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)

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.
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PMID:Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis. 318 12

The activities of individual enzymes of the isoprenoid pathway from mevalonate kinase to squalene synthetase in homogenates of seeds germinated up to 32h were assayed. Changes in the activity of each enzyme were observed and compared with the activity at the 2h germination stage. Activities of alkaline phosphatase and fructose 1,6-diphosphate aldolase were similarly measured to provide a reference for changes in the general metabolic activity of seeds during imbibition of water. Water uptake reached a plateau after 12h. The reference enzymes almost doubled in activity between 2 and 8h and thereafter their activities steadily declined. All of the enzymes of the isoprenoid pathway increased in activity between 2 and 6h and, thereafter, with the exception of the prenyltransferase, their activities remained relatively constant. With the prenyltransferase activity the initial increase was followed by a short plateau between 6 and 9h and then a second increase to a maximum between 14 and 16h. After 16h the activity declined. The relative activities of the isoprenoid enzymes at 16h of germination were mevalonate kinase>phosphomevalonate kinase>pyrophosphomevalonate decarboxylase approximately isopentenyl pyrophosphate isomerase>squalene synthetase>isopentenyl pyrophosphate/dimethylallyl pyrophosphate prenyltransferase. The finding that the prenyltransferase may be the rate-limiting enzyme in squalene synthesis from mevalonate is discussed in relation to regulation of isoprenoid synthesis during pea-seed germination.
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PMID:Development of the activities of enzymes of the isoprenoid pathway during early stages of pea-seed germination. 434 63

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.
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PMID:Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium. 1128 53

Lowering the growth temperature of HepG2 cells from 37 degrees C to 20 degrees C results in a 73% reduction in human squalene synthase (HSS) protein, a 76% reduction in HSS mRNA, and a 96% reduction in promoter activity of a secreted alkaline phosphatase-HSS reporter gene. A similar decrease in either mRNA or protein levels is observed for 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl diphosphate synthase, the LDL receptor, and fatty acid synthase. All these proteins and mRNAs show either a decrease or a complete loss of sterol-dependent regulation in cells grown at 20 degrees C. In contrast, sterol regulatory element binding proteins (SREBPs)-1 and -2 exhibit a 2- to 3-fold increase in mRNA levels at 20 degrees C. The membrane-bound form of the SREBPs is dramatically increased, but the proteolytic processing to the nuclear (N-SREBP) form is inhibited under these conditions. Overexpression of the N-SREBP or SREBP cleavage-activating protein (SCAP), but not site-1 or site-2 proteases, restores the activation of the HSS promoter at 20 degrees C, most likely by liberating the SCAP-SREBP complex so that it can move to the Golgi for processing. These results indicate that the cholesterol synthesizing machinery is down-regulated at low temperatures, and points to the transport of the SCAP-SREBP complex to the Golgi as the specific down-regulated step.
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PMID:Low-temperature effect on the sterol-dependent processing of SREBPs and transcription of related genes in HepG2 cells. 1275 79

A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cerevisiae. The strain was blocked at squalene synthase (ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E,E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E,E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography-mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCl, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15 mM MgCl(2) provided optimum conditions for the stabilization of FPP.
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PMID:Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants. 1275 56

In the present in vitro study, we tested the hypothesis that parathyroid hormone-related protein (PTHrP) might be a mediator of interleukin-6 (IL-6) and its soluble receptor (IL-6sR) in osteoblasts. We found that IL-6, within 1-20 ng/mL, added together with IL-6sR (100 ng/mL), rapidly (1 hour) increased PTHrP mRNA in human osteoblastic osteosarcoma MG-63 cells and human osteoblastic (hOB) cells from trabecular bone. PD098059, a mitogen-activated protein kinase (MAPK) kinase inhibitor, at 10 microM, and two inhibitors of protein prenylation and thus Ras activation, simvastatin (1 microM) and a farnesyltransferase (FTase) inhibitor (100 nM), but not the phosphatidylinositol 3-kinase inhibitor wortmannin, blocked the IL-6/IL-6sR-induced PTHrP expression in these cells. In addition, PD098059 as well as simvastatin and the FTase inhibitor abolished alkaline phosphatase activity and/or osteocalcin mRNA induction by the IL-6/IL-6sR in these cells. Our results support the role of the Ras/MAPK pathway as a major mechanism in the modulation of both PTHrP expression and differentiation in human osteoblasts.
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PMID:The interleukin-6/soluble interleukin-6 receptor system induces parathyroid hormone-related protein in human osteoblastic cells. 1512 68

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. Statins inhibit 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (HMGCR), the first step of the isoprenoid biosynthetic pathway, leading to the depletion of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The effects of statins on bone have previously been attributed to the depletion of GGPP, because the addition of exogenous GGPP prevented statin-stimulated osteoblast differentiation in vitro. However, in a recent report, we demonstrated that the specific depletion of GGPP did not stimulate but, in fact, inhibited osteoblast differentiation. This led us to hypothesize that isoprenoids upstream of GGPP play a role in the regulation of osteoblast differentiation. We demonstrate here that the expression of HMGCR and FPP synthase decreased during primary calvarial osteoblast differentiation, correlating with decreased FPP and GGPP levels during differentiation. Zaragozic acid (ZGA) inhibits the isoprenoid biosynthetic pathway enzyme squalene synthase, leading to an accumulation of the squalene synthase substrate FPP. ZGA treatment of calvarial osteoblasts led to a significant increase in intracellular FPP and resulted in inhibition of osteoblast differentiation as measured by osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization. Simultaneous HMGCR inhibition prevented the accumulation of FPP and restored osteoblast differentiation. In contrast, specifically inhibiting GGPPS to lower the ZGA-induced increase in GGPP did not restore osteoblast differentiation. The specificity of HMGCR inhibition to restore osteoblast differentiation of ZGA-treated cultures through the reduction in isoprenoid accumulation was confirmed with the addition of exogenous mevalonate. Similar to ZGA treatment, exogenous FPP inhibited the mineralization of primary calvarial osteoblasts. Interestingly, the effects of FPP accumulation on osteoblasts were found to be independent of protein farnesylation. Our findings are the first to demonstrate that the accumulation of FPP impairs osteoblast differentiation and suggests that the depletion of this isoprenoid may be necessary for normal and statin-induced bone formation.
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PMID:Effects of farnesyl pyrophosphate accumulation on calvarial osteoblast differentiation. 2158 55

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