©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Rat Intestinal Alkaline Phosphatase Isoforms with Different Carboxyl-terminal Peptides Are Both Membrane-bound by a Glycan Phosphatidylinositol Linkage (*)

Michael J. Engle , Akhtar Mahmood , David H. Alpers (§)

From the (1) Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

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-l 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 and + 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 D.


INTRODUCTION

Over 95% of rat intestinal alkaline phosphatase (IAP)() is localized to the apical brush border membrane, yet this same enzyme contributes >70% of the total serum alkaline phosphatase activity after fat feeding (1) . This response to fat feeding results from an increase in enzyme protein concentration, not in activity (2) . Because alkaline phosphatase is a membrane-bound enzyme, a separate secretory pathway or a soluble form of the enzyme, or both, would be needed to explain IAP in the serum. We have found such a possible pathway for a membrane-bound IAP via its association with a secreted phospholipid-rich membrane with surfactant properties, the surfactant-like particle (SLP). This SLP is characterized by enrichment for IAP, over 30 times greater than the brush border content per mg of protein (3) . In addition, evidence for two separate IAPs in rat intestinal mucosa was found, raising the question whether one isoenzyme was uniquely directed to the SLP for secretion or was secreted by a different mechanism.

Cell-free translation of rat intestinal RNA has yielded two independently regulated IAP isoforms (62 and 65 kDa) (4) that show different sugar content of glycosyl side chains (5) . This suggested that two IAP isoforms were encoded in individual and distinct RNAs. The first cDNA isolated encoding a rat IAP (IAP-I) had a predicted structure of the carboxyl-terminal peptide that differed markedly from IAP sequences predicted in other species (6) . Evidence from studies using a portion of the cDNA sequence at the 3` end of the coding region, as well as the full-length cDNA as probes, suggested that the 2.7- and 3.0-kb mRNAs encoding rat IAP each represented a different isoform (7) . Two different groups reported a second rat IAP cDNA (IAP-II), which differs most strikingly from the predicted structure of IAP-I in the carboxyl-terminal region (8, 9) . Thus, unlike the mouse and human, the rat intestine expresses two separate IAP isoforms, most likely derived from two separate genes.

Rat IAP-I appeared bound to membranes after transfection into COS-l cells via a glycan phosphatidylinositol (GPI) linkage (6) . The cDNA encoding this protein contains a carboxyl-terminal hydrophobic amino acid sequence preceded by a small amino acid domain (-NSA-) located 28 residues from the end of the predicted amino acid sequence. These features are consistent with the requirements suggested for GPI linkage (10). On the other hand, the predicted carboxyl-terminal sequence of rat IAP-II differed significantly from that of rat IAP-I and would not appear to contain the consensus requirements for GPI linkage. The 24 amino acids at the carboxyl terminus were predicted to be either neutral or hydrophilic (8) and were preceded by a distinctive segment of 17 threonine residues. These findings suggested that rat IAP-II might be attached to membranes via a non-GPI linkage or else secreted as a soluble protein.

The present experiments investigated the possibility that rat IAP-II is not linked by a GPI mechanism. Experiments were conducted to examine the anchoring of both IAP-I and IAP-II to the cell plasma membrane and to secreted SLPs. A series of DNA constructs were used with altered carboxyl-terminal sequences. The data show that both IAP-I and IAP-II are membrane-bound via a GPI linkage, and that both are also included after transfection in the secreted SLP produced by Caco-2 cells.


