Pancreatic Islets Express a Ca2+-independent Phospholipase A2 Enzyme That Contains a Repeated Structural Motif Homologous to the Integral Membrane Protein Binding Domain of Ankyrin*

(Received for publication, October 9, 1996, and in revised form, February 10, 1997)

Zhongmin Ma , Sasanka Ramanadham , Kirsten Kempe , Xiaoyuan Sherry Chi , Jack Ladenson and John Turk Dagger

From the Mass Spectrometry Resource, Divisions of Endocrinology, Diabetes, and Metabolism and of Laboratory Medicine, Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Pancreatic islets express a Ca2+-independent phospholipase A2 (CaI-PLA2) activity that is sensitive to inhibition by a haloenol lactone suicide substrate that also attenuates glucose-induced hydrolysis of arachidonic acid from islet phospholipids and insulin secretion. A cDNA has been cloned from a rat islet cDNA library that encodes a protein with a deduced amino acid sequence of 751 residues that is homologous to a CaI-PLA2 enzyme recently cloned from Chinese hamster ovary cells. Transient transfection of both COS-7 cells and Chinese hamster ovary cells with the cloned islet CaI-PLA2 cDNA resulted in an increase in cellular CaI-PLA2 activity, and this activity was susceptible to inhibition by haloenol lactone suicide substrate. The domain of the islet CaI-PLA2 from amino acid residues 150-414 is composed of eight stretches of a repeating sequence motif of approximately 33-amino acid residues in length that is highly homologous to domains of ankyrin that bind both tubulin and integral membrane proteins, including several proteins that regulate ionic fluxes across membranes. These findings complement previous pharmacologic observations that suggest that CaI-PLA2 may participate in regulating transmembrane ion flux in glucose-stimulated beta -cells.


INTRODUCTION

Glucose-induced insulin secretion from pancreatic islet beta -cells requires that glucose be transported into the beta -cell and metabolized (1). Signals derived from glucose metabolism result in inactivation of plasma membrane ATP-sensitive K+ channels (KATP),1 membrane depolarization, activation of voltage-operated Ca2+ channels, influx of Ca2+, and a rise in cytosolic [Ca2+], which triggers insulin exocytosis (2). Stimulation of islets with glucose also induces hydrolysis of arachidonic acid from islet membrane phospholipids (3), and the resultant accumulation of nonesterified arachidonic acid (4) may facilitate Ca2+ entry into beta -cells (5) and amplify depolarization-induced insulin secretion (6).

Hydrolysis of arachidonic acid from membrane phospholipids in glucose-stimulated islets appears to be mediated in part by a phospholipase A2 (PLA2) enzyme that is catalytically active in the absence of Ca2+ and that is inactivated by a haloenol lactone suicide substrate (HELSS) (7-10). Treatment of islets with HELSS results in attenuation of the glucose-induced rise in beta -cell cytosolic [Ca2+] and in inhibition of insulin secretion (8-10). The structure of islet Ca2+-independent phospholipase A2 (CaI-PLA2) is not known, but a CaI-PLA2 enzyme has recently been cloned from CHO cells (11) and its sequence determined (GenBank accession number 115470). We report here the cloning, expression, and sequence analysis of a homologous enzyme from a rat pancreatic islet cDNA library (12).


EXPERIMENTAL PROCEDURES

Materials

Enhanced chemiluminescence detection reagents, [32P]dCTP (3000 Ci/mmol), [35S]dATPS (>1000 Ci/mmol), and L-alpha -1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine (50 mCi/mmol) were purchased from Amersham Corp. Male Harlan Sprague Dawley rats (180-220 g) were obtained from Sasco (O'Fallon, MO); collagenase was from Boehringer Mannheim; tissue culture media (CMRL-1066 and MEM), penicillin, streptomycin, Hanks' balanced salt solution, heat-inactivated fetal bovine serum, and L-glutamine were from Life Technologies, Inc.; Pentex bovine serum albumin (BSA, fatty acid-free, fraction V) was from Miles Laboratories (Elkhart, IN); rodent Chow 5001 was from Ralston Purina (St. Louis, MO); ampicillin and kanamycin were from Sigma; and D-glucose was from the National Bureau of Standards. Media included KRB (Krebs-Ringer bicarbonate buffer; 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2); nKRB (KRB supplemented with 3 mM D-glucose); cCMRL-1066 (CMRL-1066 from Life Technologies, Inc. supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine and 1% (w/v) each of penicillin and streptomycin); and Hank's balanced salt solution supplemented with 0.5% penicillin/streptomycin). The HELSS is (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one and was purchased from Cayman Chemical (Ann Arbor, MI).

Isolation of Pancreatic Islets, Preparation of Islet beta -Cells and Non-beta -cells, and Culture of COS-7, CHO, and HIT-T15 Cells

Islets were isolated aseptically from male Harlan Sprague Dawley rats, as described (13), by collagenase digestion of excised, minced pancreas, density gradient isolation, and manual selection under microscopic visualization (10). Isolated islets were transferred to Falcon Petri dishes containing 2.5 ml of cCMRL-1066, placed under an atmosphere of 95% air, 5% CO2, and cultured overnight at 37 °C. Purified populations of islet beta -cells and non-beta -cells were prepared by fluorescence-activated cell sorting of dispersed cells obtained by treating islets with the enzyme dispase, as described previously (5, 7, 10, 14, 15). COS-7 and CHO cells were cultured in Dulbecco's modified Eagle's medium (MEM) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. HIT-T15 cells were obtained from ATCC (Bethesda, MD) and were cultured in Ham's F12K medium (Life Technologies, Inc.) containing 7 mM D-glucose, 10% dialyzed horse serum, and 2.5% fetal bovine serum.

