©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification, Characterization, and Partial Amino Acid Sequence of a G Protein-activated Phospholipase C from Squid Photoreceptors (*)

(Received for publication, December 2, 1993; and in revised form, August 8, 1994)

Jane Mitchell (1) Joanne Gutierrez (2) John K. Northup (2)

From the  (1)Department of Pharmacology, University of Toronto, Toronto, M5S 1A8 Canada and the (2)Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Invertebrate visual transduction is thought to be initiated by photoactivation of rhodopsin and its subsequent interaction with a guanyl nucleotide-binding protein (G protein). The identities of the G protein and its target effector have remained elusive, although evidence suggests the involvement of a phospholipase C (PLC).

We have identified a phosphatidylinositol-specific PLC from the cytosol of squid retina. The enzyme was purified to near-homogeneity by a combination of carboxymethyl-Sepharose and heparin-Sepharose chromatography. The purified PLC, identified as an approximately 140-kDa protein by sodium dodecyl sulfate-polyacrylamide gels, hydrolyzed phosphatidylinositol 4,5-bisphosphate (PIP(2)) at a rate of 10-15 µmol/min/mg of protein with 1 µM Ca. The partial amino acid sequence of the protein showed homology with a PLC cloned from a Drosophila head library (PLC21) and lesser homology with Drosophila norpA protein and mammalian PLC beta isozymes.

Reconstitution of purified squid PLC with an AlF-activated 44-kDa G protein alpha subunit extracted from squid photoreceptor membranes resulted in a significant increase in PIP(2) hydrolysis over a range of Ca concentrations while reconstitution with mammalian G(t)alpha or G(i)1alpha was without effect. These results suggest that cephalopod phototransduction is mediated by Galpha-44 activation of a 140-kDa cytosolic PLC.


INTRODUCTION

The molecular mechanism of photoexcitation in vertebrate vision is well established. Photoactivated rhodopsin interacts with a heterotrimeric G protein (^1)which in turn activates a cGMP-phosphodiesterase. The resulting decrease in cGMP levels leads to closure of cGMP-gated ion channels in the membrane and transient hyperpolarization of the cell(1, 2, 3) . Photoexcitation of rhodopsin in most invertebrates, on the other hand, leads to an increase in cation permeability that results in a transient depolarization of the cell(4, 5) . The biochemical events initiated by the photoactivation of rhodopsin and resulting in the opening of cation channels in the membrane of invertebrate photoreceptors have not yet been established.

Biochemical evidence from a number of invertebrates suggests the involvement of a G protein in the initial events of visual transduction (6, 7, 8, 9, 10) . This evidence includes reports of proteins cross-reacting with antibodies to G protein alpha subunits, toxin-mediated ADP-ribosylation, or light-dependent GTP binding to alpha subunits in squid(11, 12, 13) , octopus(14, 15) , and blowfly(16, 17) . It is not clear, however, which second messenger systems are linked to these G proteins. In Limulus, evidence of two different second messengers has emerged from electrophysiological studies in which injection of inositol 1,4,5-trisphosphate (12, 18) or cGMP (19) were shown to mimic the effect of light on ventral photoreceptors, resulting in depolarization. Further evidence for inositol 1,4,5-trisphosphate as a second messenger, at least in Drosophila, came from studies of the norpA mutant. Phospholipase C (PLC) activity is abundant in normal Drosophila eyes and drastically reduced in norpA mutants that exhibit defective phototransduction (20, 21, 22) . The deduced sequence of the norpA protein showed homology to PLC enzymes(23) , and isolated norpA protein demonstrated Ca-sensitive phospholipid hydrolysis(24) . The beta(1) subtype of mammalian PLC has recently been shown to be regulated by G protein alpha subunits of the G(q)/G family(25, 26, 27, 28, 29) . A protein highly homologous to this class of mammalian G proteins has been demonstrated in Drosophila eyes(30, 31) , yet it remains to be shown whether this G protein regulates the norpA PLC.

