scholarly journals Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

1981 ◽  
Vol 200 (3) ◽  
pp. 645-654 ◽  
Author(s):  
Graeme P. Young ◽  
Steven T. Yedlin ◽  
David H. Alpers
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Double Diffusion ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Intestinal Alkaline Phosphatase ◽  
Absolute Increase ◽  
Membrane Bound

Enzymically active intestinal alkaline phosphatase exists in both soluble and membrane-bound forms in the suckling rat. Antiserum prepared against purified soluble alkaline phosphatase (anti-AlP) was shown to be monospecific when assessed by Ouchterlony double-diffusion analysis and immunoelectrophoresis. The two forms of alkaline phosphatase were antigenically identical and possessed similar affinities for anti-AlP. To study the biosynthesis of the two forms, 14-day-old rats were injected intraperitoneally with [3H]leucine. The labelling kinetics of alkaline phosphatase, extracted from supernatant and brush-border membrane fractions with anti-AlP, was followed over 20h. Incorporation of [3H]leucine into membrane-bound alkaline phosphatase was rapid, reaching a plateau at 6h. The soluble enzyme showed slower incorporation of label and maximal radioactivity was not reached until 12h after labelling, a lag of 6h behind the membrane-bound enzyme. Soluble alkaline phosphatase could not have been a precursor of the membrane form, as there was no early peak of radioactivity in the soluble form. To determine if the soluble enzyme was irreversibly derived from the membrane enzyme, a newly developed technique of labelling brush-border membrane proteins in vivo by intraluminal injection of diazotized [125I]iodosulphanilic acid was used. The appearance of 125I in soluble and membrane alkaline phosphatase was then monitored over a 7h period, encompassing the lag between maximal leucine labelling of the two forms. The results failed to show either a proportional transfer of radioactivity from membrane to soluble alkaline phosphatase or an absolute increase in radioactivity of the soluble form during degradation of brush-border alkaline phosphatase. Therefore there does not appear to be a serial precursor/product relationship between the soluble and membrane-bound forms of suckling-rat intestinal alkaline phosphatase.

In vivo effect of calcium (Ca) on brush border intestinal alkaline phosphatase (BBIAP) in the rat

Bone ◽  
2006 ◽  
Vol 38 (5) ◽  
pp. S16-S17 ◽  
Author(s):  
M.L. Brance ◽  
R.M. Brun ◽  
A. Rigalli ◽  
L. De Candia ◽  
R.C. Puche
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Intestinal Alkaline Phosphatase

Duodenal brush border intestinal alkaline phosphatase activity affects bicarbonate secretion in rats

2007 ◽  
Vol 293 (6) ◽  
pp. G1223-G1233 ◽  
Author(s):  
Yasutada Akiba ◽  
Misa Mizumori ◽  
Paul H. Guth ◽  
Eli Engel ◽  
Jonathan D. Kaunitz
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Intestinal Alkaline Phosphatase ◽  
Glycerol Phosphate ◽  
Secretory Rate ◽  
Rat Duodenum ◽  
Ph Stat ◽  
Cystic Fibrosis Transmembrane

We hypothesized that duodenal HCO3− secretion alkalinizes the microclimate surrounding intestinal alkaline phosphatase (IAP), increasing its activity. We measured AP activity in rat duodenum in situ in frozen sections with the fluorogenic substrate ELF-97 phosphate and measured duodenal HCO3− secretion with a pH-stat in perfused duodenal loops. We examined the effects of the IAP inhibitors l-cysteine or l-phenylalanine (0.1–10 mM) or the tissue nonspecific AP inhibitor levamisole (0.1–10 mM) on AP activity in vitro and on acid-induced duodenal HCO3− secretion in vivo. AP activity was the highest in the duodenal brush border, decreasing longitudinally to the large intestine with no activity in stomach. Villous surface AP activity measured in vivo was enhanced by PGE2 intravenously and inhibited by luminal l-cysteine. Furthermore, incubation with a pH 2.2 solution reduced AP activity in vivo, whereas pretreatment with the cystic fibrosis transmembrane regulator (CFTR) inhibitor CFTRinh-172 abolished AP activity at pH 2.2. l-Cysteine and l-phenylalanine enhanced acid-augmented duodenal HCO3− secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced HCO3− secretion. Moreover, l-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased HCO3− secretion, inhibited by suramin. In conclusion, enhancement of the duodenal HCO3− secretory rate increased AP activity, whereas inhibition of AP activity increased the HCO3− secretory rate. These data support our hypothesis that HCO3− secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases HCO3− secretion via activation of P2 receptors.

