63
Biochimica et Biophysica Acta, 1160 (1992) 63-66 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34364
Recoverin, a novel calcium-binding protein from vertebrate photoreceptors Hans-Georg Lambrecht and Karl-Wilhelm Koch Institut fiir Biologische lnformationsverarbeitung, Forschungszentrum Jiilich, Jiilich, (Germany)
Key words: Photoreceptor; Guanylyl cyclase; Recoverin; Calcium-binding protein
Photoreceptor guanylyl cyclase activity is modulated by an endogenous calcium-binding protein called recoverin. A modified isolation procedure for recoverin using gel-filtration chromatography instead of a heat denaturation step is presented. The elution volume of recoverin corresponds to a monomer. Recoverin exhibits a calcium-dependent mobility shift in a native gel electrophoresis. Isoelectric focusing revealed a pI of 5.25. No subspecies of recoverin were detected.
Introduction
Calcium has been identified as the intracellular messenger of light adaptation in vertebrate photoreceptors. Electrophysiological studies demonstrated that prevention of the light-induced decrease of cytoplasmic calcium through clamping of the intracellular calcium near the dark value leads to a loss of light-adapting characteristics (for recent reviews, see Refs. 1-4). Enzymes involved in regulating the metabolism of guanosine 3',5'-cyclic monophosphate (cGMP), the intracellular messenger of visual excitation, are the possible targets of a decreased calcium level [1,4]. Cyclic GMP is synthesized by a photoreceptor-specific guanylyl cyclase [5,6] and hydrolyzed via an enzymatic cascade triggered by illumination [7]. As reported so far, calcium has a profound influence on guanylyl cyclase (GC) activity only. Decreasing calcium below the dark value of 300 nM leads to a 5-20-fold stimulation of GC activity [8,9]. This effect is highly cooperative and part of a negative feedback loop that restores the cGMP level after illumination and controls light adaptation in vertebrate photoreceptors. A novel calcium-binding protein of the EF-hand family called recoverin or modulator protein [10,11] is necessary for the activation of cyclase. Recoverin has been purified and its amino-acid
Correspondence to: K.-W. Koch, Institut fiir Biologische Informationsverarbeitung, Forschungszentrum Jiilich, Postfach 1913, 5170 Jiilich, Germany. Abbreviations: ROS, rod outer segments; PBS, phosphate-buffered saline; FPLC, fast protein liquid chromatography.
sequence determined [10-13]. It is phosphorylated at low calcium concentrations ( < 200 nM) by an unknown kinase. The phosphorylation probably modulates the activation of GC [14]. In contrast to other calcium-binding effector molecules like calmodulin [15], recoverin activates its target enzyme GC when calcium is decreased below 300 nM. We here report some biochemical properties of recoverin and describe an alternative purification procedure yielding high amounts of native recoverin. Materials and Methods
Materials. All columns, column material and molecular mass standards were from Pharmacia. All detergents were from Boehringer-Mannheim. Freund's adjuvant and 4-chloro-l-naphthol were from Sigma. Horseradish peroxidase was from Amersham. Preparation of ROS and GC assay. Rod outer segments (ROS) were prepared from freshly collected bovine eyes by a standard protocol [11]. Assays of guanylyl cyclase activity at different free calcium concentrations was performed as described previously using the calcium buffer E G T A [11]. Biological activity of recoverin is tested in a reconstitution experiment and expressed as modulator activity [11]. Purification of recoverin. Dark-kept ROS from 120170 retinae were adjusted to 100 mM NaCI, 1 mM MgCI 2 and 3 mM ATP and fully bleached for 15 min at 37°C in a waterbath. After illumination the suspension was adjusted to 10 mM PO43- using a 1 M N a z H P O a / N a H 2 P O 4 ((pH 7.2), 3: 1) stock solution and left on ice for 20 min. The suspension was centrifuged (35 000 rpm, 35 min, TI70 Beckman), the su-