RNA Isolation, Reverse Transcription, and Polymerase Chain Reaction

Total RNA was isolated from cells and tissues after solubilization in guanidinium thiocyanate by extraction (phenol/chloroform/isoamyl alcohol) and precipitation (isopropyl alcohol) (16). First strand cDNA was transcribed with avian myeloblastosis virus reverse transcriptase (RT) obtained from Boehringer Mannheim. Polymerase chain reactions (PCR) were performed on a Perkin-Elmer DNA Thermal Cycler 480. Amplification steps (25-30 cycles) included denaturation (95 °C, 1 min), annealing (50-60 °C, 1 min), and extension (72 °C, 2 min) performed in Taq DNA polymerase buffer (Life Technologies, Inc.) containing 1.5 mM MgCl2; 1 µM of each primer; 200 µM each of dATP, dGTP, dCTP, and dTTP; and 25 units/ml Taq DNA polymerase. PCR products were analyzed by 1% agarose gel electrophoresis and visualized with ethidium bromide (16).

cDNA Cloning and Sequencing

A pair of PCR primers (sense, 5'-TATGCGTGGTGTGTACTTCC-3' and antisense, 5'-TGGAGCTCAGGGCGACAGCA-3') were designed based on the cDNA sequence of the CHO cell CaI-PLA2 (11) and used in RT-PCR reactions with RNA from HIT-T15 insulinoma cells. These reactions yielded a 820-bp product. A 32P-labeled form of this product was prepared by randomly primed labeling and used to screen an islet cDNA library prepared as described elsewhere (12) in the Lambda ZAP Express system (Stratagene). Clones that hybridized with the probe were isolated and further plaque-purified. The pBK-CMV plasmid containing the CaI-PLA2 cDNA sequence as insert was excised in vivo according to the manufacturer's protocol. Insert sizes were determined after digestion with the restriction enzymes EcoRI and XhoI. The cloned cDNA in pBK-CMV was sequenced from the double strand by the method of Sanger et al. (17) using a Sequenase version 2.0 DNA sequencing kit (United States Biochemical Corp.) with T3 and T7 primers and specific synthetic oligonucleotide primers.

Expression and Purification of CaI-PLA2

After removal of the 5'-untranslated region, the full-length rat islet CaI-PLA2 cDNA was subcloned in-frame into the EcoRI and XhoI sites of pET-28c (Novagen) using standard techniques. The fidelity of the construct was verified by restriction analysis and sequencing. The pET28-CaI-PLA2 construct then was transformed into the bacterial expression host Escherichia coli strain BL21(DE3) (Novagen). This system yields a fusion protein with polyhistidine and T7 epitope tag sequences joined to the sequence encoded by the insert. Cells transformed with pET28c containing no insert served as negative controls. Expression was induced by incubation of the cells with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 h and assessed by 10% SDS-PAGE analysis of total protein followed by Coomassie Blue staining. The expressed protein was affinity-purified by nickel-chelate chromatography as described in the manufacturer's protocol (Novagen). The protein was eluted with imidazole and analyzed by 10% SDS-PAGE.

Immunoblotting Analyses

For Western blotting analyses, proteins were transferred to a nylon membrane, which was subsequently blocked (1 h, room temperature) with Tris-buffered saline-Tween (TBS-T, which contains 20 mM Tris-HCl, 137 mM NaCl, pH 7.6, and 0.1% Tween 20) containing 1% BSA and 3% milk powder. Following three washes with TBS-T, the blot was then incubated (1 h, room temperature) with a monoclonal antibody (1:50 dilution in TBS-T with 1% BSA) raised in a mouse against a synthetic peptide representing a 10-amino acid residue sequence (221TPLHLACQMG230) located at the C terminus of islet CaI-PLA2. The nylon membrane was washed three times in TBS-T and incubated (1 h, room temperature) with a goat anti-mouse IgG conjugated to horseradish peroxidase (Boehringer Mannheim) at a 1:3000 dilution in TBS-T containing 1% BSA. Detection of the secondary antibody was performed by enhanced chemiluminescence.

Examination of the Abundance of CaI-PLA2 mRNA by Competitive RT-PCR

Competitive RT-PCR (18, 19) was used to determine the abundance of CaI-PLA2 mRNA in various preparations. In this approach, a competitor DNA species is prepared that contains the same primer template sequences as the target cDNA but that contains an intervening sequence that differs from the target in size or in restriction sites so that PCR products from the target and competitor can be distinguished (18, 19). Using the competitor as an internal control, amounts of target cDNA can be determined by allowing known amounts of the competitor to compete with the target for primer binding during amplification (18, 19).

To prepare the competitor DNA, two composite primers were synthesized (sense, 5'-TGTGGGCAAGGTCATCC-3' and antisense, 5'-CTTGGAGGCCATGTAG-3'). These primers contain the CaI-PLA2 primer sequence (underlined) attached to sequences that hybridize to rat glyceraldehyde-3-phosphate dehydrogenase cDNA (20). This pair of primers was then used in PCR reactions with rat glyceraldehyde-3-phosphate dehydrogenase cDNA as template. In these reactions, the CaI-PLA2 primer sequences are incorporated into the PCR product during amplification, and the intervening sequence derives from glyceraldehyde-3-phosphate dehydrogenase. The resultant PCR product (380 bp in length) was analyzed by agarose gel electrophoresis, isolated with a QIAEX gel extraction kit (QIAGEN), and used as the competitor DNA species in subsequent PCR experiments. In these experiments, the primer pair (sense 5'--3' and antisense 5'--3') was used. These primers hybridize to the CaI-PLA2 cDNA sequence and to the competitor DNA sequence. The PCR product derived from the CaI-PLA2 cDNA is 528 bp in length and that from the competitor DNA is 380 bp in length. The products were then analyzed by 1% agarose gel electrophoresis and visualized with ethidium bromide. Product band intensity was then determined with an IS-1000 Digital Imaging System.

With a fixed amount of target and competitor, varying the PCR cycle number from 19 to 31 was found to yield a constant relative intensity of the target and competitor PCR product bands. In subsequent experiments, 28 PCR cycles were used. To construct a standard curve, the competitor DNA solution was diluted in the range of 10-2 to 10-6, and aliquots of each dilution were added to a reaction mixture containing a fixed amount of CaI-PLA2 cDNA. After PCR amplification, products were analyzed by agarose gel electrophoresis, and product band intensity was determined as above. The ratio of product band intensities was found to correspond to the amount of input DNA over the range of tested concentrations. Similar results were obtained in experiments in which the amount of competitor DNA was fixed and the amount of target DNA was varied.

