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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.
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PMID:Catalytic properties of the HhaII restriction endonuclease. 299 12

Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.
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PMID:Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane. 300 80

Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine-phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A.
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PMID:A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation. 300 39

Phosphatidylinositol-4-phosphate (PtdIns-P) kinase was purified approximately 30-fold from rat brain cytosol. No contaminating activity of PtdIns kinase or of phosphomonoesterase and phospholipase C using PtdIns-P or PtdIns-P2 as substrate could be detected in the enzyme preparation. The PtdIns-P kinase activity was severalfold higher when PtdIns-P/PtdEtn vesicles rather than PtdIns-P alone were used as substrate. This might be due to increased accessibility of the enzyme for the vesicular substrate, further indicated by the lower activity obtained when PtdCho or PtdIns, phospholipids with bulky head groups, was also present in the vesicles. The product PtdIns-P2 was a competitive inhibitor with respect to PtdIns-P and 50% inhibition of enzyme activity was observed at the same product concentration regardless of whether the substrate-product mixture was presented in vesicular or micellar form, or the substrate and product were added in separate vesicles. The polyamines spermine and spermidine enhanced PtdIns-P kinase activity severalfold. Spermine also caused a shift in the MgCl2 saturation curve from sigmoidal to hyperbolic, lowering the Mg2+ concentration required for optimum kinase activity to the physiological range. Myelin basic protein enhanced the enzyme activity when PtdIns-P/PtdEtn vesicles were used as substrate, whereas it was inhibitory when PtdIns-P was added alone. The possible role of polyamines and the product PtdIns-P2 in the regulation of PtdIns-P kinase activity is discussed.
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PMID:Phosphatidylinositol-4-phosphate kinase from rat brain. Activation by polyamines and inhibition by phosphatidylinositol 4,5-bisphosphate. 302 90

A light microscopic technique based on alkaline phosphatase histochemistry was developed to specifically quantitate Type II cells in the intact rat lung. Lungs were fixed in 4% neutral-buffered formalin containing 0.25 M sucrose and embedded in glycol methacrylate. Two micron thick sections were mounted on glass microscope slides. Alkaline phosphatase activity was localized by using naphthol AS-BI phosphate as substrate in 0.125 M 2-amino-2-methyl-1-propanol buffer containing 0.625 mM MgCl2 (pH 8.9). Sections were counterstained with Harris hematoxylin. Type II cells were the only cell type in the alveolar region containing alkaline phosphatase activity, an observation that was confirmed by using electron microscopic histochemistry. By combining the alkaline phosphatase staining technique with standard morphometric procedures, the proliferative response to a single intratracheal dose of 10 mg silica was followed as a function of time. Type II cells were significantly increased at all time points examined. Twenty eight days following silica, Type II cells had increased to (252 +/- 16) X 10(6) cells per set of lungs compared to a control value of (141 +/- 32) X 16(6) cells. The method presented is a simple and rapid technique for examining Type II cell population kinetics.
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PMID:Quantitation of silica-induced type II cell hyperplasia by using alkaline phosphatase histochemistry in glycol methacrylate embedded lung. 303 99

The development of optimal methods for preservation is important for the advancement of liver transplantation. This study compares hypothermic storage (HS) and hypothermic pulsatile perfusion (HPP) with various solutions, using an isolated normothermic perfusion model (LIPM). Canine livers were removed from mongrel dogs without warm ischemia and flushed with either heparinized Ringer's lactate (control and HPP-preserved groups) or the solution used for hypothermic storage (TP-V or modified Collins). The type of preservation and solution for each of the experimental groups was as follows: group I (n = 7), no preservation, fresh; group II (n = 7), 24-h HS with TP-V (a hyperosmolar colloid solution containing sucrose, dextrose, and ATP-MgCl2); group III (n = 7), 24-h HS with modified Collins (C-2), an intracellular crystalloid solution; group IV (n = 5), 24-h HP with TP-V; group V (n = 6), 24-h HPP with Belzer solution, containing ATP-MgCl2; group VI (n = 3), 24-h HPP with albumin. After the preservation period, livers were placed on HPP at 37 degrees C with albumin-mannitol solution for 3-h testing in an LIPM. Perfusate samples were taken at 1-h intervals to assess liver function. LDH, SGOT, alkaline phosphatase, lactic acid, LAP, GGT, pO2, pCO2, pH, osmolarity, AMP, ADP, and ATP were studied. Histologic studies were performed, as were representative HIDA scans. Using the LIPM, livers preserved by HS and HPP with TP-V solution appeared to be superior to those preserved with modified Collins, Belzer, and albumin solutions. In these non-TP-V groups, the greatest cellular and organ damage was observed. TP-V HPP appeared to give the best overall liver functional response and histologic results and is recommended as the preferred method for 24-h liver preservation.
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PMID:Liver preservation techniques for transplantation. 330 26

The 3 or 4 phosphate ester of dopamine (PD) was hydrolyzed by homogenates of rat tissues to give inorganic phosphate (Pi) and dopamine. The rate of hydrolysis of PD by kidney homogenates was increased by exogenous MgCl2 but not CaCl2 or KCl. The activity of brain, heart or liver homogenates was insensitive to the added salts. Several lines of evidence indicate that alkaline phosphatase activity contributes to the high rate of PD hydrolysis by the kidney but not brain homogenate. The intravenous infusion of PD at 12 mumole/kg in one hr to anesthetized rats increased the dopamine content of the plasma, kidney and heart without altering brain or liver dopamine. The results suggest that PD may be more effective than dopamine for increasing dopamine levels of the kidney. In addition, the hydrolysis of PD by brain homogenates, which is independent of alkaline phosphatase activity, suggests that specific enzymes exist for the metabolism of PD.
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PMID:Pharmacologic properties of a phosphate ester of dopamine. 358 52

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.
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PMID:In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine. 609 74

Incubation of rat liver nuclear envelopes with [gamma-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidylinositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.
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PMID:Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes. 609 90

Rat kidney cortex slices were homogenized with a polytron in a isoosmotic medium containing 5 mmol/l EGTA. By two precipitations with MgCl2 (12 mmol/l) and differential centrifugation, brush border membranes were purified. The brush border marker enzymes alkaline phosphatase and aminopeptidase M were found to be enriched 17.0 +/- 5.3-fold and 16.7 +/- 3.7-fold, respectively. By this method, a high yield of brush border membranes was obtained (48.3 +/- 7.9% for alkaline phosphatase; 47.0 +/- 9.5% for aminopeptidase M). The acid phosphatase was enriched 5-fold, whereas other lysosomal enzymes (glucosaminidase, glucuronidase, cathepsin D) were enriched only 0.2-fold. Acid phosphatase activity could not be washed out, but could be separated from alkaline phosphatase and leucine aminopeptidase by means of free flow electrophoresis and sucrose density gradient centrifugation. Vesicles prepared by the presently described Mg/EGTA-method show better transport properties, compared to vesicles prepared by the calcium method of Evers et al. (Evers, C., Haase, W., Murer, H. and Kinne, R. (1978) Membrane Biochem. 1, 203-219), whereas by SDS-polyacrylamide gel electrophoresis, no differences in the protein patterns were observed.
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PMID:A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers. 611 19


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