64 pernatant harvested and remaining membrane particles removed by a second centrifugation step at 80000 rpm for 20 min in a TL100.3 Beckman rotor. The cleared solution (12-16 ml) was concentrated 10-fold by ultrafiltration using a centricon 10 (Amicon). The concentrated sample was loaded on a Superdex 75 HiLoad 16/60 gel filtration column (Pharmacia). The column was equilibrated in 50 mM NaCI, 20 mM Tris-HCl (pH 8.0) (buffer A) and chromatography of proteins was performed in the same buffer at a flow rate of 1 ml/min. Fractions containing recoverin were further purified on a MonoQ H R 5 / 5 anion exchange column using a NaCl-gradient (50-275 mM) as described [11]. The Superdex 75 Hiload 16/60 column (separation range 3-70 kDa) was calibrated with the following proteins: bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). The void volume (V0) of the column ( = 44 ml) was determined with Blue-dextran. The elution volumes (V~) of proteins were used to calculate their Kav-values according to Kav = (Ve Vo)/(V t - Vo) where Vt is the total volume of the column (120 ml). Antibodies against recoverin. Purified recoverin (100 /xg) was mixed with Freund's complete adjuvant and subcutaneously injected in a New Zealand white rabbit. Immunization was boosted after 3 weeks by injecting the same amount of recoverin. Blood was collected after two weeks and the recoverin antibodies were affinity purified from the serum. For this purpose, recoverin was coupled to CNBr-activated Sepharose according to the manufacturers manual (0,75 mg recoverin per 1 ml Sepharose). The serum was diluted 10-fold with 10 mM Tris (pH 7.5) and passed over the recoverin affinity column. Specific antibodies were eluted with 0.1 M glycine (pH 2.5) and with 0.1 M triethanolamine (pH 11.5). Shortly after elution, the antibody solutions were neutralized with 1 M Tris-HC1 (pH 8.0) and concentrated by chromatography on Protein A-Sepharose [16]. Electrophoresis and Western blotting. Electrophoresis was performed after Laemmli [17] or with the PhastSystem of Pharmacia. Gels were stained with silver or Coomassie brilliant blue R-250 or were electroblotted on nitrocellulose (Schleicher & Schfill). After blotting, the nitrocellulose membrane was blocked with PBS 0.5% Tween-20 for 2 h at room temperature or overnight at 4°C. Incubation with the purified anti-recoverin antibody (dilution 1:1000 or 1:500 in PBS 0.05% Tween-20) was for 1 h at room temperature or overnight at 4°C. The membrane was washed three times in the same buffer without antibody and then incubated with the second antibody (anti-rabbit IgG coupled with horseradish peroxidase 1 : 300) for 1 h at room temperature. The washing step was repeated and
the immunoreaction was visualized with 4-chloro-1n a p h t h o l / H 2 0 2 as peroxidase substrate. Results and Discussion
Recoverin is necessary for the calcium-dependent activation of photoreceptor GC [10,11]. In order to further study its function and properties, we here present a rapid and mild isolation procedure yielding high amounts of purified recoverin. Purification to apparent homogeneity was achieved with three steps: (1), Specific elution of soluble ROS proteins after illumination using the ROS membranes as an affinity matrix [,18]; (2), FPLC gel filtration chromatography and (3), FPLC anion-exchange chromatography on a MonoQ column. Enrichment of recoverin during this isolation procedure correlated well with its ability to increase GC activity at low calcium. The procedure presented here has the advantage of using milder conditions for separation, i.e., gel-filtration chromatography instead of a heat-denaturation step [11]. This resulted in a higher yield of purified protein (about 0.25 mg per 100 retinae). Furthermore, this purification protocol also avoids the use of any detergents in the elution buffers which are part of other purification protocols [10,13]. We found that various kinds of detergents, including ndodecyl-/3-D-maltoside, octanocyl-N-methylglucamide, n-octylglucoside, Triton X-100, Nonidet P-40 and ndodecyl-N, N-dimethyl-3-amino- 1-propanesulfonate, all interfere with the activation of photoreceptor GC by recoverin.