COS-7 and CHO Cell Transfection and CaI-PLA2 Activity Assay

The full-length islet CaI-PLA2 cDNA was subcloned into pcDNA3.1 (Invitrogen) in the forward orientation. Petri dishes (100 mm diameter) containing either COS-7 cells or CHO cells at about 60-80% confluence were transfected with 20 µg of plasmid DNA by calcium phosphate precipitation. After overnight culture, fresh MEM was added to each plate, and the cells were then cultured for an additional 2 days. At 72 h after transfection, cells were harvested after washing with ice-cold phosphate-buffered saline. For CaI-PLA2 enzyme activity assays, the cells were collected by centrifugation (Beckman table-top centrifuge, 3000 rpm, 2 min, room temperature). These cells were then resuspended and washed three times with ice-cold phosphate-buffered saline and once with homogenization buffer (250 mM sucrose, 40 mM Tris-HCl, pH 7.5). The cells were pelleted again, resuspended in homogenization buffer, and disrupted by sonication. The homogenates were centrifuged (170,000 × g, 60 min, 4 °C) to obtain a cytosolic supernatant. The protein content of the cytosolic fraction was measured by Bio-Rad assay. CaI-PLA2 activity was measured in aliquots of cytosol (100 µl, approximately 25 µg of protein) added to assay buffer (200 mM Tris-HCl, pH 6.0, 10 mM EGTA, total assay volume 200 µl). As described in the appropriate figure legends, effects of preincubating (2 min, room temperature) cytosolic fractions with various concentrations of HELSS on CaI-PLA2 activity were also determined, as were effects of varying the assay solution pH and effects of including nucleotide phosphates such as ATP in the assay solution. Reactions were initiated by injection of an ethanolic solution (5 µl) of L-a-1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine substrate (specific activity 50 mCi/mmol, final concentration 5 µM). Assay mixtures were then incubated (3 min, 37 °C, with shaking), and reactions were terminated by addition of butanol (100 µl) and vortexing (8 s). After centrifugation (2000 × g, 2 min), reaction products in 25 µl of the upper (butanol) layer were analyzed by Silica Gel G thin layer chromatography in the solvent system petroleum ether/ethyl ether/acetic acid (80/20/1). The region of the TLC plate containing free fatty acid (Rf 0.58) was identified with iodine vapor and scraped into scintillation vials, and the 14C-labeled content was then determined by liquid scintillation spectrometry. The amount of [14C]linoleate released was then converted to a PLA2 specific activity value (pmol/mg protein × min) as described elsewhere (7).

Dot Matrix Analysis of the Islet CaI-PLA2 Amino Acid Sequence and Comparison to Other Sequences

Dot matrix plots (21) were constructed with the UWGCG programs COMPARE and DOTPLOT using a window of 30 and stringency of 15. Segments of 30 amino acids from the horizontal axis were compared with segments from the vertical axis, and a dot was placed in the appropriate position whenever the total score of aligned sequences exceeded a value of 15. These plots were used to examine internal repeating motifs in the islet CaI-PLA2 sequence and to compare the sequences of ankyrin and of related proteins to the CaI-PLA2 sequence.


RESULTS

Cloning the Rat Islet CaI-PLA2 cDNA

An oligonucleotide probe was generated by RT-PCR using HIT-T15 insulinoma cell RNA as template and primers designed from the CHO cell CaI-PLA2 sequence. Both CHO cells and HIT-T15 cells are hamster-derived lines, and HIT-T15 cells express a CaI-PLA2 activity similar to that in islets (22). A 32P-labeled form of the resultant 820-bp RT-PCR product was generated by randomly primed labeling and used to screen an islet cDNA library. Screening about 900,000 plaque-forming units resulted in the identification of five positive clones that were isolated, plaque-purified, and excised in vivo. The size of the cDNA inserts in the plasmids from each of the five clones was then determined after release of the inserts by digestion with the restriction enzymes EcoRI and XhoI. Each clone contained an insert of about 3.3 kilobases.

Sequencing the inserts from both 5'- and 3'-ends revealed that each contained an identical 5'-sequence and an identical 3'-sequence ending in a poly(A) tail. A putative initiation codon (ATG) was observed 475 bp downstream of the 5'-end. Nucleotide sequencing of the entire 3288-bp insert from one clone revealed a single long open reading frame (2256 bp), which was predicted to encode a protein with 751 amino acid residues. The insert also contained 474 bp of 5'-untranslated region and 558 bp of 3'-untranslated region including a poly(A) tail. A presumptive polyadenylation signal (AATAAA) was present 14 bp upstream of the poly(A) tail (Fig. 1).



Fig. 1. Nucleotide sequence and deduced amino acid sequence of cloned rat pancreatic islet CaI-PLA2 cDNA. The putative methionine start codon (ATG) and the presumptive polyadenylation signal (AATAAA) present 14-bp upstream of the poly(A) tail are displayed in bold type. Regions of amino acid sequence homologous to ankyrin are underlined. Arrowheads (R1-R8) mark the beginning of each of the eight consecutive strings of repeating sequence motif.
[View Larger Versions of these Images (57 + 62K GIF file)]


Deduced Amino Acid Sequence of the Rat Islet CaI-PLA2

The encoded protein has a calculated molecular mass of 83,591 daltons and a predicted isoelectric point of 6.6. The amino acid sequence of islet CaI-PLA2 deduced from the nucleotide sequence of the cDNA does not exhibit significant homology to the sequences of known Ca2+-dependent PLA2 enzymes, including the 85-kDa cytosolic PLA2 or secretory PLA2 (23). The islet CaI-PLA2 exhibits 90% identity of nucleotide sequence and 95% identity of amino acid sequence to the CaI-PLA2 recently cloned from CHO cells (11). One absolutely conserved region is that between His421 and Glu551, which flanks and includes a GXSXG lipase consensus sequence (11, 24, 25). This sequence occurs in the islet CaI-PLA2 as 462TT466.

Examination of the islet CaI-PLA2 amino acid sequence using dot matix analysis revealed (Fig. 2A) that the domain from amino acid residues 150-414 is composed of eight strings of a repetitive sequence motif of approximately 33 amino acid residues in length. The alignment of the repeats in the islet CaI-PLA2 sequence is illustrated in Fig. 2B. A BLAST search of the National Biomedical Research Foundation protein data base revealed significant homology of the repetitive sequence of the islet CaI-PLA2 to a number of ankyrin-related proteins (ARP), including the Caenorhabditis elegans ARP (26) and both murine (27) and human (28) red cell ankyrin. Dot matrix analysis revealed that the repetitive sequence motif of the islet CaI-PLA2 is highly homologous to that of an 89-kDa domain of murine red cell ankyrin (Fig. 3A) that contains binding sites for tubulin and for several integral membrane proteins (28-30). Alignment of the repeating domain of islet CaI-PLA2 with the repeating domain of ankyrin indicated that both repeating motifs have very similar consensus sequences (Fig. 3B). The CHO cell CaI-PLA2 is also known to contain ankyrin-like repeats (11).


