A novel phosphatase upregulated inAkp3knockout mice

Reexamination of the Akp3−/−mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the Akp3−/−gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel murine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner ( gIAP) as opposed to duodenum-specific IAP ( dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant ( kcat) and a 30% drop in Km, leading to a 4-fold reduction kcat/ Kmcompared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of Akp3−/−mice. Developmentally, Akp3 expression is turned on during postnatal days 13–15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in Akp3−/−mice fed a high-fat diet.

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Differential regulation of mRNAs encoding for rat intestinal alkaline phosphatase

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1990.259.1.g93 ◽

1990 ◽

Vol 259 (1) ◽

pp. G93-G98 ◽

Cited By ~ 2

Author(s):

R. Eliakim ◽

S. Seetharam ◽

C. C. Tietze ◽

D. H. Alpers

Keyword(s):

Alkaline Phosphatase ◽

Amino Acid ◽

Cdna Probe ◽

Fold Increase ◽

Maximal Activity ◽

Amino Acid Residues ◽

Intestinal Alkaline Phosphatase ◽

Mrna Accumulation ◽

Dihydroxyvitamin D3 ◽

Fat Feeding

A cDNA probe encoding the entire structural region of the 62-kDa rat intestinal alkaline phosphatase from amino acid residues 1 to 531 detected multiple mRNA species (3.0, 2.7, and 2.2 kb) in rat intestinal RNA. The 3.0-kb species was most evident in duodenum but could be easily detected in jejunum using a 48-mer oligonucleotide encoding amino acid residues 492-508. This 48-mer oligonucleotide bound preferentially to the 3.0-kb mRNA, suggesting that the 2.7-kb mRNA differed in this region. To determine whether each of the mRNAs encoding rat intestinal alkaline phosphatase responded coordinately to physiological stimuli, the full-length cDNA and the 48-mer oligonucleotide were used as probes for the 2.7- and 2.2-kb and the 3.0-kb mRNAs, respectively. Intestinal mRNA concentration was measured by Northern blot analysis in acute (single feed, 17 kcal) and chronic (3 wk, 30% fat diet) fat feeding and in rachitic rats after 1,25-dihydroxyvitamin D3 therapy. There was a large increase (8- to 25-fold) in the 3.0-kb mRNA 7 h after acute fat feeding, with a much smaller increase (1.4- to 5.0-fold) in the 2.7- and 2.2-kb species. The peak in 3.0-kb mRNA accumulation correlated in time with the maximal activity of serum phosphatase activity after acute fat feeding (4- to 5-fold increase). In contrast, there was a much smaller increase in all mRNAs and in tissue and serum enzyme activity after chronic fat feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

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Cloning and expression of the bovine intestinal alkaline phosphatase gene: biochemical characterization of the recombinant enzyme

Biochemical Journal ◽

10.1042/bj2900503 ◽

1993 ◽

Vol 290 (2) ◽

pp. 503-508 ◽

Cited By ~ 29

Author(s):

H Weissig ◽

A Schildge ◽

M F Hoylaerts ◽

M Iqbal ◽

J L Millán

Keyword(s):

Alkaline Phosphatase ◽

Biochemical Characterization ◽

Sequence Similarity ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Regulatory Elements ◽

Eukaryotic Expression ◽

Cloning And Expression ◽

Coding Region ◽

Intestinal Alkaline Phosphatase

A complete genomic clone and a full-length cDNA coding for bovine intestinal alkaline phosphatase have been isolated and sequenced. The gene (5.4 kb) contains 11 exons separated by ten small introns at positions identical to those other members of the eukaryotic tissue-specific alkaline phosphatase family. In addition, 1.5 kb of upstream sequences contain putative regulatory elements showing sequence similarity to human and mouse intestinal alkaline phosphatase promoter sequences. To achieve recombinant bovine intestinal alkaline phosphatase expression, the coding region of the gene was subcloned into the pcDNA I eukaryotic expression vector and transfected into Chinese hamster ovary cells. Recombinant bovine intestinal alkaline phosphatase displays enzymatic properties comparable with those of purified native bovine intestinal alkaline phosphatase, a slightly increased thermal stability and, upon desialylation, it shows a homogeneous behaviour in agarose gel electrophoresis and isoelectric focusing. The availability of the recombinant bovine intestinal alkaline phosphatase and the elucidation of its primary sequence will help to accelerate our efforts to obtain the first crystallographic model of a eukaryotic alkaline phosphatase molecule.

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The Two mRNAS Encoding Rat Intestinal Alkaline Phosphatase Represent Two Distinct Nucleotide Sequences

Clinical Chemistry ◽

10.1093/clinchem/38.12.2506 ◽

1992 ◽

Vol 38 (12) ◽

pp. 2506-2509 ◽

Cited By ~ 21

Author(s):

M J Engle ◽

D H Alpers

Keyword(s):

Polymerase Chain Reaction ◽

Alkaline Phosphatase ◽

Duodenal Mucosa ◽

Differential Regulation ◽

Untranslated Regions ◽

Terminal Sequence ◽

Intestinal Alkaline Phosphatase ◽

Chain Reaction ◽

Polymerase Chain ◽

Fat Feeding

Abstract Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.