Gel-filtration chromatography Gel filtration chromatography on the Superdex 75 column was an effective step in separating recoverin from other retinal proteins. Recoverin containing fractions are 80% pure (Fig. 1A, B). One of the main contaminants is a 29-kDa protein (p29), but it can be separated from recoverin along with other contaminants by a subsequent anion-exchange chromatography. Fractions of the gel-filtration step containing recoverin exhibited the same modulator activity as purified recoverin. For example, partially purified recoverin obtained after the gel-filtration step (Fig. 1) was reconstituted with GC in washed ROS membranes and the GC activity was determined at high (563 nM) and low (4 nM) calcium. GC activity increased from 1.8 _+ 0.1 n m o l / m i n per mg to 4.2 _+ 0.5 n m o l / m i n per mg (three determinations). Almost identical values were obtained using purified recoverin as shown in Fig. 2A (1.47 n m o l / m i n per mg at high free calcium and 4.7 n m o l / m i n per mg at low free calcium) and as we have described previously [11]. In contrast, fractions of soluble ROS proteins without recoverin did only show the basal GC activity as it is found in washed ROS membranes [9-11]. The main portion of the p29 eluted
65 A 1,5 O
~Re
¢
O
IdW(kDa)
B
1,0
c-~4TI~"
0,5
-
67
-
49 34 SO
0,0 ,
0
,
,
20
~0
i
i
,
,
l
8O
60
i
100
Fraction (ml) Fig. 1. Gel-filtration chromatography of soluble ROS proteins. (A), An extract of soluble ROS proteins obtained after illumination (1.5 ml) was applied on a Superdex 75 HiLoad 16/60 FPLC column. The first 28 ml of the elution volume are not displayed (no elution of protein peaks), fractions containing recoverin eluted between 65 and 73 ml (see bold line). (B), SDS-PAGE of the fraction containing recoverin (Rec) marked by the bold line. The gel (10-15% acrylamide) was silver stained.
A
B MW(kDa)
%o
E
O
.E
-
94
-
67
-
43
-
30
-
20
shortly before recoverin on the gel filtration column. F u r t h e r purification and initial characterization (Lainbrecht, H.-G. and Koch, K.-W., unpublished results) revealed that this protein is not a calcium-binding protein (as, e.g., calbindin or calretinin [19] which have about the same molecular mass). We used a purified preparation of the p29 as a control in o u r reconstitution assay to show that the c a l c i u m - d e p e n d e n t activation of G C specifically depends on the presence of recoverin (Fig. 2A) and is not mimicked by other soluble retinal proteins (Fig. 2B). W e also used the gel-filtration c h r o m a t o g r a p h y for a molecular weight estimation. T h e logarithms of molecular mass standards (see Materials and M e t h o d s ) were plotted as a function of their Kav values. W e determined a Kay value of 0.35 for recoverin which would correspond to a molecular mass of 32 kDa. This indicates that recoverin is probably a m o n o m e r . Recoverin as a calcium-binding protein Calcium-binding properties of recoverin have been d e d u c e d from its amino-acid sequence which contains at least 3 E F - h a n d s [20] and have been directly d e m o n strated by 45Ca-autoradiography [10,11]. A n o t h e r characteristic feature of calcium binding proteins is their calcium d e p e n d e n t mobility shift during gel electrophoresis. Interestingly, recoverin displayed a different mobility in native electrophoresis (greater mobility in the presence of 1 m M E G T A than in the presence of 1 m M Ca2÷; see Fig. 3B) but not in S D S - P A G E (Fig. 3A), whereas many other calcium-binding proteins like calmodulin show a mobility shift in both gel systems [21]. Recoverin has an isoelectric point of 5.25 (Fig. 4A) which is close to the p I of visinin (5.1). Visinin, a calcium-binding protein from chicken cones
_E ao 0
E e>
"N 2,0 -
Co
EGTA
O 1,0i
o,o
I I h
I
h
I
[Ca2+] Fig. 2. Calcium-dependent GC activity at high (h = 563 nM) and low (l = 4 nM) free calcium. Washed ROS membranes were either reconstituted with purified recoverin (A) or purified p29 (B) and assayed for GC activity (expressed in nmol cGMP produced per minute and per mg rhodopsin). SDS-PAGE (12.5% acrylamide) of 1 /zg recoverin and 1/zg p29 is shown in (a) and (b).
ii!!iii@ ~ CO
EGTA
--
i li!iiiiiiii!i!~iii!