Fig. 2. Repeating sequences in islet CaI-PLA2. A, dot matrix analysis. Dot matrix analyses of the islet CaI-PLA2 sequence was performed with the UWGCG programs COMPARE and DOTPLOT with a window of 30 and stringency of 15 to search for regions with internal homology. Such regions are represented by the more densely populated regions of the plot, corresponding to residues 150-414. B, comparison of the eight aligned strings of repeating sequence motif in islet CaI-PLA2. Identical residues (black boxes) and conservative changes (open boxes) are indicated for the corresponding amino acid residues. The consensus sequence is identified in the lower portion of the figure.
[View Larger Version of this Image (34K GIF file)]



Fig. 3. Alignment of the islet CaI-PLA2 amino acid sequence with repeating domains of ankyrin. A, dot matrix analysis of the amino acid sequence of CaI-PLA2 versus that of mouse erythrocyte ankyrin. The dot matrix analysis was performed using high stringency parameters (>20), as described in Fig. 2. B, alignment of the repeating domain of CaI-PLA2 with repeats 11-18 in the sequences of ankyrin from C. elegans and mouse erythrocytes. Identical residues (black boxes) and conservative changes (open boxes) are indicated for the corresponding amino acids.
[View Larger Version of this Image (64K GIF file)]


Bacterial Expression of the Protein Encoded by the Cloned Islet CaI-PLA2 cDNA

The open reading frame of the putative islet CaI-PLA2 cDNA was subcloned in-frame into the bacterial expression vector pET-28c (Novagen) to generate a fusion protein containing the islet CaI-PLA2 sequence and both polyhistidine and T7 epitope tag sequences. Upon isopropyl-1-thio-beta -D-galactopyranoside induction of bacterial cultures transfected with vector containing the islet CaI-PLA2 insert, strong expression of the expected 87-kDa fusion protein occurred, and this material was not observed in control cultures (Fig. 4). The expressed protein was recognized by a monoclonal antibody raised against a synthetic peptide contained within the islet CaI-PLA2 sequence.


Fig. 4. Bacterial expression of the protein encoded by the cloned islet CaI-PLA2 cDNA. Proteins were analyzed by 10% SDS-PAGE and visualized by Coomassie Blue staining (lanes 1, 2, and 5) or by Western blotting with anti-CaI-PLA2 antibody (lanes 3 and 4). Lanes 1 and 3 represent negative controls in which the expression host cell E. coli strain BL21(DE3) was transformed with pET28c vector containing no insert. Lanes 2 and 4 represent transformation of the host cell with the pET28-CaI-PLA2 construct. For Western blotting analyses (lane 3 and 4), proteins were transferred to a nylon membrane and probed with a monoclonal anti-CaI-PLA2 antibody, as described under "Experimental Procedures." Lane 5 represents expressed protein, which contains a polyhistidine tag sequence, after affinity purification by nickel-chelate chromatography.
[View Larger Version of this Image (45K GIF file)]


Expression of CaI-PLA2 Activity in COS-7 and CHO Cells After Transient Transfection with the Cloned Islet CaI-PLA2 cDNA

For eukaryotic expression, the islet CaI-PLA2 cDNA was inserted into the vector pcDNA3.1 (Invitrogen), and this construct (pcDNA3.1-CaI-PLA2) was used to transfect both COS-7 and CHO cells. At 72 h after transfection with this construct or with vector containing no insert, CaI-PLA2 activity was measured in cytosolic fractions prepared from these cells. No measurable CaI-PLA2 activity was observed in the cytosol of either untransfected COS-7 cells (not shown) or COS-7 cells transfected with vector containing no insert (Fig. 5A). In contrast, COS-7 cells transfected with the pcDNA3.1-CaI-PLA2 construct exhibited substantial amounts of cytosolic CaI-PLA2 activity (Fig. 5A). This activity was inhibited by pretreatment of cytosol with a suicide substrate (HELSS) (Fig. 5A) that has previously been demonstrated to inhibit CaI-PLA2 activity in islet cytosol (7). Similar results were obtained with CHO cells. Both untransfected CHO cells (not shown) and CHO cells transfected with vector containing no insert exhibited measurable CaI-PLA2 activity (Fig. 5B). This was expected because a CaI-PLA2 enzyme has been cloned from CHO cells (11). Transient transfection of CHO cells with the pcDNA3.1-CaI-PLA2 construct, however, resulted in a 5.5-fold increase in cytosolic CaI-PLA2 activity, and this activity was inhibited by treatment of cytosol with HELSS (Fig. 5B).


Fig. 5. Eukaryotic expression of the protein encoded by the cloned islet CaI-PLA2 cDNA. A, CaI-PLA2 activity expressed in transfected COS-7 cells. CaI-PLA2 activity assays were performed with cytosol obtained form COS-7 cells transfected either with pcDNA3.1 vector containing no insert (1st three lanes) or with pcDNA3.1-CaI-PLA2 (last three lanes), which contains the islet CaI-PLA2 cDNA as insert. All assays were performed in buffer containing 10 mM EGTA. In the 1st (VO) and 4th (VI) lanes, cytosol was not pretreated before CaI-PLA2 activity was measured. In the 3rd (VO+H) and 6th (VI+H) lanes, cytosol was incubated with the CaI-PLA2 inhibitor HELSS before CaI-PLA2 activity was measured. In the 2nd (VO+E) and 5th (VI+E) lanes, cytosol was incubated with ethanolic vehicle that did not contain HELSS before CaI-PLA2 activity was measured. The meanings of the designations on the x axis of the panel are as follows: VO, vector only; VO+E, vector only plus ethanol; VO+H, vector only plus HELSS; VI, vector with insert; VI+E, vector with insert plus ethanol; VI+H, vector with insert plus HELSS. B, CaI-PLA2 activity expressed in transfected CHO cells. In B, the left bar (VO) represents CaI-PLA2 enzymatic activity in cytosol prepared from CHO cells that had been transfected with pcDNA3.1 vector only. The middle bar (VI) represents CaI-PLA2 activity in cytosol prepared from CHO cells that had been transfected with the pcDNA3.1-CaI-PLA2 construct. The right bar (VI+H) represents CaI-PLA2 activity in cytosol that was treated with HELSS after it was prepared from CHO cells that had been transfected with the pcDNA3.1-CaI-PLA2 construct. Data represent mean values (n = 3), and S.E. are indicated. The meanings of the designations on the x axis of B are the same as in A.
[View Larger Version of this Image (24K GIF file)]