Alkaline phosphatase: A marker enzyme for brush border membrane

1972 ◽  
Vol 28 (4) ◽  
pp. 385-386 ◽  
Author(s):  
U. Schmidt ◽  
U. C. Dubach ◽  
I. Bieder ◽  
B. Funk
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Marker Enzyme

Potential differences influence amino acid/Na+ symport rates in larval Manduca sexta midgut brush-border membrane vesicles.

1994 ◽  
Vol 189 (1) ◽  
pp. 55-67
Author(s):  
R Parthasarathy ◽  
W R Harvey
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Rate Determining Step ◽  
Half Saturation Constant

The time-dependent fluorescence intensity of an intravesicular potential-sensitive dye was used to probe the real-time kinetics of potential difference (PD)-dependent amino acid/Na+ symport at pH9 into brush-border membrane vesicles obtained from larval Manduca sexta midgut. Neutral amino acids (alanine, proline) are symported at higher rates as the vesicles are hyperpolarized. The symport rates of acidic (glutamate) and basic (arginine) amino acids are almost PD-independent. The half-saturation constant of alanine is PD-independent between -108 and -78 mV, although the maximal symport velocity increases by half as the voltage is increased. Amino acid throughput is evidently enhanced as the relatively high transmembrane PDs (> 150 mV, lumen positive) measured in vivo are approached. The half-saturation concentrations of Na+ were in the range 15-40 mmol l-1 for most of the amino acids examined and increased with voltage for alanine. The Vmax observed as a function of cation or amino acid concentration increased as the vesicle was hyperpolarized in the case of leucine and alanine. The data support the hypothesis that carrier and substrates are at equilibrium inasmuch as substrate translocation seems to be the rate-determining step of symport.

The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes

2000 ◽  
Vol 3 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Q. XIE ◽  
D. H. ALPERS
Keyword(s):  
Alkaline Phosphatase ◽  
Transcriptional Start Site ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Start Site ◽  
Intestinal Alkaline Phosphatase ◽  
Promoter Regions ◽  
Flanking Regions

Xie, Q., and D. H. Alpers. The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes. Physiol Genomics 3: 1–8, 2000.—Rat intestinal alkaline phosphatases (IAP-I and -II) differ in primary structure, substrate specificity, tissue localization, and response to fat feeding. This study identifies two distinct genes (∼5–6 kb) corresponding to each isozyme and containing 11 exons of nearly identical size. The exon-intron junctions are identical with those found in IAP genes from other species. The 1.7 and 1.2 bp of 5′ flanking regions isolated from each gene, respectively, contain Sp1 and gut-enriched Kruppel-like factor (GKLF) binding sites, but otherwise show little identity. There is a potential CAAT-box 14 bp 5′ to the transcriptional start site, 36 bp upstream from IAP-I, and a TATA-box 31 bp 5′ to the transcriptional start site, 55 bp upstream from IAP-II. Transfection of these promoter regions (linked to luciferase as a reporter gene) into a kidney cell line, COS-7, produced the differential response to oleic acid expected from in vivo studies, i.e., threefold increase using the 5′ flanking region of IAP-II, but not IAP-I. This response was not reproduced by 5,8,11,14-eicosatetraynoic acid (ETYA) or clofibrate, suggesting that peroxisome proliferator response elements are not involved. Isolation of the IAP-II gene will allow determination of the sequences responsible for dietary fat response in the enterocyte.

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