'~'% ~iiiii !i
~ i i!
~
Fig. 3. Electrophoretic mobility of recoverin in the presence of calcium or EGTA. Recoverin obtained after MonoQ anion-exchange chromatography was subjected to either SDS-PAGE (A) or a native PAGE (B). 2 Izg of recoverin were electrophoresed on a 12.5% gel. Either 1 mM CaCl 2 or 1 mM EGTA was added to the sample buffer.
66
B
A
ratio of 1 : 160 (taking an average of 70 mg rhodopsin in an ROS preparation of 100 retinae). This quantitation confirms our previous estimation that recoverin is one of the major soluble proteins and thus may also serve as an intracellular calcium buffer beside its role as the endogenous activator of photoreceptor GC [4,11].
ii!i iiii!~ ¸iill~ii¸i iii~il ~i~ii ~~ii~~
pH ,i~i~!~i!
5.85 Acknowledgements
Rec
-'-Rec
5.20 -
1,.55
We thank Mrs. D. H6ppner-Heitmann for technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
References -
1
2
Fig. 4. Isoelectric focusing. (A), lane 1 shows a p l standard and lane 2 shows 0.5 Izg of purified recoverin. (B), Western blot of soluble ROS proteins. After isoelectric focusing of an extract of ROS proteins samples were electrotransferred on a nitrocellulose membrane. Proteins were probed with a purified antibody to recoverin.
i22], has about 60% sequence homology to recoverin. Isoelectric focusing of an extract of soluble ROS proteins and detection of recoverin with an affinity-purified antibody revealed only one band at p I 5.25 (Fig. 4B). The presence of two 26 kDa proteins was recently described in amphibian ROS [23], and at least one protein with p I 5.8 seemed to influence the activity of cGMP phosphodiesterase in a calcium-dependent way. Partial sequence analysis of this protein revealed a similarity to both visinin and calbindin [23]. It is unclear, however, whether one of the 26 kDa proteins is identical to recoverin or whether both proteins represent subspecies or completely different polypeptides. Polymorphism has recently been described for two other photoreceptor proteins, arrestin [24] and GC [6]. We here show that native recoverin from mammalians is only present in one form and that a protein with a similar molecular mass, the p29, does not share any characteristic properties with recoverin. Based on the recovery of biological activity we previously calculated a molar ratio of recoverin to rhodopsin of about 1:100. The extractable amount of 0.25 mg from 100 retinae would correspond to a similar molar
1 Pugh, E.N., Jr. and Lamb, T.D. (1990) Vision Res. 30, 1923-1948. 2 Fain, G.L. and Matthews, H.R. (1990) Trends Neurosci. 13, 378-384. 3 Yau, K.-W. (1991) Curr. Opin. Neurobiol. 1, 252-257. 4 Kaupp, U.B. and Koch, K.-W. (1992) Annu. Rev. Physiol. 54, 153-175. 5 Koch, K.-W. (1991) J. Biol. Chem. 266, 8634-8637. 6 Hayashi, F. and Yamazaki, A. (1991) Proc. Natl. Acad. Sci. USA 88, 4746-4750. 7 Stryer, L. (1991) J. Biol. Chem. 266, 10711-10714. 8 Pepe, I.M., Panfoli, I. and Cugnoli, C. (1986) FEBS Lett. 203, 73-76. 9 Koch, K.-W. and Stryer, L. (1988) Nature 334, 64-66. 10 Dizhoor, A.M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K.A., Philipov, P.P., Hurley, J.B. and Stryer, L. (1991) Science 251,915-918. 11 Lambrecht, H.-G. and Koch, K.-W. (1991) EMBO J. 10, 793-798. 12 Polans, A.S., Buczylko, J., Crabb, J. and Palczewski, K. (1991) J. Cell Biol. 112, 981-989. 13 Kutuzov, M.A., Shmukler, B.E., Suslov, O.N., Dergachev, A.E., Zagarov, A.A. and Abdulaev, N.G. (1991) FEBS Lett. 293, 21-24. 