MATERIALS AND METHODS

IAP cDNA Constructs

Rat IAP-I was ligated into the BamHI site of the mammalian transfection vector pSG5 (Pharmacia Biotech Inc.) (11) . All other modifications were made from this construct (Fig. 1). (a) For chimeric IAP, the 3` end of the IAP-I insert was removed between NarI (bp 1532) and BglII in the multiple cloning region of pSG5. A similar digest (NarI-EcoRI) was made using the IAP-II DNA which had been produced by PCR and subcloned into the TA cloning vector (Invitrogen, San Diego, CA). This portion of IAP-II includes the most 3` 170 bp of the coding region as well as 1000 bp of the untranslated region. The chimeric DNA produced by ligating this NarI EcoRI fragment into the restricted pSG5/IAP-I vector encodes for the first 1531 bp of IAP-I and the final 170 bp of IAP-II. The protein resulting from translation of this DNA should have both alkaline phosphatase activity from IAP-I and 57 amino acids supporting the IAP-II carboxyl-terminal signal. (b) For truncated IAP, the chimeric IAP DNA was digested with AvaI and NheI (Fig. 1). This digestion removes the nucleotides encoding the carboxyl-terminal 24 amino acids, but leaves intact the unique stretch of 17 threonine residues found in IAP-II, as well as 350 bp of the 3`-untranslated region, including the poly(A) tail. The single-point mutations were constructed by PCR using the chimeric DNA as template and 5`-oligomers containing the desired base changes as well as the AvaI restriction sequence at bp 1633. The 3`-oligomer was derived from a sequence in the untranslated region and included the NheI restriction site at bp 2243. The chimeric DNA and the PCR products were digested with AvaI and NheI, and the PCR fragments were subsequently ligated into the chimeric DNA. All constructs were verified by restriction digests and DNA sequencing.


Figure 1: The carboxyl termini of the rat intestinal alkaline phosphatase constructs indicating changes and mutations described under ``Materials and Methods.''



Cell Culture and Transfection

Caco-2 cells (courtesy of Dr. Jeffrey Field, University of Iowa) were used between passages 40 and 45. The cells were grown in 100-mm diameter plastic dishes as described previously (11) . COS-1 cells (courtesy of Dr. John Brady, National Cancer Institute, NIH) were grown in DMEM with the same antibiotics used for Caco-2 cells. COS-1 cells were used between passages 36 and 57. Transfections were performed either with the calcium phosphate precipitation method (Caco-2 cells) (11) or by electroporation (COS-1 cells). Optimal transfection conditions for electroporation were 1000 µF, 300 V at resistance, using the Invitrogen Electroporator (Invitrogen Corp.). Cells were electroporated in 750 µl in 0.4-cm cuvettes. Control transfections contained either pSG5 alone or no added DNA.

Treatment with Phosphatidylinositol-specific Phospholipase C (PI-PLC)

PI-PLC prepared from Staphylococcus aureus culture medium was generously provided by Dr. Martin G. Low (Columbia University, New York, NY). COS-1 cells were washed three times with serum-free DMEM and then incubated for 60 min at 37 ° with 3 ml of serum-free DMEM. The medium was removed, centrifuged at 600 g to remove cellular debris, and stored at -20 °C. The cells were refed with 3 ml of serum-free DMEM containing 60-80 milliunits/ml PI-PLC. Following a 60-min incubation the medium was removed, centrifuged, and stored at -20 °C. Alkaline phosphatase activity was measured with 10 mMp-nitrophenyl phosphate as substrate. One unit of activity equals the amount of enzyme needed to hydrolyze 1 mmol of substrate per min at 37 °C. After assay for enzyme activity, the media from replicates of the same transfection were pooled, concentrated with an Amicon YM-10 membrane, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots were performed as described previously (12) , using an antibody to the trypanosome-variable surface glycoprotein cross-reacting determinant (CRD), kindly supplied by Dr. John Englund (Johns Hopkins University, Baltimore, MD). The ECL detection system with linked horseradish peroxidase (Amersham Corp.) was used for visualization of antigen-antibody complexes. Media from nontransfected cells (containing no rat IAP) was used as a control.

Ethanolamine Labeling Studies

Cells were incubated overnight (16-20 h) in DMEM supplemented with 1% fetal bovine serum and 100 µCi/ml of [1-H]ethan-1-ol-2-amine HCl, 15 Ci/mmol (Amersham Corp.). Following the labeling period cells were scraped into 1 ml of immunoprecipitation buffer to which 15 µl of antiserum raised against native rat IAP were added (11) . Following denaturing polyacrylamide gel electrophoresis, the gels were treated with ENHANCE (DuPont NEN) and exposed to X-Omat film (Eastman Kodak Co.) for 20-24 h. Apparent protein size was determined by comparing the protein of interest to a set of molecular weight markers (Bio-Rad).