Recent identification of the principle G protein from squid photoreceptors as a 44-kDa protein closely related to mammalian G(q)alpha has led to speculation that this G protein is coupled to a PLC(32) . We present evidence in this report for the isolation and characterization of a novel phospholipase from squid photoreceptors that is activated by Ca and regulated by the 44-kDa G protein alpha subunit purified from the same photoreceptors. Our results strongly suggest that G protein-activated PLC is the major signal transduction pathway in cephalopod vision.


EXPERIMENTAL PROCEDURES

Materials

CM-Sepharose and heparin-Sepharose were purchased from Pharmacia Biotech Inc. Phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PIP(2) were purchased from Sigma. Phosphatidyl[2-^3H]inositol 4,5-bisphosphate (16.3Ci/mmol), phosphatidyl[2-^3H]inositol (1 Ci/mmol), and anti-G(q)alpha antiserum were purchased from DuPont NEN. Frozen squid eyes were purchased from Calamari Inc., Woods Hole, MA.

Phospholipase C Assay

Phosphatidylinositol hydrolyzing activity was measured, unless otherwise stated, by incubation for 10 min and 37 °C of a mixture containing 10 mM Hepes, pH 7.0, 10 mM NaCl, 4 mM MgSO(4), 100 mM KCl, 0.1% deoxycholate, 50 µM PIP(2), 250 µM PE containing 25,000 dpm of [^3H]PIP(2), 10 µM free Ca in the presence of 2 mM EGTA in a total volume of 100 µl. The reaction was terminated by the addition of 500 µl of chloroform, methanol, 0.1 M HCl (200:100:0.6) followed by the addition of 150 µl of 1 N HCl, 5 mM EGTA. A 0.2-ml aliquot of the upper aqueous phase was removed for measurement of radioactivity. Free Ca concentrations were calculated as in (33) .

Squid Photoreceptor Fractionation

20 frozen squid eyes were thawed into 40 ml of 10 mM Mops, pH 7.5, 2 mM EGTA, 1 mM dithiothreitol (MED buffer) at room temperature, vortexed for 60 s and filtered to remove eye cups, and then homogenized by 5 strokes of a loose-fitting pestle in a Dounce homogenizer. Membranes were separated from the cytosol by centrifugation at 30,000 times g for 20 min at 4 °C. The membrane pellet was resuspended with 34% sucrose in MED buffer, and photoreceptor membranes floated at 28,000 rpm in an SW28 rotor for 30 min. The floating membrane layer was washed twice further with MED buffer containing 100 mM NaCl.

PLC Purification

Cytosol from the 30,000 times g sedimentation was diluted with an equal volume of 100 mM Mes, pH 6.0, 100 mM NaCl, 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, and applied to a column (1.6 times 15 cm) of CM-Sepharose previously equilibrated in 50 mM Mes, pH 6.0, 100 mM NaCl. The column was washed with 2 column volumes of 50 mM Mes, 200 mM NaCl, and the PLC was eluted with a 120-ml linear gradient of 200-500 mM NaCl in 50 mM Mes, pH 6.0. Fractions containing PLC activity from the CM-Sepharose column were pooled and diluted with an equal volume of 100 mM Hepes, pH 7.0, 1 mM EGTA, 1 mM dithiothreitol (HED buffer) and applied to a column (1.6 times 25 cm) of heparin-Sepharose equilibrated in HED, 100 mM NaCl. PLC activity was eluted from the column in a 200-ml linear gradient of 100-500 mM NaCl in HED.

Amino Acid Sequencing

A fraction of highly purified PLC from heparin-Sepharose was chromatographed over G-25 in 100 mM NH(4)HCO(3), lyophilized, and resuspended to about 10 mg/ml with 70% formic acid for CNBr digestion (34) . The CNBr digest was then chromatographed on an AquaPore butyl column (ABI Brownlee) with a 0-80% acetonitrile gradient. Peaks identified by A were rechromatographed isocratically to verify purity prior to sequencing on an Applied BioSystems model 470/900 gas-phase sequencer. Sequences were compared with known DNA and protein sequences in the GenBank using the tFASTa utility.