14 Lambrecht, H.-G. and Koch, K.-W. (1991) FEBS Lett. 294, 207209. 15 Klee, C.B., Crouch, T.H. and Richman, P.G. (1980) Annu. Rev. Biochem. 49, 489-515. 16 Harlow, E. and Cone, D. (1988) Antibodies, a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbour. 17 Laemmli, U.K. (1970) Nature 227, 680-685. 18 Kiihn, H. (1984) in Progress in Retinal Research, Vol. 3. (Osborne, N. and Chader, J. eds.), pp. 123-156, Pergamon Press, Oxford. 19 Pastells, B., Rogers, J., Blachier, F. and Pochet, R. (1990) Vis. Neurosci. 5, 1-16. 20 Moncrief, N.D., Kretsinger, R.H. and Goodman, M. (1990). J. Mol. Evol. 30, 522-562. 21 Burgess, W.H., Jemiolo, D.K. and Kretsinger, R.H. (1980) Biochim. Biophys. Acta 623, 257-270. 22 Yamagata, K., Goto, K., Kuo, C.H., Kondo, H. and Miki, N. (1990) Neuron 2, 469-476. 23 Kawamura, S. and Murakami, M. (1991) Nature 349, 420-423. 24 Weyand, I. and Kiihn, H. (1990) Eur. J. Biochem. 193, 459-467.
Biochimica et Biophysica Acta, 1160 (1992) 63-66 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34364
Recoverin, a novel calcium-binding protein from vertebrate photoreceptors Hans-Georg Lambrecht and Karl-Wilhelm Koch Institut fiir Biologische lnformationsverarbeitung, Forschungszentrum Jiilich, Jiilich, (Germany)
Key words: Photoreceptor; Guanylyl cyclase; Recoverin; Calcium-binding protein
Photoreceptor guanylyl cyclase activity is modulated by an endogenous calcium-binding protein called recoverin. A modified isolation procedure for recoverin using gel-filtration chromatography instead of a heat denaturation step is presented. The elution volume of recoverin corresponds to a monomer. Recoverin exhibits a calcium-dependent mobility shift in a native gel electrophoresis. Isoelectric focusing revealed a pI of 5.25. No subspecies of recoverin were detected.
Introduction
Calcium has been identified as the intracellular messenger of light adaptation in vertebrate photoreceptors. Electrophysiological studies demonstrated that prevention of the light-induced decrease of cytoplasmic calcium through clamping of the intracellular calcium near the dark value leads to a loss of light-adapting characteristics (for recent reviews, see Refs. 1-4). Enzymes involved in regulating the metabolism of guanosine 3',5'-cyclic monophosphate (cGMP), the intracellular messenger of visual excitation, are the possible targets of a decreased calcium level [1,4]. Cyclic GMP is synthesized by a photoreceptor-specific guanylyl cyclase [5,6] and hydrolyzed via an enzymatic cascade triggered by illumination [7]. As reported so far, calcium has a profound influence on guanylyl cyclase (GC) activity only. Decreasing calcium below the dark value of 300 nM leads to a 5-20-fold stimulation of GC activity [8,9]. This effect is highly cooperative and part of a negative feedback loop that restores the cGMP level after illumination and controls light adaptation in vertebrate photoreceptors. A novel calcium-binding protein of the EF-hand family called recoverin or modulator protein [10,11] is necessary for the activation of cyclase. Recoverin has been purified and its amino-acid
Correspondence to: K.-W. Koch, Institut fiir Biologische Informationsverarbeitung, Forschungszentrum Jiilich, Postfach 1913, 5170 Jiilich, Germany. Abbreviations: ROS, rod outer segments; PBS, phosphate-buffered saline; FPLC, fast protein liquid chromatography.