Characterization of CaI-PLA2 Activity in COS-7 Cells Transiently Transfected with the Islet CaI-PLA2 cDNA Construct

The concentration dependence of inhibition by HELSS of CaI-PLA2 activity in COS-7 cells transfected with the islet CaI-PLA2 cDNA construct was found to be very similar to that of the CaI-PLA2 activity in native HIT insulinoma cell cytosol (Fig. 6). Like the CaI-PLA2 activity in islet and HIT cell cytosol (7, 22), the CaI-PLA2 activity in cytosol from transiently transfected COS-7 cells was optimal near neutrality and was stimulated by including 1 mM ATP in the assay solution (Fig. 7). The effects of ATP to stimulate cytosolic CaI-PLA2 activity in transiently transfected COS-7 cells, however, differed in some respects from those observed with islet (7) or HIT cell (22) CaI-PLA2 activity. First, the ATP concentration dependence for stimulation of CaI-PLA2 activity from transiently transfected COS-7 cells was bell-shaped, and the stimulatory effect decreased at ATP concentrations exceeding 1 mM (not shown). CaI-PLA2 activity in cytosol from islets (7) or HIT cells (22) is stimulated by ATP concentrations as high as 10 mM. Second, there was less nucleotide phosphate selectivity with the CaI-PLA2 activity from transfected COS-7 cells than with islet (7) or HIT cell (22) activity. Although ATP was more effective than AMP or ADP, ADP did exert some activating influence on CaI-PLA2 activity from transfected COS-7 cells (not shown). ADP does not stimulate HIT cell CaI-PLA2 activity and suppresses the stimulatory effect of ATP (22). In addition, GTP and UTP were nearly as effective as ATP in stimulating CaI-PLA2 activity from transfected COS-7 cells (not shown), whereas GTP has little effect on HIT cell CaI-PLA2 activity (22). Finally, the non-hydrolyzable ATP analog AMP-PCP stimulates CaI-PLA2 activity from transfected COS-7 cells less effectively than ATP (not shown), whereas the two compounds are equipotent stimulators of HIT cell CaI-PLA2 activity (22).


Fig. 6. HELSS concentration dependence of inhibition of CaI-PLA2 activity in native HIT insulinoma cell cytosol compared with that for CaI-PLA2 activity in transfected COS-7 cell cytosol. Cytosol was prepared from native (non-transfected) HIT-T15 cells and from COS-7 cells that had been transfected with the pcDNA3.1-CaI-PLA2 construct, as described under "Experimental Procedures." Cytosol was preincubated at room temperature for 2 min in assay buffer containing no added Ca2+, 5 mM EGTA, and either no HELSS (control) or varied concentrations of HELSS (10-14 to 10-6 M). After the preincubation, the temperature of the assay solution was increased to 37 °C, and enzymatic reactions were initiated by addition of substrate. CaI-PLA2 activity was measured as described as under "Experimental Procedures" and is plotted as a percent of control activity observed in cytosol that was not exposed to HELSS.
[View Larger Version of this Image (20K GIF file)]



Fig. 7. Influence of pH and of ATP on CaI-PLA2 activity expressed in transfected COS-7 cells. Cytosol was prepared from COS-7 cells that had been transfected with the pcDNA3.1-CaI-PLA2 construct, and CaI-PLA2 activity was measured as in Fig. 6. For pH values from 7 to 9, assay solution was buffered with 100 mM Tris-HCl. For pH values from 4 to 6, assay solution was buffered with 100 mM sodium acetate. Open symbols reflect assays performed without ATP, and closed symbols reflect assays performed with ATP (1 mM). CaI-PLA2 activity is plotted as dpm of [14C]linoleate released from L-alpha -1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine substrate.
[View Larger Version of this Image (17K GIF file)]


Expression of CaI-PLA2 mRNA in Rat Islet Cells

To determine whether islet CaI-PLA2 mRNA is specifically expressed in beta -cells within islets, a competitive RT-PCR (18, 19) method was developed. A competitor DNA was prepared that shares with CaI-PLA2 cDNA sequences recognized by the primers but that yields a smaller product than that derived from the CaI-PLA2 cDNA (Fig. 8A). The ratio of signals from the target and competitor was found to correspond to the input DNA over a wide range of concentrations (Fig. 8B). Single cell suspensions were then prepared from isolated islets and analyzed by fluorescence-activated cell sorting (5, 7, 10, 14, 15) to yield two populations of cells. One population contains over 90% beta -cells, and the second contains 10-15% beta -cells and about 70% alpha -cells (5, 7, 10, 14, 15). RNA was then prepared from the two populations of cells and used as template in RT-PCR reactions with CaI-PLA2-specific primers and the competitor DNA. At equivalent amounts of input RNA, a substantially higher target to competitor ratio was observed with RNA from the beta -cell-enriched population than with that from the alpha -cell-enriched population (Fig. 9). This suggests that the RNA species amplified in RT-PCR reactions with the beta -cell-enriched population did not arise from contaminating non-beta -cells and that beta -cells contain CaI-PLA2 mRNA.