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Effect of fat feeding on intestinal alkaline phosphatase activity in tissue and serum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1981.241.6.g461 ◽

1981 ◽

Vol 241 (6) ◽

pp. G461-G468 ◽

Cited By ~ 7

Author(s):

G. P. Young ◽

S. Friedman ◽

S. T. Yedlin ◽

D. H. Allers

Keyword(s):

Alkaline Phosphatase ◽

Enzyme Activity ◽

Small Bowel ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Intestinal Alkaline Phosphatase ◽

Serum Enzyme ◽

Small Bowel Mucosa ◽

Fat Feeding ◽

Bowel Mucosa

Serum intestinal alkaline phosphatase activity is increased by fat feeding, but the mechanism of this increase is not fully understood. Fasting rats were fed a single feed of either corn oil (12 kcal) or an isocaloric elemental feed (Vivonex 100 HN). Changes in enzyme activity in the small bowel mucosa and serum were followed for 20 h. Only the fat-fed rats had increased serum enzyme activity, being maximal at 7 h and three times the fasting level. This resulted from an increase in the amount of enzyme protein in the serum and not from an increase in its catalytic efficiency. The serum biological half-life of 125I-labeled intestinal alkaline phosphatase was the same in fasted (2.51 min) and fat-fed rats (2.55 min). Both types of feed caused a quantitatively similar increase in brush-border-bound alkaline phosphatase activity. However, levels of soluble intracellular alkaline phosphatase in intestinal mucosa were affected differently: the elemental diet caused a substantial rise, whereas no significant change was seen after fat feeding. The isoelectric pattern of phosphatase activity in serum after fat feeding was identical to that of soluble intracellular and not membranous alkaline phosphatase. Therefore, serum intestinal alkaline phosphatase activity rises in response to a single fat feed as a result of increased delivery of the enzyme to the blood and not as a result of an increase in its normally short biological half-life. This rise cannot be directly linked to an increase in the amount of brush-border-bound enzyme, and it appears that the serum enzyme is derived directly from a pool of soluble intracellular enzyme in the small bowel mucosa.

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Synthesis and parallel secretion of rat intestinal alkaline phosphatase and a surfactant-like particle protein

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1995.268.6.e1205 ◽

1995 ◽

Vol 268 (6) ◽

pp. E1205-E1214 ◽

Cited By ~ 15

Author(s):

D. H. Alpers ◽

Y. Zhang ◽

D. J. Ahnen

Keyword(s):

Alkaline Phosphatase ◽

Lamina Propria ◽

Extracellular Enzyme ◽

Apical Surface ◽

Intestinal Alkaline Phosphatase ◽

Alkaline Phosphatases ◽

Parallel Patterns ◽

Precursor Pool ◽

Enhanced Secretion ◽

Fat Feeding

Rat intestinal microvillous alkaline phosphatases are secreted bidirectionally from the enterocyte attached to a phospholipid-rich membrane (surfactant-like particle). To determine the intracellular pathways for newly synthesized alkaline phosphatases and for the extracellular enzyme-particle complex in the intestinal mucosa, pulse-chase experiments were performed. Synthesis of both isoforms of alkaline phosphatase in fasted rats peaked in the Golgi at 15–30 min and in the microvillous membrane at 60 min, without intermediate localization in the basolateral membranes. A second peak of incorporation was found at 15–30 min in scrapings obtained from the apical surface of the enterocytes. These results demonstrate a dominant direct Golgi-to-microvillous membrane transport for newly synthesized alkaline phosphatase. An additional precursor pool(s) appears responsible for the early appearance of enzyme in the lumen. Newly synthesized alkaline phosphatase isoforms and the 97-kDa protein of surfactant-like particles showed parallel patterns of appearance in enterocytes, luminal washings, and lamina propria after triacylglycerol feeding and were preferentially secreted into the lumen and lamina propria at times (5-7 h) when enterocyte content of these newly synthesized proteins had declined toward basal rates. Enhanced secretion of newly synthesized proteins for hours after fat feeding could explain the prolonged rise in serum and luminal washings of both the enzyme and the particle.