sequence determined [10-13]. It is phosphorylated at low calcium concentrations ( < 200 nM) by an unknown kinase. The phosphorylation probably modulates the activation of GC [14]. In contrast to other calcium-binding effector molecules like calmodulin [15], recoverin activates its target enzyme GC when calcium is decreased below 300 nM. We here report some biochemical properties of recoverin and describe an alternative purification procedure yielding high amounts of native recoverin. Materials and Methods
Materials. All columns, column material and molecular mass standards were from Pharmacia. All detergents were from Boehringer-Mannheim. Freund's adjuvant and 4-chloro-l-naphthol were from Sigma. Horseradish peroxidase was from Amersham. Preparation of ROS and GC assay. Rod outer segments (ROS) were prepared from freshly collected bovine eyes by a standard protocol [11]. Assays of guanylyl cyclase activity at different free calcium concentrations was performed as described previously using the calcium buffer E G T A [11]. Biological activity of recoverin is tested in a reconstitution experiment and expressed as modulator activity [11]. Purification of recoverin. Dark-kept ROS from 120170 retinae were adjusted to 100 mM NaCI, 1 mM MgCI 2 and 3 mM ATP and fully bleached for 15 min at 37°C in a waterbath. After illumination the suspension was adjusted to 10 mM PO43- using a 1 M N a z H P O a / N a H 2 P O 4 ((pH 7.2), 3: 1) stock solution and left on ice for 20 min. The suspension was centrifuged (35 000 rpm, 35 min, TI70 Beckman), the su-
64 pernatant harvested and remaining membrane particles removed by a second centrifugation step at 80000 rpm for 20 min in a TL100.3 Beckman rotor. The cleared solution (12-16 ml) was concentrated 10-fold by ultrafiltration using a centricon 10 (Amicon). The concentrated sample was loaded on a Superdex 75 HiLoad 16/60 gel filtration column (Pharmacia). The column was equilibrated in 50 mM NaCI, 20 mM Tris-HCl (pH 8.0) (buffer A) and chromatography of proteins was performed in the same buffer at a flow rate of 1 ml/min. Fractions containing recoverin were further purified on a MonoQ H R 5 / 5 anion exchange column using a NaCl-gradient (50-275 mM) as described [11]. The Superdex 75 Hiload 16/60 column (separation range 3-70 kDa) was calibrated with the following proteins: bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). The void volume (V0) of the column ( = 44 ml) was determined with Blue-dextran. The elution volumes (V~) of proteins were used to calculate their Kav-values according to Kav = (Ve Vo)/(V t - Vo) where Vt is the total volume of the column (120 ml). Antibodies against recoverin. Purified recoverin (100 /xg) was mixed with Freund's complete adjuvant and subcutaneously injected in a New Zealand white rabbit. Immunization was boosted after 3 weeks by injecting the same amount of recoverin. Blood was collected after two weeks and the recoverin antibodies were affinity purified from the serum. For this purpose, recoverin was coupled to CNBr-activated Sepharose according to the manufacturers manual (0,75 mg recoverin per 1 ml Sepharose). The serum was diluted 10-fold with 10 mM Tris (pH 7.5) and passed over the recoverin affinity column. Specific antibodies were eluted with 0.1 M glycine (pH 2.5) and with 0.1 M triethanolamine (pH 11.5). Shortly after elution, the antibody solutions were neutralized with 1 M Tris-HC1 (pH 8.0) and concentrated by chromatography on Protein A-Sepharose [16]. Electrophoresis and Western blotting. Electrophoresis was performed after Laemmli [17] or with the PhastSystem of Pharmacia. Gels were stained with silver or Coomassie brilliant blue R-250 or were electroblotted on nitrocellulose (Schleicher & Schfill). After blotting, the nitrocellulose membrane was blocked with PBS 0.5% Tween-20 for 2 h at room temperature or overnight at 4°C. Incubation with the purified anti-recoverin antibody (dilution 1:1000 or 1:500 in PBS 0.05% Tween-20) was for 1 h at room temperature or overnight at 4°C. The membrane was washed three times in the same buffer without antibody and then incubated with the second antibody (anti-rabbit IgG coupled with horseradish peroxidase 1 : 300) for 1 h at room temperature. The washing step was repeated and