Fig. 8. Determination of the abundance of CaI-PLA2 mRNA by competitive RT-PCR. A, agarose gel electrophoresis of products obtained from competitive PCR reactions. Competitive RT-PCR was performed as described under "Experimental Procedures," and products were analyzed by 1% agarose gel electrophoresis and visualized with ethidium bromide. Lane 1 represents the product obtained when only CaI-PLA2 cDNA and no competitor was included in the reaction mixture. Lane 10 represents the product obtained when only competitor DNA and no CaI-PLA2 cDNA was included in the reaction mixture. Lanes 2-8 represent products obtained when a fixed amount of competitor DNA and serial 10-fold dilutions of CaI-PLA2 cDNA were included in the reaction mixture. B, relationship of target to competitor signal ratio to input DNA. The relative intensities of the product bands from the gel in A were determined with an IS-1000 Digital Imaging System and plotted as a function of input target DNA.
[View Larger Version of this Image (17K GIF file)]



Fig. 9. Relative abundance of CaI-PLA2 mRNA in pancreatic islet beta -cells and in non-beta -cells cells as estimated by competitive RT-PCR. Single cell suspensions were prepared from isolated pancreatic islets with the enzyme dispase and subjected to fluorescence-activated cell sorting to yield a population of nearly homogeneous beta -cells and a population containing about 70% beta -cells, 10-15% beta -cells, and 15-20% other cells. The second population is designated "non-beta -cells" in the figure. RNA was then prepared from the two populations of cells and used as template in competitive RT-PCR reactions with CaI-PLA2-specific primers and the competitor DNA species described under "Experimental Procedures." Products were analyzed, and the relative intensity of the target to competitor bands was determined as in Fig. 8.
[View Larger Version of this Image (13K GIF file)]


Expression of CaI-PLA2 mRNA was also examined in other tissues by competitive RT-PCR. At equivalent amounts of input RNA, the highest target to competitor ratio (TCR) among tissues examined was observed in brain (TCR 1.3). Target signal was also observed in lung (TCR 0.6), spleen (TCR 0.4), kidney (TCR 0.4), liver (TCR 0.2), heart (TCR 0.1), and skeletal muscle (TCR 0.1).


DISCUSSION

The studies described here indicate that pancreatic islet beta -cells express an 84-kDa CaI-PLA2 enzyme that is highly homologous to an enzyme recently cloned from CHO cells (11). The occurrence of ankyrin-like repeats in the islet CaI-PLA2 sequence raises interesting possibilities about the function of this protein in beta -cells. The domain of ankyrin that contains similar repeats is responsible for the binding of ankyrin to a number of integral membrane proteins that govern ionic fluxes across cellular membranes (29), including the Na+/K+-ATPase of renal basolateral membranes (31-33), a renal amiloride-sensitive Na+ channel (34), the red cell anion exchanger (35, 36), a cerebellar inositol-trisphosphate receptor (37), and a voltage-dependent Na+ channel in myelinated neurons (29).

Regulation of ionic fluxes at the beta -cell plasma membrane is critical in the control of insulin secretion, and both inactivation of KATP channels and activation of voltage-operated Ca2+ channels are required for the induction of insulin secretion by glucose (2). One product of the action of PLA2 on islet phospholipids is nonesterified arachidonic acid. Arachidonic acid is the vastly predominant sn-2 fatty acid substituent in islet phospholipids and comprises 36% of the total fatty acyl mass in islet phospholipids (9, 10). Nonesterified arachidonic acid facilitates Ca2+ entry into cells through both voltage-operated (38) and receptor-operated (39) Ca2+ channels and induces a rise in beta -cell cytosolic [Ca2+] that is dependent on Ca2+ entry from the extracellular space (5). Both exogenous PLA2 and the products of its action suppress KATP channel activity in excised and cell-attached patches of plasma membranes from HIT insulinoma cells (40).

Taken together, the observations summarized in the preceding two paragraphs raise the possibility that the ankyrin-like repeat domains of the islet CaI-PLA2 might serve to attach the enzyme to beta -cell KATP channels or to voltage-operated Ca2+ channels or to anchor the enzyme in the membrane in close proximity to such channels. The alpha -subunits of voltage-operated Ca2+ channels exhibit striking similarities in sequence to voltage-operated Na+ channels (41, 42) that interact with ankyrin through its repeating domain (29). Ankyrin is distributed in close proximity to voltage-operated Ca2+ channels in the triad junctional structures of skeletal myocytes, although direct interaction of ankyrin with these channels has not been demonstrated (43).

The repeat sequences in ankyrin are also responsible for its binding to the cytoskeletal component tubulin (29). Although the precise role of the cytoskeletal apparatus in insulin secretion is not clearly established, a number of agents that interfere with microtubule assembly or disassembly inhibit insulin secretion (44-47), and morphological evidence suggests a direct association of microtubules with insulin secretory vesicles (48). Electron microscopic evidence demonstrates secretory granules associated with microtubules and directed toward the plasma membrane (48). An association of the islet PLA2 with microtubular structures mediated by its ankyrin-like repeat domain might facilitate interaction of the enzyme with secretory granule membranes and/or plasma membranes. It is of interest in this regard that treatment of parotid acinar secretory granules with PLA2 greatly increases their tendency to fuse with plasma membranes (49), which is the final event in exocytosis.

The sensitivity of the islet CaI-PLA2 to inhibition by the HELSS suggests that this enzyme could be the target within beta -cells that is responsible for the ability of HELSS to inhibit the glucose-induced rise in beta -cell cytosolic [Ca2+] and insulin secretion (7-10), although HELSS has also recently been demonstrated to inhibit an isozyme of phosphatidate phosphohydrolase (50). HELSS does not inhibit any known Ca2+-dependent PLA2 of groups I through IV (51-54), but Ca2+-independent PLA2 activities from several sources are sensitive to inhibition by HELSS. These include the 84-kDa CHO cell CaI-PLA2 (11) and a PLA2 from the macrophage-like P388D1 cell line (51, 52, 55, 56), which has recently been demonstrated to represent the mouse homolog of the CHO cell CaI-PLA2 (61). In addition, Ca2+-independent, HELSS-sensitive PLA2 activities thought to reside in 40-kDa proteins have been described in myocardium (53), HIT cells (22), and renal proximal tubular cells (57).

It is not yet known whether these 40-kDa proteins are related to the 84-kDa CaI-PLA2 enzymes, although generation of the former from the latter by proteolytic processing is a formal possibility. It is also possible that the 40-kDa proteins are separate gene products that share sequence homology with the 84-kDa CaI-PLA2 enzymes, perhaps including the serine lipase consensus sequence that is likely to confer sensitivity to HELSS. The 40-kDa proteins from myocardium (58) and HIT cells (22, 59) are thought to be associated with 85-kDa protein subunits that are homologous to phosphofructokinase and that confer sensitivity to activation by ATP and to modulation of this effect by ADP. Experiments with the CaI-PLA2 enzymes from CHO cells (62) and P388D1 cells (51) have failed to demonstrate association with phosphofructokinase-like proteins, although both enzymes behave as 300-350-kDa multimers on gel filtration chromatography, as do CaI-PLA2 activities from myocardium (58) and HIT cells (59). Whether the 84-kDa CaI-PLA2 enzymes may exist as hetero-oligomers consisting of distinct catalytic and regulatory proteins in some cells but not others is a question that will require further examination.