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Secretion and distribution of rat intestinal surfactant-like particles after fat feeding

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.5.g944 ◽

1994 ◽

Vol 266 (5) ◽

pp. G944-G952 ◽

Cited By ~ 11

Author(s):

F. Yamagishi ◽

T. Komoda ◽

D. H. Alpers

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Lamina Propria ◽

Serum Alkaline Phosphatase ◽

Buoyant Density ◽

Apical Surface ◽

Intestinal Alkaline Phosphatase ◽

Particle Content ◽

Serum Alkaline Phosphatase Activity ◽

Fat Feeding

Triacylglycerol feeding increases serum intestinal alkaline phosphatase (IAP) activity and leads to the appearance of an alkaline phosphatase-containing particle in the luminal washings over the apical surface of the rat enterocyte and in the blood (J. Clin. Invest. 84: 1355-1361, 1989). To examine the coordinate appearance of these particles and the enzyme and to follow their distribution in vivo after feeding, an enzyme-linked immunoabsorbent assay (ELISA) was developed, using antisera raised against the purified intact surfactant-like particle. Tissue compartments that were examined for phosphatase activity and particle content included isolated enterocytes, lamina propria, intestinal luminal washings, and serum. Alkaline phosphatase activity peaked earliest in the lamina propria (3 h), followed by the enterocyte and the luminal washings (5 h) and serum (7 h). Surfactant-like particle content peaked in the enterocyte and lamina propria at 3 h, followed by the serum (3-5 h) and the luminal washings (5 h). The buoyant density of the particle in the enterocyte (d = 1.08-1.09) and serum (d = 1.07-1.08) after fat feeding was similar to that of the isolated particle (d = 1.07-1.08). The density of particle proteins detected by ELISA in fasted serum was more diffuse and > 1.10, consistent with partial degradation of the particle and/or its proteins. These data confirm that the particle and its bound IAP are secreted from the enterocyte after triacylglycerol feeding and that they appear in compartments adjacent to both the basolateral (serum) and apical (luminal wash) surfaces of the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

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Both tissue and serum phospholipases release rat intestinal alkaline phosphatase

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1990.259.4.g618 ◽

1990 ◽

Vol 259 (4) ◽

pp. G618-G625 ◽

Cited By ~ 4

Author(s):

R. Eliakim ◽

M. J. Becich ◽

K. Green ◽

D. H. Alpers

Keyword(s):

Alkaline Phosphatase ◽

Brush Border Membrane ◽

Surface Glycoprotein ◽

Cell Fractionation ◽

Intestinal Alkaline Phosphatase ◽

Cytosol Fraction ◽

Variable Surface Glycoprotein ◽

Variable Surface ◽

Fat Feeding ◽

Brush Border Membrane Enzymes

Rat intestinal alkaline phosphatase (IAP) is unique among the brush-border membrane enzymes in that it is released bidirectionally (lumen and blood) and exists in either soluble (serum) or particulate (cellular) form. To elucidate the mechanism of membrane release, we examined the effects of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and serum anchor-specific phospholipase D (PLD) on the solubility of the various tissue forms of IAP. The "solubility" of cytosol IAP could be explained in part by intracellular PtdIns-PLC activity, detected by production of acidic IAP isomers, and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-sensitive PtdIns hydrolysis. Contamination with serum (abundant with anchor-specific PLD) was responsible for the complete or partial solubilization of IAP that was found during processing of light mucosal scrapings. Anchor-specific PLD activity was increased after fat feeding, and the IAP released did not react with antiserum that recognizes the PtdIns-PLC-released phospholipid portion of trypanosomal variable surface glycoprotein. These data are consistent with the hypothesis that, after secretion from the enterocyte bound to a phospholipid-rich membranous particle, IAP release into serum is mediated by serum anchor-specific PLD. The soluble forms of IAP in the lumen and the cytosol fraction appear to be due to a combination of endogenous PtdIns-PLC activity and anchor-specific PLD contamination that occurs during cell fractionation.

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Translation of rat intestinal RNA yields two alkaline phosphatases

Biochemical Journal ◽

10.1042/bj2340563 ◽

1986 ◽

Vol 234 (3) ◽

pp. 563-568 ◽

Cited By ~ 11

Author(s):

N L Sussman ◽

S Seetharam ◽

M C Blaufuss ◽

D H Alpers

Keyword(s):

Alkaline Phosphatase ◽

Translation Product ◽

Intestinal Alkaline Phosphatase ◽

Alkaline Phosphatases ◽

Rat Intestine ◽

Entire Small Intestine ◽

Monospecific Antiserum ◽

Rna Immunoprecipitation ◽

Molecular Masses ◽

Fat Feeding

After translation of total rat intestinal RNA, immunoprecipitation using monospecific antiserum against rat intestinal alkaline phosphatase yielded two polypeptides in the adult duodenum and jejunum (molecular masses 62 and 65 kDa). Immunoprecipitation of both bands was blocked by a single purified alkaline phosphatase. In the adult ileum and in the entire small intestine of suckling pups, only the 62 kDa translation product was found. After fat feeding, translated alkaline phosphatase increased by an amount proportionate to the increase in enzyme activity previously seen in the serum. A small fraction of nascent alkaline phosphatase was translocated into microsomal vesicles, producing peptides of 65 and 69 kDa. Tunicamycin-treated membranes demonstrated a different signal peptide for each translation product. N-Terminal sequencing of the translation products showed leucine residues at similar positions, but overlap with the mature protein sequence was not demonstrated. On the basis of these data, we propose the presence of two mRNAs encoding alkaline phosphatase in the rat intestine.

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