G Protein Isolation and Reconstitution Assays

G protein alpha subunits were extracted by incubating washed membranes on ice for 30 min in 10 mM Mops, pH 7.5, 10 mM NaF, 20 µM AlCl(3), 10 mM MgSO(4) (AMF), followed by centrifugation at 100,000 times g for 30 min at 4 °C. AMF-activated membranes were then further incubated on ice for 30 min in 10 mM Mops, pH 7.5, 2 M KCl, followed by centrifugation at 100,000 times g for 30 min at 4 °C. G(i)1alpha was purified from rat brain, and G(t)alpha was purified from bovine rod outer segments using previously published methods(35, 36, 37) . For measurement of G protein-regulated PLC activity, aliquots of Galpha-44 extract (0-5 µl), or equivalent molar concentrations of G(t)alpha or G(i)1alpha, were mixed on ice with 5 µl of PIP(2)/PE vesicles (400 µM PIP(2), 8 mM PE containing 25,000 dpm of [^3H]PIP(2) in 10 mM Hepes, pH 7.0, 1% sodium cholate), this mixture was diluted to a final volume of 100 µl containing 10 mM Hepes, pH 7.0, 100 mM NaCl, 4 mM MgSO(4), 0.1 µM free Ca, 2 mM EGTA. The reaction was started by the addition of 2 ng of purified PLC and continued for 10 min at 30 °C. Reactions were terminated as for the PLC assay. In some assays, the specificity of G protein activation of PLC was tested by preincubating Galpha-44 samples with 4 µl of G(q)alpha antisera for 30 min on ice followed by addition of 10 µl of Pansorbin (Calbiochem) and further incubation for 30 min. Absorbed proteins were removed by centrifugation of the samples at 10,000 times g for 10 min, and unabsorbed proteins in supernatants were tested for PLC activation as before. Control immunoprecipitations were performed using the same procedure, but using 1 µl of nonimmune rabbit serum.

Other Methods

GTPS binding to squid photoreceptor membranes was performed as described previously(38) . SDS-PAGE was carried out by the method of Laemmli(39) . Protein bands on gels were visualized by Coomassie Blue, and protein concentrations in extracts were determined using the amido black staining procedure(40) . Proteins were transferred to nitrocellulose for identification by Western blotting with anti-G(q)alpha antisera as described previously (41) , and blots were developed with a horseradish peroxidase-conjugated secondary antibody and visualized using an enhanced chemiluminescent substrate (ECL, Amersham).


RESULTS

Distribution of Phospholipase C and GTP-binding Activity in Squid Photoreceptors

Hydrolysis of phosphatidylinositol could be demonstrated in homogenates of squid photoreceptors. When membranes were sedimented from this homogenate, the majority of PLC activity was recovered in the supernatant fraction and washing the membranes in the presence of 100 mM NaCl removed 95% of the PLC activity (Table 1). Conversely, the majority of GTPS binding in squid retinal homogenates was associated with the membrane fraction (Table 1). Squid eye homogenates and extracts separated on SDS gels and transferred to nitrocellulose were probed with an antibody to mammalian G(q)alpha (Fig. 1). A protein migrating at 44 kDa, previously identified as a GTP-binding protein having a protein sequence similar to that of mammalian G(q)alpha(23) , was recognized on our Western blots by the G(q)alpha antibody. This 44-kDa G protein alpha subunit (termed Galpha-44) was associated with the membrane fraction, but could be solubilized by incubating the membranes in 2 M KCl (Fig. 1, lane 3). Unlike bovine photoreceptor G(t)alpha that dissociates from the membrane in the presence of GTP, Galpha-44 solubility in high salt buffers was not dependent on the presence of nucleotide. Rhodopsin and G protein beta subunits remained associated with the membrane even in the presence of high salt concentrations and were solubilized only in the presence of detergent.