the immunoreaction was visualized with 4-chloro-1n a p h t h o l / H 2 0 2 as peroxidase substrate. Results and Discussion
Recoverin is necessary for the calcium-dependent activation of photoreceptor GC [10,11]. In order to further study its function and properties, we here present a rapid and mild isolation procedure yielding high amounts of purified recoverin. Purification to apparent homogeneity was achieved with three steps: (1), Specific elution of soluble ROS proteins after illumination using the ROS membranes as an affinity matrix [,18]; (2), FPLC gel filtration chromatography and (3), FPLC anion-exchange chromatography on a MonoQ column. Enrichment of recoverin during this isolation procedure correlated well with its ability to increase GC activity at low calcium. The procedure presented here has the advantage of using milder conditions for separation, i.e., gel-filtration chromatography instead of a heat-denaturation step [11]. This resulted in a higher yield of purified protein (about 0.25 mg per 100 retinae). Furthermore, this purification protocol also avoids the use of any detergents in the elution buffers which are part of other purification protocols [10,13]. We found that various kinds of detergents, including ndodecyl-/3-D-maltoside, octanocyl-N-methylglucamide, n-octylglucoside, Triton X-100, Nonidet P-40 and ndodecyl-N, N-dimethyl-3-amino- 1-propanesulfonate, all interfere with the activation of photoreceptor GC by recoverin.
Gel-filtration chromatography Gel filtration chromatography on the Superdex 75 column was an effective step in separating recoverin from other retinal proteins. Recoverin containing fractions are 80% pure (Fig. 1A, B). One of the main contaminants is a 29-kDa protein (p29), but it can be separated from recoverin along with other contaminants by a subsequent anion-exchange chromatography. Fractions of the gel-filtration step containing recoverin exhibited the same modulator activity as purified recoverin. For example, partially purified recoverin obtained after the gel-filtration step (Fig. 1) was reconstituted with GC in washed ROS membranes and the GC activity was determined at high (563 nM) and low (4 nM) calcium. GC activity increased from 1.8 _+ 0.1 n m o l / m i n per mg to 4.2 _+ 0.5 n m o l / m i n per mg (three determinations). Almost identical values were obtained using purified recoverin as shown in Fig. 2A (1.47 n m o l / m i n per mg at high free calcium and 4.7 n m o l / m i n per mg at low free calcium) and as we have described previously [11]. In contrast, fractions of soluble ROS proteins without recoverin did only show the basal GC activity as it is found in washed ROS membranes [9-11]. The main portion of the p29 eluted
65 A 1,5 O
~Re
¢
O
IdW(kDa)
B
1,0
c-~4TI~"
0,5
-
67
-
49 34 SO
0,0 ,
0
,
,
20
~0
i
i
,
,
l
8O
60
i
100
Fraction (ml) Fig. 1. Gel-filtration chromatography of soluble ROS proteins. (A), An extract of soluble ROS proteins obtained after illumination (1.5 ml) was applied on a Superdex 75 HiLoad 16/60 FPLC column. The first 28 ml of the elution volume are not displayed (no elution of protein peaks), fractions containing recoverin eluted between 65 and 73 ml (see bold line). (B), SDS-PAGE of the fraction containing recoverin (Rec) marked by the bold line. The gel (10-15% acrylamide) was silver stained.