Assays for Membrane-bound Status of IAP Isoenzymes

Three additional methods were used to document whether the IAP isoforms were membrane-bound. Phase partitioning with Triton X-114 and separation of free from membrane-bound IAP by NaBr density gradient centrifugation have been described previously (11) . Chromatography with Sepharose 6B (Pharmacia) to separate the intact SLP from solubilized IAP was performed as reported earlier (12) .

Fluorescence Microscopy

COS-1 cells were transfected and grown in Chamber Slide culture chambers (Nunc Inc., Naperville, IL). Control and transfected cells were washed three times with phosphate-buffered saline (pH 7.3) containing 0.1 mM CaCl and 1 mM MgCl (PBS-CM), followed by a 15 min incubation at room temperature with 2% paraformaldehyde. The cells were again washed three times with PBS-CM and then incubated for 10 min at room temperature with 50 mM NHCl in PBS to quench free aldehyde groups. After two washes with PBS-CM plus 0.2% gelatin (PBS-CMG), the cells were exposed to rat IAP antibody (diluted 1:100 in PBS-CMG) for 30 min at room temperature. The cells were next washed four times with PBS-CMG for 5 min each and incubated for 30 min at room temperature with fluorescein isothiocyanate-conjugated second antibody (Sigma) diluted 1:10 in PBS-CMG. Cells were rinsed four times, 5 min each, with PBS-CMG and two times for 5 min with PBS alone. Slides were mounted with 50% glycerol in PBS, sealed with nail varnish, and stored at 4 °C in the dark until examined. Slides were examined using a Nikon fluorescent microscope.

RESULTS

Secretion of Membrane-bound Alkaline Phosphatase from Caco-2 Cells

To determine whether both IAPs were membrane-bound or whether IAP-II might be secreted in soluble form, the DNA constructs encoding IAP-I and the chimeric IAP were transfected into Caco-2 cells. These cells were chosen because alkaline phosphatase secretion resulting from transfection with IAP-I DNA is mediated by a secreted membrane, the SLP (11) . Transfection with IAP-I DNA resulted in a 29-fold increase in medium alkaline phosphatase activity (). When chimeric DNA was used, the increase in activity was approximately 220-fold. Enzyme activity alone, however, cannot distinguish between soluble and SLP-bound IAP. The secreted product of IAP-I was found mostly in the detergent-rich fraction of the Triton X-114 partition assay (); the same result was found for the secreted product of chimeric DNA transfection.

Because IAP-II and the chimeric IAP contained no hydrophobic carboxyl-terminal peptide (8) , two further assays were used to document the membrane-bound nature of the expressed product of the chimeric DNA. The peak of IAP activity on NaBr gradient was at d = 1.08 (Fig. 2), as expected for SLP, not at d = 1.06 as seen with solubilized IAP (3) . When concentrated media from chimeric transfections were applied to a Sepharose 6B column, the peak of IAP activity eluted with the void volume (Fig. 3), well before the Rof soluble IAP. Identical results were seen using media from IAP-I cDNA transfection (data not shown). Thus, in the Caco-2 cell system, transfection with DNA encoding either IAP-I or the chimeric IAP produced a secreted IAP which appeared to be membrane-bound. We next examined the intracellular targeting of both IAP isoenzymes in a system which did not produce a secreted form of IAP.


Figure 2: NaBr gradient fractionation of media from Caco-2 cells transfected with the chimeric IAP construct. Media were collected 72 h after transfection and prepared for the gradient as described under ``Materials and Methods.'' One-ml fractions were collected and assayed for alkaline phosphatase activity.




Figure 3: Sepharose 6B column chromatography of media from Caco-2 cells transfected with the chimeric IAP construct. Media from transfected cells were collected 72 h after transfection and prepared as described under ``Materials and Methods.'' A 37 1.8-cm column was eluted with Tris-HCl buffer, pH 7.8. Void volume fractions were numbers 8-12.