Figure 1: SDS-polyacrylamide gel and immunoblot analysis of squid retinal fractions. Samples of squid retinal membrane preparations and extracted proteins were prepared as described under ``Experimental Procedures'' and subjected to electrophoresis through 10% polyacrylamide. a, the gel was stained with Coomassie Blue; b, the proteins were transferred to nitrocellulose and immunoblotted with antisera specific for mammalian G(q)/G. Lane 1, 5 µg of squid eye homogenate; lane 2, 5 µg of squid retinal membranes; lane 3, 2 µg of 2 M KCl membrane extract.



Purification of PLC

Unlike all other reported phospholipase C enzymes, the soluble PLC extracted from squid retinas did not adsorb to either DEAE or Mono Q but bound tightly to anionic media. The chromatographic profile of a squid eye supernatant fraction when applied to CM-Sepharose is represented in Fig. 2a and shows that the majority of the soluble proteins did not adsorb to this media and passed through the column in low salt buffers. The PIP hydrolyzing activity was eluted from the column as a single peak in approximately 0.3 M NaCl and was associated with a mixture of proteins as seen on SDS-PAGE (Fig. 2b). This mixture of proteins was further resolved by purification over heparin-Sepharose (Fig. 3a). In this second purification step, we identified a protein migrating at approximately 140,000 Da on SDS gels (Fig. 3b) as the major protein associated with PIP hydrolysis activity in the chromatogram. The purity of the early fractions migrating in the activity peak from the heparin-Sepharose column were estimated by Coomassie Blue staining to be greater than 95% phospholipase and were used for subsequent characterization and sequencing without further purification. This purification scheme allowed us to obtain 450 µg of PLC from 20 squid eyes. The purified enzyme has a maximal specific activity of 10-15 µmol of PIP hydrolyzed per min per mg of protein in the presence of 1 µM Ca and 0.1% deoxycholate. A summary of the purification is presented in Table 2.


Figure 2: a, chromatography of soluble fraction from squid retina through CM-Sepharose. See ``Experimental Procedures'' for explanation. b, SDS-polyacrylamide gel electrophoresis of fractions obtained from CM-Sepharose. Aliquots (10 µl) of the indicated fractions were subjected to electrophoresis through 7.5% polyacrylamide gel. Proteins were visualized with Coomassie Blue. Numbers at the side of the gel indicate the migration of protein standards.




Figure 3: a, chromatography of squid retinal PLC through heparin-Sepharose. See ``Experimental Procedures'' for explanation. b, SDS-polyacrylamide gel electrophoresis of fractions obtained from heparin-Sepharose. Aliquots (10 µl) of the indicated fractions were subjected to electrophoresis as described in Fig. 2.





pH and Ca Dependence of the PLC

The effect of pH on the PIP(2) and PI hydrolyzing activity was measured in the presence of 1 µM and 1 mM free Ca, respectively (data not shown). The enzyme exhibited activity over a broad pH range from 6 to 8 with PI as substrate showing slightly higher activity at pH 6.5. When PIP(2) was used as substrate, the enzyme had an even broader range of activity from pH 5 to 8, again slightly higher around neutral pH. The Ca dependence of hydrolysis of both PI and PIP(2) was also examined (data not shown). PI hydrolysis by the purified enzyme shallowly increased with increasing Ca up to 10 mM, whereas with PIP(2) vesicles as substrate, the PLC activity increased steeply to a maximum level at 3-10 µM and then rapidly decreased at higher calcium concentrations.