Although the 84-kDa islet CaI-PLA2 expressed in transiently transfected COS-7 cells is sensitive to activation by ATP under the assay conditions employed in this study, the nature of this effect differs in several respects from that of CaI-PLA2 activities in islet and HIT cell cytosol and is in general quite similar to effects observed with the P388D1 cell CaI-PLA2 (51). The possibility that the differences in nucleotide phosphate sensitivity between the islet CaI-PLA2 expressed in transfected COS-7 cells and that in native islet cytosol might reflect the influence of an interacting protein that is expressed in islets but that is not abundantly expressed in COS-7 cells is under study. It is of interest that the CHO cell CaI-PLA2, after expression in an Sf9 cell host, has recently been demonstrated to interact avidly and selectively with both ATP affinity matrices and with calmodulin affinity matrices (60), suggesting that the enzyme may bind ATP and interact with other cytosolic proteins.


FOOTNOTES

*   This research was supported by Grant R37-DK-34388 from the National Institutes of Health and Grant 42674 from the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U51898[GenBank].


Dagger    To whom correspondence should be addressed: Box 8127, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
1   The abbreviations used are: KATP, ATP-sensitive K+ channel; BSA, bovine serum albumin; CaI, Ca2+-independent; CHO, Chinese hamster ovary; HELSS, haloenol lactone suicide substrate; KRB, Krebs-Ringer bicarbonate buffer; MEM, modified Eagle's medium; PCR, polymerase chain reaction; PLA2 phospholipase A2; RT, reverse transcriptase; TBS-T, Tris-buffered saline-Tween; bp, base pair(s); AMP-PCP, adenosine 5'-(beta ,gamma -methylenetriphosphate); TCR, target to competitor ratio.

ACKNOWLEDGEMENTS

We are grateful to Alan Bohrer, Bingbing Li, Zhiqing Hu, and Dr. Mary Mueller for excellent technical assistance.