Transfection of IAP Isoforms in COS-1 Cells

COS-1 cells have low cellular levels of alkaline phosphatase activity and do not secrete the enzyme under basal conditions (6) . Thus, any media IAP activity would result either from secreted protein or from the action of serum proteases or lipases on membrane-bound IAP. COS-1 cells were initially transfected with DNA encoding IAP-I, the chimeric IAP, the truncated IAP, and the chimeric IAP in which the amino acids at position 533 and 535 had been mutated to prolines. Cells and media were examined for alkaline phosphatase activity 72 h later (). Cellular alkaline phosphatase activity was increased 143-, 122-, 97-, and 37-fold when transfected with DNA encoding IAP-I, chimeric IAP, truncated IAP, and mutated chimeric IAP, respectively. The four constructs produced increases in alkaline phosphatase activity in the medium by 41-, 104-, 840-, and 189-fold. When expressed as a percent of total activity (cell + medium), the cell-associated activity was 78, 44, 7, and 13%, respectively (). These data suggested that the carboxyl-terminal portion of both IAP-I and IAP-II (chimeric IAP) were directed to membranes, whereas removal of the carboxyl-terminal peptide or mutation of the probable GPI-linkage site produced a soluble and secreted protein.

Localization of IAP Activity by Immunocytochemistry

Immunofluorescence using anti-IAP antibody and fluorescein isothiocyanate-labeled second antibody localized the protein products of the transfected cDNAs. Fig. 4demonstrates that the proteins expressed after transfection of cDNA encoding IAP-I and the chimeric IAP were found predominantly on the external plasma membrane, whereas control cells and cells transfected with the truncated IAP were unlabeled.


Figure 4: Immunofluorescent labeling of nonpermeabilized COS-1 cells transfected with the following cDNAs: A, control; B, IAP-I; C, chimeric construct; D, truncated construct. Cells were fixed and incubated with anti-IAP-I antibody as described under ``Materials and Methods.''



Demonstration of GPI Linkage for IAP-I and IAP-II

IAP-I has been demonstrated to be membrane-bound by a GPI linkage (6) and was used as a positive control for the experiments that examined the possibility that the chimeric IAP might also be anchored by a similar mechanism. COS-1 cells were transfected with cDNA encoding IAP-I, the chimeric IAP, the truncated IAP, or a series of mutated chimeric IAPs (see I). After 72 h cells were incubated for 1 h either with or without PI-PLC, and the amount of alkaline phosphatase activity released into the medium was assessed (I). PI-PLC led to a 37-38-fold increase in medium activity for both IAP-I and chimeric IAP-transfected cells. In contrast, medium alkaline phosphatase activity was unchanged following PI-PLC treatment of cells transfected with either the truncated IAP DNA or the proline substituted chimeric IAP DNA.

In the chimeric IAP the putative GPI linkage site is -NSA-, 4-6 residues beyond the oligothreonine sequence and 19-21 residues from the predicted end of the peptide. An Asn Thr change in the putative (attachment) site nearly eliminated IAP linkage that was sensitive to PI-PLC, as did the mutation Ala Pro in the + 2 site. Mutation to Thr in the + 1, + 2, + 3, or + 4 sites did not much affect PI-PLC sensitive linkage. Threonine is known to support GPI linkage, although less strongly than some other amino acid residues, whereas proline does not support GPI linkage at all (10) .

Direct demonstration of the GPI linkage for the chimeric IAP was obtained by documenting the appearance of the trypanosome variable surface glycoprotein cross-reacting determinant (CRD) after PI-PLC treatment and by incorporation of ethanolamine into the newly synthesized product of the chimeric IAP DNA. Fig. 5demonstrates that anti-CRD antibody, which recognizes the inositol phosphate moiety exposed by PI-PLC hydrolysis, also recognizes the product of both IAP-I and the chimeric IAP DNA expressed in COS-l cells. The apparent molecular mass of IAP-I was 65 kDa, as previously reported (6) . The chimeric IAP product was larger (about 90 kDa), despite the fact that most of the coding region of the two constructs was identical. Media from nontransfected COS-1 cells showed no reaction with anti-CRD antiserum. Transfected cells were also incubated with [H]ethanolamine to determine incorporation into the GPI moiety. Fig. 6shows that in cells transfected with chimeric IAP DNA a single band of radioactive immunoprecipitated IAP was seen, with an apparent molecular mass of 93 kDa; IAP resulting from transfection with IAP-I cDNA had an molecular mass of 69 kDa. These data support previous observations of two different IAP isoforms in rat intestinal tissue, in which the product of the IAP-I gene corresponded with the 68-kDa isoform, and that of IAP-II with the 90-kDa isoform (4, 13) .