Peptide Sequence Identification of Squid Retinal PLC

Initial experiments failed to identify any amino-terminal sequence of the full-length PLC from heparin-Sepharose. Therefore, the PLC was subjected to CNBr degradation, and sequences from eight peptide fragments were obtained. Seven of these were independent sequences, with an eighth fragment fully contained in a larger sequence. The four longest independent polypeptides are presented in Fig. 4. This figure compares the amino acid sequence obtained for these fragments with homologous sequences identified from three PLCs, Drosophila neuronal PLC21, Drosophila visual norpA, and a mammalian PLC beta rat1. Squid eye PLC sequences 1-3 matched PLC21 as the highest match in GenBank for sequence homology. Peptide sequence 4 did not match any PLC when compared against the entire sequence data base. When compared only against the identified PLC sequences, this peptide aligns as shown in Fig. 6. In all cases, these sequences were matched best to the PLC21 clone.


Figure 4: Comparison of the amino acid sequences of peptide fragments obtained from squid eye PLC with those deduced for PLC21, norpA, and Rat1. The preparation and sequencing of the peptide fragments from purified squid eye PLC are described under ``Experimental Procedures.'' The sequences are given in one-letter code. The sequences showing identity with those in the squid PLC are shown in bold letters. Amino acid residues are numbered at the beginning of each line.




Figure 6: Effect of Galpha-44 on PLC activity at varying calcium concentrations. Purified PLC (2 ng) was incubated with phospholipid vesicles containing PIP(2) and indicated concentrations of free Ca in the presence (bullet) or absence (circle) of 8 ng of squid retinal Galpha-44. Assays were performed as described under ``Experimental Procedures.''



G Protein Regulation of PLC Activity

AMF-activated Galpha-44 extracted from squid photoreceptor membranes or mammalian G protein alpha subunits were reconstituted with purified PLC (Fig. 5). The squid Galpha-44 protein markedly stimulated PIP(2) hydrolysis by the enzyme. Half-maximal and maximal activation were achieved with a molar ratio of Galpha-44:PLC of 2:1 and 14:1, respectively, whereas addition of either G(i)alpha or G(t)alpha, even at an 18:1 ratio over PLC, was without effect. Purified squid PLC could be activated approximately 3-fold by Galpha-44 in the absence of AMF, indicative of the presence of activated Galpha-44 in our preparation; however, addition of AMF increased this activation further to approximately 9 times that seen under basal conditions (Table 3). The PLC activation by Galpha-44 was sensitive to boiling and could be eliminated by immunoprecipitation with the anti-G(q)alpha antibody (Table 3). Galpha-44 activated squid PLC over a range of Ca concentrations and did not appreciably alter the enzyme's sensitivity to calcium (Fig. 6).


Figure 5: Effect of G proteins on PLC activity. Purified PLC was incubated with phospholipid vesicles containing PIP(2) and indicated concentrations of purified squid eye Galpha-44 (bullet), bovine retinal G(t)alpha (up triangle), or rat brain G(i)1alpha () in the presence of AlF and 1 µM free Ca. Results shown are representative of independent experiments using three separate PLC preparations.






DISCUSSION

The results of our experiments presented here clearly demonstrate the presence of a PLC enzyme in squid photoreceptors. The 140-kDa enzyme was readily purified from the cytosolic fraction of squid photoreceptors and shared similar properties with several mammalian phospholipase C subtypes. The catalytic properties of the enzyme toward PI were very different from those toward PIP(2). Over all concentrations of calcium up to 1 mM, the purified enzyme had a higher specific activity for PIP(2) than PI and was 10 times more active with PIP(2) as substrate at 1 µM free calcium than with PI at 1 mM free calcium. The squid protein was active over a broad pH range from 5.5 to 8 but was almost inactive at pH 9. With the exception that squid PLC was more active at neutral pH, this substrate preference most resembles that reported for the mammalian PLC beta subtype(42) , but is quite distinct from that reported for the only other invertebrate PLC purified to date, namely Drosophila norpA gene product, that is maximally active at pH 9 and inactive at pH 6. Squid PLC appears to be a uniquely basic protein that is not recognized by antisera to either the beta, , or classes of vertebrate PLC isozymes. (^2)Sequence data obtained from CNBr fragments of the purified protein showed best homology to the Drosophila neuronal PLC clone, PLC21, with lesser homology to the norpA gene product. This latter finding was unexpected in that norpA clearly functions importantly in fly vision, and it may signify an evolutionary divergence among invertebrate photoreceptors. Additional neuronal signaling function(s) for norpA based upon cellular localization has recently been suggested, and our findings may further signify that additional PLC isozymes participate in visual signaling. Full analysis of the relationship of this protein to other members of the PLC family of proteins, however, must await elucidation of the complete amino acid sequence of the squid PLC and studies of its cellular distribution.