REFERENCES

  1. Meglasson, M. D., and Matschinsky, F. M. (1986) Diabetes Metab. Rev. 2, 163-214 [Medline] [Order article via Infotrieve]
  2. Misler, S., Falke, L. C., Gillis, K., and McDaniel, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7119-7123 [Abstract]
  3. Turk, J., Wolf, B. A., Lefkowith, J. B., Stump, W. T., and McDaniel, M. L. (1986) Biochim. Biophys. Acta 879, 399-409 [Medline] [Order article via Infotrieve]
  4. Wolf, B. A., Turk, J., Sherman, W. R., and McDaniel, M. L. (1986) J. Biol. Chem. 261, 3501-3511 [Abstract/Free Full Text]
  5. Ramanadham, S., Gross, R., and Turk, J. (1992) Biochem. Biophys. Res. Commun. 184, 647-653 [Medline] [Order article via Infotrieve]
  6. Wolf, B. A., Pasquale, S. M., and Turk, J. (1991) Biochemistry 30, 6372-6379 [Medline] [Order article via Infotrieve]
  7. Gross, R. W., Ramanadham, S., Kruszka, K. K., Han, X., and Turk, J. (1993) Biochemistry 32, 327-336 [Medline] [Order article via Infotrieve]
  8. Ramanadham, S., Gross, R. W., Han, X., and Turk, J. (1993) Biochemistry 32, 337-346 [Medline] [Order article via Infotrieve]
  9. Ramanadham, S., Bohrer, A., Mueller, M., Jett, P., Gross, R. W., and Turk, J. (1993) Biochemistry 32, 5339-5351 [Medline] [Order article via Infotrieve]
  10. Ramanadham, S., Bohrer, A., Gross, R. W., and Turk, J. (1993) Biochemistry 32, 13499-13509 [Medline] [Order article via Infotrieve]
  11. Jones, S. S., Tang, J., Kriz, R., Shafer, M., Knopf, J., and Seehra, J. (1996) FASEB J. 10, L15 (abstr.)
  12. Ma, Z., Ramanadham, S., Kempe, K., Hu, Z., Ladenson, J., and Tuk, J. (1996) Biochim. Biophys. Acta 1308, 151-163 [Medline] [Order article via Infotrieve]
  13. McDaniel, M. L., Colca, J. R., Kotagal, N., and Lacy, P. E. (1983) Methods Enzymol. 98, 182-200 [Medline] [Order article via Infotrieve]
  14. Pipeleers, D. G. (1984) in Methods in Diabetes Research (Larner, J., ed), pp. 185-211, John Wiley & Sons, Inc., New York
  15. Wang, J.-L., and McDaniel, M. L. (1990) Biochem. Biochem. Biophys. Res. Commun. 66, 813-818
  16. Davis, L. G., Kuehl, W. M., and Battery, J. F. (1994) in Basic Methods in Molecular Biology (Wonsiewicz, M., and Greenfield, S., eds), 2nd Ed., pp. 335-338, Appleton & Lange, East Norwalk, CT
  17. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  18. Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2725-2729 [Abstract]
  19. Siebert, P. D., and Larrick, J. W. (1992) Nature 359, 557-558 [CrossRef][Medline] [Order article via Infotrieve]
  20. Tso, J. Y., Sun, X. H., Kao, T. H., Reece, K. S., and Wu, R. (1985) Nucleic Acids Res. 13, 2485-2502 [Abstract]
  21. Maizel, J. J., and Lenk, R. P. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 7665-7669 [Abstract]
  22. Ramanadham, S., Wolf, M. J., Jett, P. A., Gross, R. W., and Turk, J. (1994) Biochemistry 33, 7442-7452 [Medline] [Order article via Infotrieve]
  23. Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. (1991) Cell 65, 1043-1051 [Medline] [Order article via Infotrieve]
  24. Brenner, S. (1988) Nature 334, 528-530 [CrossRef][Medline] [Order article via Infotrieve]
  25. Sugimoto, H., Hayashi, H., and Yamashita, S. (1996) J. Biol. Chem. 271, 7705-7711 [Abstract/Free Full Text]
  26. Otsuka, A. J., Franco, R., Yang, B., Shim, K. H., Tang, L. Z., Zhang, Y. Y., Boontrakulpoontawee, P., Jeyaprakash, A., Hedgecock, E., Wheaton, V. I., and Sobery, A. (1995) J. Cell Biol. 129, 1081-1092 [Abstract]
  27. White, R. A., Birkenmeier, C. S., Peters, L. L., Barker, J. E., and Lux, S. E. (1992) Mamm. Genome 3, 281-285 [Medline] [Order article via Infotrieve]
  28. Lux, S. E., John, K. M., and Bennett, V. (1990) Nature 344, 36-42 [CrossRef][Medline] [Order article via Infotrieve]
  29. Peters, L. L., and Lux, S. E. (1993) Semin. Hematol. 30, 85-118 [Medline] [Order article via Infotrieve]
  30. Lambert, S., Yu, H., Prchal, J. T., Lawler, J., Ruff, P., Speicher, D., Cheung, M. C., Kan, Y. W., and Palek, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1730-1734 [Abstract]
  31. Drenckhahn, D., and Bennett, V. (1987) Eur. J. Cell Biol. 43, 479-486 [Medline] [Order article via Infotrieve]
  32. Morrow, J. S., Cianci, C. D., Ardito, T., Mann, A. S., and Kashgarian, M. (1989) J. Cell Biol. 108, 455-465 [Abstract]
  33. Nelson, W. J., and Hammerton, R. W. (1989) J. Cell Biol. 108, 893-902 [Abstract]
  34. Smith, P. R., Saccomani, G., Joe, E. H., Angelides, K. J., and Benos, D. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6971-6975 [Abstract]
  35. Bennett, V., and Stenbuck, P. J. (1979) Nature 280, 468-473 [Medline] [Order article via Infotrieve]
  36. Thevenin, B. J.-M., and Low, P. S. (1990) J. Biol. Chem. 265, 16166-16172 [Abstract/Free Full Text]
  37. Davis, L. H., Otto, E., and Bennett, V. (1991) J. Biol. Chem. 266, 11163-11169 [Abstract/Free Full Text]
  38. Vacher, P., McKenzie, J., and Dufy, B. (1989) Am. J. Physiol. 257, E203-E211 [Abstract/Free Full Text]
  39. Miller, B., Sarantis, M., Traynelis, S. F., and Attwell, D. (1992) Nature 355, 722-725 [CrossRef][Medline] [Order article via Infotrieve]
  40. Eddlestone, G. T. (1995) Am. J. Physiol. 268, C181-C190 [Abstract/Free Full Text]
  41. Tanabe, B., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kanagawa, K., Kojima, M., Hirose, T., and Numa, S. (1987) Nature 328, 313-318 [CrossRef][Medline] [Order article via Infotrieve]
  42. Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H., and Numa, S. (1986) Nature 320, 188-192 [Medline] [Order article via Infotrieve]
  43. Flucher, B. E., Morton, M. E., Froehner, S. C., and Daniels, M. P. (1990) Neuron 5, 339-351 [Medline] [Order article via Infotrieve]
  44. Lacy, P. E., Howell, S. L., Young, D. A., and Fink, C. J. (1968) Nature 219, 1177-1179 [Medline] [Order article via Infotrieve]
  45. Lacy, P. E., Klein, H. J., and Fink, C. J. (1973) Endocrinology 92, 1458-1468 [Medline] [Order article via Infotrieve]
  46. Stuctchfield, S., and Howell, S. L. (1980) FEBS Lett. 175, 393-396 [CrossRef]
  47. Howell, S. L., and Tyhurst, M. (1984) Experientia (Basel) 40, 1098-1105 [Medline] [Order article via Infotrieve]
  48. Boquist, S. L., and Tyhurst, M. (1985) in The Diabetic Pancreas (Volk, B. W., and Arquilla, E. R., eds), pp. 127-212, Plenum Publishing Corp., New York
  49. Nagao, T., Kubo, T., Fujimoto, R., Nishio, H., Takeuchi, T., and Hata, F. (1995) Biochem. J. 307, 563-569 [Medline] [Order article via Infotrieve]
  50. Balsinde, J., and Dennis, E. A. (1996) J. Biol. Chem. 271, 31937-31941 [Abstract/Free Full Text]
  51. Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233 [Abstract/Free Full Text]
  52. Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde, F. K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8527-8531 [Abstract]
  53. Hazen, S. L., Zupan, L. A., Weiss, R. H., Getman, D. P., and Gross, R. W. (1991) J. Biol. Chem. 266, 7227-7232 [Abstract/Free Full Text]
  54. Zupan, L. A., Weiss, R. H., Hazen, S. L., Parnas, B. L., Aston, K. W., Lennon, P. J., Getman, D. P., and Gross, R. W. (1993) J. Med. Chem. 36, 95-100 [Medline] [Order article via Infotrieve]
  55. Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) J. Biol. Chem. 270, 445-450 [Abstract/Free Full Text]
  56. Ackermann, E. J., and Dennis, E. A. (1995) Biochim. Biophys. Acta 1259, 125-136 [Medline] [Order article via Infotrieve]
  57. Portilla, D., Shah, S. V., Lehman, P. A., and Creer, M. H. (1994) J. Clin. Invest. 93, 1609-1615 [Medline] [Order article via Infotrieve]
  58. Hazen, S. L., and Gross, R. W. (1993) J. Biol. Chem. 268, 9892-9900 [Abstract/Free Full Text]
  59. Ramanadham, S., Wolf, M. J., Ma, Z., Li, B., Wang, J., Gross, R. W., and Turk, J. (1996) Biochemistry 35, 5464-5471 [CrossRef][Medline] [Order article via Infotrieve]
  60. Wolf, M. J., and Gross, R. W. (1996) J. Biol. Chem. 271, 30879-30885 [Abstract/Free Full Text]
  61. Balboa, M. A., Balsinde, J., Jones, S. S. & Dennis, E. A. (1997) J. Biol. Chem. 272, in press
  62. Tang, J., Kriz, R. W., Hoffman, N., Shaffer, M., Seehra, J. & Jones, S. S. (1997) J. Biol. Chem. 272, in press

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.