Figure 5: Expression of the trypanosome-variable cross-reacting determinant after PI-PLC treatment. COS-1 cells were transfected with the following constructs: no cDNA (-); IAP-I (I); chimeric construct with the IAP-II carboxyl terminus (II); the truncated construct (Tr). Cells were treated witih PI-PLC 72 h after transfection. Western blots on the concentrated media were performed as described under ``Materials and Methods.''




Figure 6: Immunoprecipitation of [H]ethanolamine-labeled COS-1 cells. Cells were transfected with either IAP-I (I) or the chimeric construct containing the IAP-II carboxyl terminus (II). Cell labeling and immunoprecipitation were carried out as described under ``Materials and Methods.''



DISCUSSION

The data in this report demonstrate that IAP-II, like IAP-I, is bound to membranes via a GPI anchor. The evidence for such a conclusion is as follows. The products resulting from transfection with cDNAs encoding these two proteins are bound to COS-1 cell membranes and released from the cells by PI-PLC treatment, are not released by PI-PLC after mutation of the putative -NSA- GPI linkage site, demonstrate reactivity with anti-CRD antiserum after PI-PLC treatment, and incorporate ethanolamine into the immunoprecipitated protein. Moreover, when transfected into Caco-2 cells that secrete IAP attached to a membranous SLP, both IAP isoforms are secreted in a form that segregates with the detergent-rich phase of the Triton X-114 separation system, consistent with their attachment to the SLP.

The deduced sequence of IAP-I (8) fits the current requirements for GPI linkage. It contains a 22-amino acid hydrophobic carboxyl-terminal peptide, a hinge region of 5 residues, including two charged ones (-NN-), and a 3-amino acid sequence with high probability for linkage (-NSA-) at positions -28-30. Another possibility is the sequence one residue shifted (-SAI-), although the probability of isoleucine being active in the + 2 position is only 10% that of alanine (14) . The predicted amino acid sequence deduced from the IAP-II cDNA, however, should not support a GPI-linked mechanism, if the same requirements are present. The carboxyl-terminal peptide of IAP-II and of the chimeric IAP lacks the apparently necessary hydrophobic span of 8-20 amino acids (10) . As a result, no hinge region can be identified, although a proline residue, often found in hinge regions, is present at positions -10 and -14.

Most evidence to date suggests that a hydrophobic signal peptide is necessary for GPI linkage (10, 15) . In fact, amino-terminal signal peptides have been substituted for the native carboxyl-terminal sequence with retention of GPI linkage (16) . There is one example of presumed GPI linkage without a hydrophobic carboxyl-terminal signal peptide. A truncated version of platelet derived growth factor receptor was produced that removed the transmembrane and cytoplasmic domains and terminated with the predicted carboxyl-terminal sequence -VTSGHCHEERVDRHDGE (17) . Surprisingly, this construct was membrane-bound and was released by PI-PLC treatment. This protein was not naturally occurring, as is IAP-II, and the truncated receptor was not normally targeted, with only 20% of the protein found on the cell surface, the rest being intracellular. The product of IAP-II cDNA transfection was located as expected for a GPI-linked protein at the cell surface, as demonstrated by immunocytochemistry and by PI-PLC release.

Although a hydrophobic carboxyl-terminal peptide is not present in IAP-II, there is a possible linkage site (-NSA-), similar to that found in IAP-I, but located in IAP-II at positions -17-21. Another possible linkage site (-NST-) is located just at the amino-terminal side of the poly-threonine sequence. Threonine in the + 2 position produces only low GPI-linkage activity (14) , but at least one natural protein, decay accelerating factor, has a threonine in this position (14) . Although this -NST- sequence in IAP-II lines up nearly exactly in paired amino acid analysis with the putative -NSA- sequence that is the most likely linkage site in IAP-I, the -NSA- site in IAP-II seems likely to be utilized based on the mutational studies.