A number of guanyl nucleotide-binding proteins have been identified in invertebrate photoreceptors, but the role of these proteins in visual transduction has not been clearly elucidated. The Galpha-44 that we have isolated from squid photoreceptors appears to be the same as a protein previously reported as the major Galpha subunit in squid photoreceptors that is closely related in amino acid sequence to mammalian G(q)alpha(32) . Indeed, our Galpha-44 was shown here to cross-react with antibodies raised against mammalian G(q)alpha. The homology of G protein alpha subunits in both squid and Drosophila eyes to the G(q)/G family of vertebrate G proteins that modulate PLC enzymes of the beta subtype led us to investigate the ability of Galpha-44 to regulate the squid PLC. The results of our experiments reported here demonstrate that the effects of Galpha-44 on squid PLC are very similar to those reported for G(q)alpha on PLC beta(1)(25, 26, 27, 28, 29) . The presence of Galpha-44 effectively increased PIP(2) hydrolysis by the enzyme over a range of Ca concentrations from 30 nM to 10 µM. This activity appeared to be specific to this alpha subunit, and we have not been able to modify the enzyme's activity with other G protein alpha subunits or the purified beta subunits. Our experiments were performed using Galpha-44 extracted from photoreceptors treated with either GTPS or AlF in order to maximally activate the G protein. We have also observed substantial activity using Galpha-44 extracted in the absence of added nucleotide or activators. A possible explanation of this basal activity is the presence of some other PLC-activating factor in the Galpha-44 preparation that affects PLC activity independent of guanyl nucleotides. Our data showing complete inhibition of PLC activation following immunoprecipitation with G(q)alpha antisera, however, indicate that the activity in the samples was attributed to Galpha-44 rather than a contaminant. We consider it more probable that this basal activity is the result of intrinsic GTP binding to Galpha-44 in our membranes which were prepared under full illumination, and we are currently addressing this issue using dark-adapted animals. Several groups have reported proteins modified by both cholera and pertussis toxins in squid, and one report has shown a protein identified by antisera raised against G(t)alpha(13) . In our preparations we did not detect proteins using polyclonal antisera raised against bovine G(t)alpha. However, this may have been the result of different extraction conditions used in our experiments, and further work would be needed to examine the role of other G proteins in invertebrate photoreceptors.

Data from several laboratories have reported that PLC enzymes are modified during purification and storage possibly by contaminating proteases. Park et al.(43) have recently reported that the Ca-dependent enzyme calpain can cleave PLC beta(1) from 150 kDa to 100- and 45-kDa fragments that retain full PIP(2) hydrolyzing activity and sensitivity to Ca but are devoid of G(q) activation. In squid retina we saw evidence of only a single species of PLC activity associated with the 140-kDa protein characterized here. Indirect evidence of proteolysis of the 140-kDa protein by a Ca-dependent enzyme was found during our early preparations when impure PLC samples were applied to hydroxyapatite; we observed rapid and complete loss of the 140-kDa protein and PLC activity associated with fractions containing 94- and 40-kDa fragments. We are currently examining the regulation of these PLC hydrolysis fragments by Galpha-44.

