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 role of phosphatidylinositol-specific phospholipase C (PIase C) in a) the enigmatic phosphatidylinositol (PI) turnover and b) in our understanding of membrane enzyme-PI interactions is the subject matter of this article. PIase C is present in both procaryotes and eukaryotes. This enzyme is considered to be involved in the cells PI breakdown which occurs in response to several external stimuli. Recent information on the physical properties, Ca2+ requirement, cellular localization and modulation of the activity of PIase C of mammalian systems can help to evaluate the PI turnover from a new angle. Existing evidence suggests that Ca2+-dependent PI breakdown is probably mediated through the cytosolic and particulate PIase C while a Ca2+ independent pathway is catalyzed by a lysosomal enzyme. Apparently PI turnover may be operating through more than one mechanism. The association of this phenomenon with a membrane receptor event linked with "Ca2+ gating" may have to be reconsidered. Modulation of the PIase C activity by unsaturated amphiphiles or the presence of this enzyme in different physico-chemical forms could be a potential regulatory feature. Hydrolysis of membrane PI of a number of cells and tissues by the bacterial PIase C has been shown to cause substantial release of acetylcholinesterase, alkaline phosphatase and 5'-nucleotidase in free, soluble form. Other membrane enzymes, e.g., alkaline phosphodiesterase I, L-leucyl-beta naphthyl amidase and Ca2+ or Mg2+ ATPase are not affected. These results indicate a specific interaction between PI and certain enzymes in membranes. The chemical nature of this linkage, whether it is covalent or non-covalent, has also been explored and has provided intriguing insight into this phenomenon. New findings also indicate that hydrolysis of PI by PIase C also can cause modifications in membrane-enzyme activities, e.g., adenylate cyclase.
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PMID:Minireview. Phosphatidylinositol specific phospholipases C. 708 67

The mechanism of release of alkaline phosphatase from membranes by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was studied. Alkaline phosphatase was readily released from pig kidney microsomes by phospholipase C but not by a variety of other treatments, e.g., high ionic strength, extremes of pH, divalent cations, chelating agents, or analogues of the polar head group of phosphatidylinositol. Alkaline phosphatase released from microsomes by phospholipase C did not bind to phospholipid vesicles containing phosphatidylinositol. Alkaline phosphatase solubilized from microsomes by butanol extraction, however, was able to bind phospholipid vesicles even when they contained no phosphatidylinositol. The ability of butanol-extracted alkaline phosphatase to bind to phospholipid vesicles was destroyed by added phosphatidylinositol-specific phospholipase C. Hydrolysis of added phosphatidylinositol by endogenous phospholipase activity in butanol extracts was also accompanied by loss of binding ability. Loss of binding ability was paralleled by a decrease in the apparent molecular weight of alkaline phosphatase. These results indicate that alkaline phosphatase is attached to membranes by a strong interaction with phosphatidylinositol.
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PMID:Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes. 740 77

