Purification and Molecular Cloning of a Novel Acidic Calmodulin-binding Protein from Rat Brain*

An acidic protein was identified among the insoluble proteins of a growth cone-enriched fraction, and this protein was purified from the Triton- and high NaC1- insoluble fraction of newborn rat brain using several column chromatographies after solubilization at an al- kaline condition. The purified protein showed a Ca2+-dependent calmodulin binding activity. This protein showed an anomalous behavior in SDS-polyacrylamide gel electrophoresis as that observed in case of GAP-43 (neuromodulin, F1, pp46, p57, B-50) and MARCKS (p87, pSO), namely shifts of apparent molecular weights under different acrylamide concentrations. Its physicochemical characteristics, such as heat stability, acidic isoelectric point, and solubility in a 2.6% perchloric acid solution, also resemble the properties of these proteins. cDNA cloning of this protein showed that the NHz-terminal 50-amino acid sequence was almost identical to CAP-23, a previously reported chicken protein of unknown function. Since the COOH-terminal half-regions of these proteins are less similar, the entire sequences of these proteins have 65% homology (52% identity), suggesting that these proteins belong to a family. Immunoblotting of several tissue extracts using a monoclonal antibody against this pro- tein showed its specific expression in brain. at a flow of ml/min. fractionated of the

(p87, pSO), namely shifts of apparent molecular weights under different acrylamide concentrations. Its physicochemical characteristics, such as heat stability, acidic isoelectric point, and solubility in a 2.6% perchloric acid solution, also resemble the properties of these proteins. cDNA cloning of this protein showed that the NHz-terminal 50-amino acid sequence was almost identical to CAP-23, a previously reported chicken protein of unknown function. Since the COOHterminal half-regions of these proteins are less similar, the entire sequences of these proteins have 65% homology (52% identity), suggesting that these proteins belong to a family. Immunoblotting of several tissue extracts using a monoclonal antibody against this protein showed its specific expression in brain.
Extracellular signals such as growth factors and neuronal transmitters are accepted at the receptors on the cell membrane, and their information is converted into various intracellular signals. The growth cone is a specialized neuronal structure having very high mobility positioned at the tip of an extended neurite (1,2). Elongation or retraction of the process and the formation of synapses with other neurons are regulated according to the external signals accepted at this position, and the change of the intracellular Ca2+ concentration is suggested to be an important intracellular signal for these processes (3)(4)(5)(6)(7)(8)(9). The molecular composition of this part is, hence, very interesting.
After establishing the method for isolation of this structure, it has been shown that various proteins specifically localize * This work was supported in part by Grant-in-Aid 03833007 for Scientific Research from the Ministry of Education, Science, and Culture of Japan (to S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We are also interested in molecules present in the growth cone fraction, and as a first step, we analyzed protein components using two-dimensional gel electrophoresis after biochemical fractionation of this structure. An enrichment of an acidic protein was detected in a high salt and Triton-insoluble fraction. In this paper we describe the biochemical purification and cDNA cloning of this protein. Here we call this protein NAP-22 from its character such as Neuronal tissueenriched acidic protein having a molecular mass of 22 kDa.

MATERIALS AND METHODS
Preparation of the Growth Cone Fraction-The growth cone fraction was prepared from embryonic rat brains (E17-El9) according to the method of Pfenninger et al. (10). Recovered growth cone fraction was collected by centrifugation at 200,000 X g for 60 min after %fold dilution with 0.32 M sucrose. The pellet fraction was suspended and homogenized with a solution containing 10 mM Tris-HC1, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF),' 0.03 mg/ml leupeptin, 1% aprotinin, and Triton X-100, pH 7.8, at 4 "C. After centrifugation at 200,000 X g for 60 min, the pellet fraction was further extracted with a solution containing 10 mM Tris-HC1, 0.8 M NaCl, 1 mM EGTA, 1 mM dithiothreitol, pH 7.8, for 30 min on ice. After centrifugation at 200,000 X g for 45 min, the pellet fraction was recovered and suspended in solution A (10 mM Tris-HC1, 0.2 mM EGTA, pH 7.8) containing 0.1 mM dithiothreitol and stored at -80 "C until use. This fraction was referred as the growth cone-insoluble membrane fraction.
Preparation of an Alkaline Extract from the Whole Brain-insoluble Fraction-All procedures were done at 0-4 "C unless otherwise described. Whole brains dissected from newborn rats (P3-P8) were homogenized in three volumes of a solution containing 10 mM Tris-HC1, 1 mM EGTA, 2 mM MgCl,, and 1 mM PMSF, pH 7.8, on ice. After centrifugation at 1,000 X g for 10 min, the supernatant was recovered and further centrifuged at 30,000 X g for 40 min. The pellet fraction was recovered and homogenized in solution A containing 1 M NaC1. After standing on ice for 30 min, the homogenate was centrifuged at 200,000 X g for 40 min, and the pellet was recovered.