The solubility of squid PLC, in contrast to G protein-regulated mammalian PLC beta which is mostly membrane-associated in brain, may reflect the association of the two enzymes with their respective G protein activators. PLC beta(1) interacts with a membrane-bound activator G(q)alpha, while our data indicate that squid Galpha-44 may be soluble under some conditions. Solubility has thus far been uniquely associated with retinal transducins in vertebrates, and this may indicate greater similarities between mammalian G(t)alpha and invertebrate Galpha-44 than is readily apparent from their amino acid sequences. This raises the possibility that Galpha-44 dissociates from the disc membrane after interaction with photoactivated rhodopsin and associates with the cytosolic PLC. Our data presented here clearly demonstrate that such an interaction can occur. The detailed examination of the molecular interaction of Galpha-44 with rhodopsin and PLC will be the subject of further reports.

A growing body of evidence suggests that light-dependent activation of PLC does take place in invertebrate photoreceptors and our data are the first demonstration of G protein regulation of PLC in such a system. As we have shown, under the regulation of Galpha-44, PLC is activated, and the concentration of inositol 1,4,5-trisphosphate and presumably diacylglycerol in invertebrate photoreceptors is increased. It remains to be demonstrated how these second messengers regulate the opening of cation channels that leads to the depolarization of the photoreceptor membrane.


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.

(^1)
The abbreviations used are: G protein, guanine nucleotide-binding protein; PLC, phospholipase C; PIP(2), phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PI, phosphatidylinositol; Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; GTPS, guanosine 5`-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. Mitchell, unpublished data.