A
B MW(kDa)
%o
E
O
.E
-
94
-
67
-
43
-
30
-
20
shortly before recoverin on the gel filtration column. F u r t h e r purification and initial characterization (Lainbrecht, H.-G. and Koch, K.-W., unpublished results) revealed that this protein is not a calcium-binding protein (as, e.g., calbindin or calretinin [19] which have about the same molecular mass). We used a purified preparation of the p29 as a control in o u r reconstitution assay to show that the c a l c i u m - d e p e n d e n t activation of G C specifically depends on the presence of recoverin (Fig. 2A) and is not mimicked by other soluble retinal proteins (Fig. 2B). W e also used the gel-filtration c h r o m a t o g r a p h y for a molecular weight estimation. T h e logarithms of molecular mass standards (see Materials and M e t h o d s ) were plotted as a function of their Kav values. W e determined a Kay value of 0.35 for recoverin which would correspond to a molecular mass of 32 kDa. This indicates that recoverin is probably a m o n o m e r . Recoverin as a calcium-binding protein Calcium-binding properties of recoverin have been d e d u c e d from its amino-acid sequence which contains at least 3 E F - h a n d s [20] and have been directly d e m o n strated by 45Ca-autoradiography [10,11]. A n o t h e r characteristic feature of calcium binding proteins is their calcium d e p e n d e n t mobility shift during gel electrophoresis. Interestingly, recoverin displayed a different mobility in native electrophoresis (greater mobility in the presence of 1 m M E G T A than in the presence of 1 m M Ca2÷; see Fig. 3B) but not in S D S - P A G E (Fig. 3A), whereas many other calcium-binding proteins like calmodulin show a mobility shift in both gel systems [21]. Recoverin has an isoelectric point of 5.25 (Fig. 4A) which is close to the p I of visinin (5.1). Visinin, a calcium-binding protein from chicken cones
_E ao 0
E e>
"N 2,0 -
Co
EGTA
O 1,0i
o,o
I I h
I
h
I
[Ca2+] Fig. 2. Calcium-dependent GC activity at high (h = 563 nM) and low (l = 4 nM) free calcium. Washed ROS membranes were either reconstituted with purified recoverin (A) or purified p29 (B) and assayed for GC activity (expressed in nmol cGMP produced per minute and per mg rhodopsin). SDS-PAGE (12.5% acrylamide) of 1 /zg recoverin and 1/zg p29 is shown in (a) and (b).
ii!!iii@ ~ CO
EGTA
--
i li!iiiiiiii!i!~iii!
'~'% ~iiiii !i
~ i i!
~
Fig. 3. Electrophoretic mobility of recoverin in the presence of calcium or EGTA. Recoverin obtained after MonoQ anion-exchange chromatography was subjected to either SDS-PAGE (A) or a native PAGE (B). 2 Izg of recoverin were electrophoresed on a 12.5% gel. Either 1 mM CaCl 2 or 1 mM EGTA was added to the sample buffer.
66
B
A
ratio of 1 : 160 (taking an average of 70 mg rhodopsin in an ROS preparation of 100 retinae). This quantitation confirms our previous estimation that recoverin is one of the major soluble proteins and thus may also serve as an intracellular calcium buffer beside its role as the endogenous activator of photoreceptor GC [4,11].
ii!i iiii!~ ¸iill~ii¸i iii~il ~i~ii ~~ii~~
pH ,i~i~!~i!
5.85 Acknowledgements
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We thank Mrs. D. H6ppner-Heitmann for technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
References -
1
2
Fig. 4. Isoelectric focusing. (A), lane 1 shows a p l standard and lane 2 shows 0.5 Izg of purified recoverin. (B), Western blot of soluble ROS proteins. After isoelectric focusing of an extract of ROS proteins samples were electrotransferred on a nitrocellulose membrane. Proteins were probed with a purified antibody to recoverin.
i22], has about 60% sequence homology to recoverin. Isoelectric focusing of an extract of soluble ROS proteins and detection of recoverin with an affinity-purified antibody revealed only one band at p I 5.25 (Fig. 4B). The presence of two 26 kDa proteins was recently described in amphibian ROS [23], and at least one protein with p I 5.8 seemed to influence the activity of cGMP phosphodiesterase in a calcium-dependent way. Partial sequence analysis of this protein revealed a similarity to both visinin and calbindin [23]. It is unclear, however, whether one of the 26 kDa proteins is identical to recoverin or whether both proteins represent subspecies or completely different polypeptides. Polymorphism has recently been described for two other photoreceptor proteins, arrestin [24] and GC [6]. We here show that native recoverin from mammalians is only present in one form and that a protein with a similar molecular mass, the p29, does not share any characteristic properties with recoverin. Based on the recovery of biological activity we previously calculated a molar ratio of recoverin to rhodopsin of about 1:100. The extractable amount of 0.25 mg from 100 retinae would correspond to a similar molar
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