteristics of the specific binding of labelled insulin to turtle thyroid microsomes were investigated. Binding experiments were performed in Krebs-Ringer bicarbonate buffer (pH 7.4) at 25 or 4 degrees C for different periods of time. Dissociation of the labelled insulin from the binding sites was also evaluated. It was found that the binding is dependent on time, temperature and microsomal protein concentration, with an optimum pH of 8.0. Unlabelled insulin and pro-insulin competed with the labelled insulin, binding in direct proportion to their biological activities, while glucagon and growth hormones did not compete for the binding sites. Scatchard plot analysis established the presence of binding sites of high and low affinities, and the rate of dissociation of bound insulin was considerably increased by the addition of unlabelled insulin. Both results are compatible with a negative co-operativity site-site interaction model. Trypsin abolished the insulin binding. These findings indicate that the microsomes from the turtle thyroid gland contain specific binding sites for insulin. However, pre-incubation of microsomes with phospholipase C or S-adenosyl-L-methionine (SAM), or incubation in the presence of 2 mol NaCl/l did not increase the specific insulin binding. Therefore, the binding properties are similar to those observed in mammalian insulin-responsive tissues except for the absence of the effects of 2 mol NaCl/l, phospholipase C or SAM, which suggests the absence of masked insulin-binding sites.
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PMID:Characterization of the insulin-binding sites in turtle thyroid microsomes. 351 Nov 68

Epididymal 4-ene steroid 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal fractions, and the activity can be altered by modifying the phospholipids in the membrane environment. To investigate the membrane dependence of 4-ene steroid 5 alpha-reductase, we have treated nuclear and microsomal membranes with combinations of phospholipase A2 and phospholipase C, and examined the effects on 4-ene steroid 5 alpha-reductase activity. Sequential addition of phospholipase A2 and phospholipase C to the nuclear fraction, reduced the 4-ene steroid 5 alpha-reductase activity to approx 25% of the control level. Neither the nature of the phospholipase, nor the sequence of addition altered the inhibition. When both phospholipases were added simultaneously, nuclear 4-ene steroid 5 alpha-reductase activity was inhibited in a linear fashion, and in tests for cooperativity, the effects of phospholipase A2 and phospholipase C were clearly additive. The microsomal enzyme responded differently to sequential phospholipase treatments; if phospholipase A2 was followed by phospholipase C, or phospholipase C followed by phospholipase A2, the 4-ene steroid 5 alpha-reductase activity was, respectively, 13 and 27% of the control. In contrast, sequential addition of the same phospholipase reduced the activity of 4-ene steroid 5 alpha-reductase to approx 40% of the control level. Furthermore, simultaneous addition of phospholipase A2 and phospholipase C to the microsomal fraction, resulted in non-linearity of 4-ene steroid 5 alpha-reductase activity with time, whereas when added individually, linearity of 4-ene steroid 5 alpha-reductase was maintained. Consequently, it was not possible to test for cooperative effects of phospholipases on the microsomal 4-ene steroid 5 alpha-reductase. These findings suggest that for the nuclear 4-ene steroid 5 alpha-reductase, the polar and non-polar regions of the membrane environment have similar functions, which are most likely involved in the maintenance of the structural integrity of the enzyme. For the microsomal enzyme, the polar and non-polar regions of the membrane appear to have different functions, not only for the maintenance of enzyme integrity, but also in the mechanism at the active site.
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PMID:Differential effects of combinations of phospholipase A2 and phospholipase C on the activity of rat epididymal nuclear and microsomal 4-ene steroid 5 alpha-reductase. 358 75

Previous attempts in several laboratories, including ours, to purify oligosaccharyl-transferase have met with limited success because of the lability of the membrane-associated enzyme after solubilization with detergents. In an effort to identify the enzyme in face of this lability, we recently developed a photoaffinity reagent to label the active site [J. K. Welply, P. Shenbagamurthi, F. Naider, H. R. Park, and W. J. Lennarz (1985) J. Biol. Chem. 260, 6459-6465]. In this report, the preparations of a more sensitive selective labeling probe, 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys-(N epsilon-p-azidobenzoyl)-Thr-NH2, is described. Using this new probe, we have confirmed, independently of catalytic activity, that hen oviduct oligosaccharyltransferase is tightly associated with the endoplasmic reticulum membrane. The 125I-labeled oligosaccharyltransferase was released from the membrane by detergent and strong alkali treatments but not by sonication, high salt, or hypotonic shock. However, all procedures that released the enzyme from the membrane resulted in a dramatic loss of enzyme activity. Treatment of sealed microsomal membrane vesicles with phospholipase A resulted in nearly complete enzyme inactivation; in contrast, phospholipase C or D had moderate or little effect, respectively. Taken together, these results suggest that the hydrophobic environment of the membrane is required for oligosaccharyltransferase activity. Trypsin treatment of intact vesicles diminished enzyme activity by nearly 70%, but it had no effect on the binding affinity of the enzyme for the 125I-labeled photoaffinity probe. This result suggests that the polypeptide acceptor portion of oligosaccharyltransferase is lumenally disposed, and that a trypsin-sensitive, cytoplasmically oriented domain or another subunit binds the carbohydrate donor, dolichol-PP-oligosaccharide.
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PMID:Studies on properties of membrane-associated oligosaccharyltransferase using an active site-directed photoaffinity probe. 370 33


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