REFERENCES

  1. Liebman, P. A., Parker, K. R., and Dratz, E. A. (1986) Annu. Rev. Physiol. 49, 765-791 [CrossRef][Medline] [Order article via Infotrieve]
  2. Chabre, M., and Dteerre, P. (1989) Eur. J. Biochem. 179, 255-266 [Medline] [Order article via Infotrieve]
  3. Stryer, L. (1991) J. Biol. Chem. 266, 10711-10714 [Free Full Text]
  4. Wong, F. (1978) Nature 276, 76-79 [Medline] [Order article via Infotrieve]
  5. Bacigalupo, J., and Lisman, J. (1983) Nature 304, 268-270 [Medline] [Order article via Infotrieve]
  6. Fein, A., and Corson, D. W. (1979) Science 204, 77-79 [Medline] [Order article via Infotrieve]
  7. Fein, A., and Corson, D. W. (1981) Science 212, 555-557 [Medline] [Order article via Infotrieve]
  8. Bolsover, S. R., and Brown, J. E. (1982) J. Physiol. (Lond.) 332, 325-342 [Medline] [Order article via Infotrieve]
  9. Fein, A. (1986) Science 232, 1543-1545 [Medline] [Order article via Infotrieve]
  10. Kirkwood, A., Weiner, D., and Lisman, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3872-3876 [Abstract]
  11. Vandenberg, C. A., and Montal, M. (1984) Biochemistry 23, 2339-2347 [Medline] [Order article via Infotrieve]
  12. Brown, J. E., Rubin, L. J., Ghalayini, A. J., Tarver, A. P., Irvine, R. F., Berridge, M. J., and Anderson, R. E. (1984) Nature 311, 160-163 [Medline] [Order article via Infotrieve]
  13. Robinson, P. R., Wood, S. F., Szuts, E. Z., Fein, A., and Hamm, H. E. (1990) Biochem. J. 272, 79-85 [Medline] [Order article via Infotrieve]
  14. Tsuda, M., Tsuda, T., Teragama, Y., Fukada, Y., Akimo, T., Yamanaka, G., Stryer, L., Katada, T., Ui, M., and Ebrey, T. E. (1986) FEBS Lett. 198, 5-10 [CrossRef]
  15. Tsuda, M. (1987) in Retinal Proteins (Ouchininikou, Y., ed) pp. 393-404, VNU Science Press, The Netherlands
  16. Paulsen, R., and Bentrop, J. (1986) Prog. Zool. 33, 299-319
  17. Bentrop, J., and Paulsen, R. (1986) Eur. J. Biochem. 161, 61-67 [Abstract]
  18. Fein, A., Payne, R., Corson, D. W., Berridge, M. J., and Irvine, R. F. (1984) Nature 311, 157-160 [Medline] [Order article via Infotrieve]
  19. Johnson, E. C., Robinson, P. R., and Lisman, J. E. (1986) Nature 324, 468-470 [Medline] [Order article via Infotrieve]
  20. Yoshika, T., Inoue, H., and Hotta, Y. (1985) J. Biochem. (Tokyo) 97, 1251-1254 [Abstract]
  21. Inoue, H., Yoshika, T., and Hotta, Y. (1985) Biochem. Biophys. Res. Commun. 132, 513-519 [Medline] [Order article via Infotrieve]
  22. Inoue, H., Yoshika, T., and Hotta, Y. (1988) J. Biochem. (Tokyo) 103, 91-94 [Abstract]
  23. Bloomquist, B. T., Shortridge, R. D., Schneuwly, S., Perdew, M., Montell, C., Steller, H., Rubin, G., and Pak, W. L. (1988) Cell 54, 723-733 [Medline] [Order article via Infotrieve]
  24. Toyoshima, S., Matsumoto, N., Wang, P., Inoue, H., Yoshioka, T., Hotta, Y., and Osawa, T. (1990) J. Biol. Chem. 265, 14842-14848 [Abstract/Free Full Text]
  25. Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507 [Abstract/Free Full Text]
  26. Taylor, S. J., Smith, J. A., and Exton, J. H. (1990) J. Biol. Chem. 265, 17150-17156 [Abstract/Free Full Text]
  27. Smrcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991) Science 252, 804-807
  28. Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991) Nature 350, 516-518 [CrossRef][Medline] [Order article via Infotrieve]
  29. Wange, R. L., Smrcka, A. V., Sternweis, P. C., and Exton, J. H. (1991) J. Biol. Chem. 266, 11409-11412 [Abstract/Free Full Text]
  30. Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117 [Abstract]
  31. Lee, Y., Dobbs, M. B., Veradi, M. L., and Hyde, D. R. (1990) Neuron 5, 889-898 [Medline] [Order article via Infotrieve]
  32. Pottinger, J. D. D., Ryba, N. J., Keen, L. N., and Findlay, J. B. C. (1991) Biochem. J. 279, 323-326 [Medline] [Order article via Infotrieve]
  33. Raafluab, J. (1960) Methods Biochem. Anal. 3, 301-325
  34. Charbonneau, H. (1989) A Practical Guide to Protein and Peptide Purification and Microsequencing (Matsudaira, P., ed) Academic Press, New York
  35. Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813 [Abstract/Free Full Text]
  36. Fawzi, A. B., and Northup, J. K. (1990) Biochemistry 29, 3804-3812 [Medline] [Order article via Infotrieve]
  37. Fung, B. K.-K. (1983) J. Biol. Chem. 258, 10495-10402 [Abstract/Free Full Text]
  38. Northup, J. K., Smigel, M. D., and Gilman, A. G. (1982) J. Biol. Chem. 257, 11416-11423 [Free Full Text]
  39. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  40. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514 [Medline] [Order article via Infotrieve]
  41. Towbin, H., Staehelin, T., and Gordon, T. (1986) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354
  42. Ryu, S. H., Suh, P.-G., Cho, K. S., Lee, K.-Y., and Rhee, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6649-6653 [Abstract]
  43. Park, D., Jhon, D.-Y., Lee, C.-W., Ryu, S. H., and Rhee, S. G. (1993) J. Biol. Chem. 268, 3710-3714 [Abstract/Free Full Text]

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