Membrane proteins can be attached to the plasma membrane in several ways. Recently, a mechanism has been described, by which a number of cell surface proteins are anchored to the exoplasmic side of the plasma membrane by covalent linkage to glycosyl-phosphatidylinositol (GPI). The growth properties of renal epithelial cells in tissue culture enable free access to apical cell surface and brush border membrane proteins. To study the nature of membrane anchoring of apical plasma membrane enzymes in cultured renal epithelial cells, confluent LLC-PK1, OK, NRK, and MDCK epithelia were treated in tissue culture dishes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the PI-PLC-specific release into the tissue culture medium of the apical membrane enzymes alkaline phosphatase (AP), gamma-glutamyl transpeptidase, leucine aminopeptidase, trehalase, and maltase was determined. Of the five enzymes tested, AP and trehalase, already described as GPI-anchored membrane proteins, were specifically released by PI-PLC from intact cell monolayers. Of the four cell lines investigated, LLC-PK1 cells express AP and trehalase which were released by PI-PLC. In OK cells, which lack AP activity, only trehalase was found to have PI-PLC-releaseable enzyme activity. MDCK cells, on the other hand, express AP activity, releaseable by PI-PLC, but no trehalase activity. In studies on the time course of synthesis and reinsertion of AP into the apical membrane of LLC-PK1 cells after removal by PI-PLC, a 60% recovery of AP activity was obtained only after 7 days. Analysis of protein release by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture supernatants after surface labeling with biotin and subsequent Western blotting with streptavidin revealed four protein bands at approximately 130, 90, 30, and 20 kD in LLC-PK1 cells and five GPI-anchored proteins at 110, 85, 65, 40, and 26 kD in OK cultures. The finding of a PI-PLC-specific release of apical membrane enzymes from renal tubular cell lines of different species (pig, opossum, rat, and dog) and of different nephron origin indicates a high conservation of the GPI anchor of renal brush border membrane proteins and further proves the high degree of differentiation retained by the cell lines in tissue culture. In addition, this method may provide a possible tool for isolating GPI-anchored apical membrane proteins from intact epithelial monolayer cultures.
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PMID:Selective release of apical membrane enzymes from cultured renal epithelia by phosphatidylinositol-specific phospholipase C. 750 39

In rat jejunal brush-border membranes (BBM), ATP hydrolysis activity was specifically stimulated by CaCl2 and by MgCl2, allowing to identify Ca(2+)-ATPase and Mg(2+)-ATPase activities with a broad pH optimum near 8.0. Nonspecific ATPase activity (in the absence of cations) had a pH optimum above 9.5 as alkaline phosphatase. The effects of Ca2+ and Mg2+ concentrations on ATPase activity evidenced two apparent KA for each cation. At high concentrations, a similar affinity for both cations was recorded (KA: 0.35 mM). At low concentrations, the affinity for Mg2+ was greater than for Ca2+ (KA: 0.02 mM and 0.07 mM respectively). In an attempt to differentially solubilize alkaline phosphatase and ATPase activities, eleven different detergents were assayed. They more or less successfully released Ca(2+)-ATPase and Mg(2+)-ATPase activities from BBM but the more membranes were solubilized by a detergent, the more activities were lost, suggesting a close dependence on integration in BBM. As to alkaline phosphatase and nonspecific ATPase, they almost co-solubilized with Ca(2+)-ATPase and Mg(2+)-ATPase but their total activity was little affected. After treatment of BBM with phosphatidylinositol-specific phospholipase C (E.C. 3.1.4.10), 58% of alkaline phosphatase activity and 45% of nonspecific ATPase activity were released in the supernatant while Ca(2+)-ATPase and Mg(2+)-ATPase activities remained totally incorporated in BBM pellets. These last results definitively demonstrate that Ca(2+)-ATPase and Mg(2+)-ATPase activities are not manifestations of alkaline phosphatase, as earlier suggested, but rather result from the existence of one or several intrinsic membrane enzymes.
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PMID:Ca(2+)-ATPase and Mg(2+)-ATPase activities distinct from alkaline phosphatase in rat jejunal brush-border membranes. 751 33

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.
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PMID:Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase. 751 12

In human neutrophils, alkaline phosphatase (AlkPase), a low-affinity receptor for IgG (FcRIIIB), and complement decay accelerating factor (DAF) are glycosyl-phosphatidylinositol (GPI)-anchored proteins. Varying greatly in biological function these three integral membrane proteins exhibit regulated cell surface expression in neutrophils. Defined by their common membrane-linkage motif, AlkPase, FcRIIIB, and DAF can be released from the lipid bilayer by the action of phosphatidylinositol-specific phospholipase C and are relatively resistant to low temperature extraction with Triton X-100 (TX-100). In this study we show that neutrophil AlkPase, FcRIII, and DAF display differential extractibility; they are relatively insensitive to TX-100 solubilization at 4 degrees C, but are readily extracted with TX-100 at 37 degrees C or by the detergent octyl glucoside at 4 degrees C. The differential extractibility of these GPI-anchored proteins is the same in unstimulated cells, where these proteins exist primarily in an intracellular pool, and stimulated cells, where they are expressed principally at the cell surface. However, no differential extraction effect is observed with two neutrophil transmembrane proteins, complement receptor 1 (CD35, CR1) and MHC Class I in either stimulated or unstimulated cells.
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PMID:Solubilization of glycosyl-phosphatidylinositol-anchored proteins in quiescent and stimulated neutrophils. 753 73