The truncated IAP isoform removed the putative GPI linkage site at positions -19-21 in IAP-II, while retaining the poly-threonine sequence and the other putative GPI linkage site, -NST- (Fig. 1). The product of this transfected cDNA was not membrane-bound and was secreted in a form not associated with membranes. Thus, the most likely predicted site for GPI linkage in IAP-II is -NSA-. The experiments utilizing mutations in the -NSASS- region further support this conclusion. The double mutation, Ala Pro/Ser Pro, resulted in a secreted protein. Proline was chosen for this substitution based on studies (14) , which found that proline was unable to support GPI attachment when present in the , + 1, or + 2 positions. Single amino acid mutations in the region of amino acids 532-535 (-SASS-) did not alter the linkage mechanism of the chimeric protein, indicating that none of these amino acids is the attachment site. Substitution of amino acid 531 (Asn to Thr), however, sharply reduced the amount of enzyme released by PI-PLC treatment, thus identifying this asparagine as the most likely candidate for GPI anchor attachment. The mutation of asparagine 531 to threonine did not completely eliminate IAP release by PI-PLC, as did the truncated or double-mutated chimeric IAP. The reason for this is not entirely clear, although mutating only one amino acid in this region may shift the GPI reading frame and permit attachment of the anchor to another, less desirable, amino acid in this region.

The unique feature of the IAP-II sequence is the region of 17 threonine residues. Although there is no direct evidence that these form the carboxyl terminus of the protein, the results of transfections with the truncated IAP and mutated chimeric IAP suggest that they do, because the putative GPI linkage site (-NSA-) would be carboxyl-terminal to the threonine sequence. These threonines could be the site for O-linked glycosylation or some other modification. Such modification could result in the increased apparent size of IAP-II compared with IAP-I (93 versus 69 kDa, Fig. 6). N-Linked glycosylation seems an unlikely single explanation, because the products of cell-free translation of rat duodenal mRNA are 62 and 65 kDa and after incubation with microsomal membranes 69 and 65 kDa, respectively (4) . O-Linked glycosylation or other modification of carboxyl-terminal threonine(s) is possible, but evidence for or against such an explanation is not available.

It is clear, however, that two IAPs are found in the rat intestinal mucosa, as detected either by purification (5) or Western blotting (13). By studying the appearance of IAP isoforms during postnatal development or after precocious induction of development by cortisone and thyroxine, the appearance of the 69-kDa IAP appears to be directed by the 2.7-kb mRNA that encodes IAP-I, whereas the 93-kDa IAP is directed by the 3.0-kb mRNA that encodes IAP-II (13) . The present data support the assignment of the 69- and 93-kDa IAP isomers to the IAP-I and IAP-II, respectively.

It is difficult to believe that IAP-II is a unique naturally occurring protein, i.e. the only one which is GPI-linked yet lacking a hydrophobic carboxyl-terminal signal peptide. It seems more likely that the requirement for GPI attachment will be modified to include a variety of sequences which remain to be elucidated. For the present, the carboxyl-terminal sequence of IAP-II provides a unique naturally occurring model to be used for targeting and for stimulation of SLP production.

  
Table: Effect of transfection of alkaline phosphatase cDNAs on enzyme secretion from Caco-2 cell


  
Table: Effect of transfection of alkaline phosphatase cDNAs on enzyme localization in COS-1 cells

Results are expressed as the mean ± S.E. of three to seven experiments.


  
Table: Effect of PI-PLC treatment on transfected COS-1 cell alkaline phosphatase activity



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Washington University School of Medicine, Campus Box 8124, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8940; Fax: 314-362-8959.

The abbreviations used are: IAP, intestinal alkaline phosphatase; GPI, glycan phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; CRD, trypanosome variable surface glycoprotein cross-reacting determinant; SLP, surfactant-like particle; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair.


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