Two cDNAs encode rat intestinal alkaline phosphatases having completely different carboxyl-terminal peptides; one is hydrophobic and fulfills the consensus requirements for glycan phosphatidylinositol linkage, and the other is neither hydrophobic nor hydrophilic, but contains a small amino acid domain (-NSASS-) just distal to a region of 17 threonine residues. Constructs were created using 80% of the amino-terminal portion of one alkaline phosphatase and the carboxyl-terminal portions of each of the isoforms. Both of the carboxyl-terminal peptides supported glycan phosphatidylinositol linkage as demonstrated by the following criteria: 1) plasma membrane targeting in transfected COS-1 cells, 2) release of transfected alkaline phosphatase by phosphatidylinositol-specific phospholipase C, 3) appearance of the trypanosome variable glycoprotein cross-reacting determinant after phospholipase C treatment, 4) ethanolamine incorporation into newly synthesized enzyme, 5) loss of phospholipase C release after mutation of the omega and omega + 2 positions in the putative linkage site, -NSA-, and 6) evidence of surface membrane localization by immunofluorescence using antibody against rat intestinal alkaline phosphatase. These data demonstrate that a predicted hydrophobic carboxyl-terminal sequence is not essential for glycan phosphatidylinositol linkage. Moreover, because both isomers are membrane-bound, the origin of soluble enzyme in the serum is likely to arise from the action of serum phosphatidylinositol-specific phospholipase C.
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PMID:Two rat intestinal alkaline phosphatase isoforms with different carboxyl-terminal peptides are both membrane-bound by a glycan phosphatidylinositol linkage. 774 44

The isoenzymic properties of the alkaline phosphatase (ALP) of the gingival crevicular fluid (GCF) were investigated and compared with those in other cells, such as human polymorphonuclear leukocytes (PMNs), and human periodontal ligament cells (PDLs), and with those of three species of periodontopathic bacteria: Porphyromonas gingivalis 381 (P. gingivalis), Prevotella intermedia ATCC25611 (P. intermedia), and Capnocytophaga sputigena ATCC33123 (C. sputigena). The biochemical properties of the isoenzymes were analyzed by the following methods: enzyme assays, inhibition pattern using three chemical inhibitors, 4 to 20% gradient polyacrylamide gel electrophoresis, thermostability, immunological specificity, and phosphatidylinositol-specific phospholipase C (PI-PLC) treatment. The inhibition experiment showed that ALP of the PMNs and PDLs possessed almost the same enzymatic properties of tissue-nonspecific ALP (bone/liver/kidney; TNSALP), and the ALP of the three species of periodontopathic bacteria possessed specific properties that were different from those of TNSALP, intestinal, or placental ALP. The ALP of the GCF was only slightly susceptible to levamisole (1 mM), L-phenylalanine (20 mM), and SDS (1%). An electrophoresis thermostability test demonstrated that the enzyme activity of the GCF was separated into one or two bands. The main heat-labile slow band contained the phosphatidylinositol (PI)-moiety-anchored ALP and possessed immunological specificity against anti-bone type ALP. The minor fast band was heat stable and showed mobility similar to that in P. gingivalis. These results indicated that the ALP of the GCF consisted of several ALP isoenzyme types whose possible origins are considered to be derived from phosphatidylinositol (PI) anchored ALP and periodontopathic bacterial ALP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties of alkaline phosphatase in the gingival crevicular fluid. 779 88

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.
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PMID:Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C. 798 Apr 26

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited "Michaelian" kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.
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PMID:Osseous plate alkaline phosphatase is anchored by GPI. 808 Dec 65


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