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The pellet was further extracted with solution A containing 1% Triton X-I00 and centrifuged at 200,000 x g for 45 min after incubation on ice for 30 min. This extraction with Triton X-100 was repeated Once again. The remaining precipitates were finally suspended in a solution containing 50 mM Tris-HC1, 2 mM EDTA, 10% glycerol, 10 mM 2mercaptoethanol, and 1 mM PMSF, pH 9.0, and kept on ice for 2 h with stirring. The sample was subjected to centrifugation, and the supernatant (the alkaline extract) was saved.
Preparation of a Perchloric Acid-soluble Fraction from Tritonextracted Membrane Fraction-When specified, the Triton-solubilized supernatant fractions described above were mixed, and perchloric acid (PCA) was added to bring 2.5% concentration. After 10 min of incubation on ice, the mixture was centrifuged at 20,000 X g for 20 min, and the supernatant was recovered. To this fraction, solid ammonium sulfate was added to bring 55% saturation, and the solution was stirred for 20 min. The precipitates were recovered after centrifugation at 20,000 X g for 40 min and washed once with a solution of 55% saturated ammonium sulfate. The pellet was then dissolved in solution A and dialyzed against the same solution. After centrifugation at 100,000 X g for 60 min, the supernatant was recovered as the PCA-soluble membrane protein fraction (31).
Purification of NAP-22-The alkaline extract was applied to a DEAE-cellulose column (1.4 X 7 cm, Whatman) which has been equilibrated with solution A. After washing with solution A, proteins were eluted with a linear gradient of NaCl from 0 to 400 mM in solution A, and the column was further washed with 800 mM NaCl. Proteins were analyzed by SDS-PAGE, and fractions containing NAP-22 were collected. To this sample, ammonium sulfate was added to bring 40% saturation. After removing the resultant precipitates by centrifugation at 20,000 X g for 20 min, the supernatant was applied to a phenyl-Sepharose column (1.4 X 7 cm, Pharmacia) preequilibrated with solution A containing 40% saturated ammonium sulfate. After washing the column with the same solution, proteins were eluted with solution A containing 2 M NaCl, then with solution A containing 1.5 M NaC1. Some portion of NAP-22 was eluted with 2 M NaC1, and the other was eluted with 1.5 M NaCl. Fractions containing NAP-22 were pooled and dialyzed against solution A. After addition of 0.3 mM CaC12, the dialysate was applied to a hydroxyapatite column (1.2 x 5 cm), and proteins were eluted with a linear gradient of potassium phosphate buffer from 20 to 350 mM. NAP-22 was eluted at about 160 mM phosphate concentration. NAP-22 was further purified by the second DEAE-cellulose column chromatography (1.2 X 4 cm) employing a linear NaCl gradient (0-300 mM). Fractions containing pure NAP-22 were pooled, dialyzed against a solution containing 10 mM MES-KOH, 0.2 mM EGTA, 50 mM NaCl, pH 6.8, and stored at -80 "C until use. In some experiments, the sample after the first DEAE-cellulose column chromatography was immersed in boiling water for 5 min, and the aggregated proteins were removed by centrifugation after cooling on ice. This procedure increased the purity of NAP-22 and improved its recovery.
Amino Acid Sequencing of the Tryptic Digests of NAP-22-Purified NAP-22 (50 pg) was digested with 0.5 pg of L-I-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin at 37 "C for 60 min, and the digests were applied to a reverse-phase high pressure liquid chromatography column (TSK-GEL ODS-I20T, 4.6 X 250 mm, Toyo-Soda, Co., Ltd., Japan). Peptides were eluted with a linear gradient of acetonitrile from 0 to 60% in 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. Peptide peaks were fractionated and vacuum evaporated. The amino acid sequence of the peptides was analyzed with a liquid-gas peptide sequencer (AB1 477A Peptide Sequencer).
cDNA Cloning of NAP-22-cDNA library of neonatal rat brain (XgtlO) was purchased from Clontech (Clontech Laboratories, Inc., Palo Alto, CA). Three oligonucleotides corresponding to two peptide sequences were kindly arranged and synthesized by Dr. F. Matsuzaki (Division of Molecular Genetics, National Institute of Neuroscience, National Center of Neuroscience and Psychiatry). Two of these probes correspond to a peptide, One is a 20-mer containing 5'-GG<ACGT>GC<AGCT>CC<ACGT>CA<AG>GA<AG> GA<AG>GG-3' (peptide sequence of GAPQEEG (single letter code)), and the other is a 35-mer containing 5"ACXGGXGC-

X G C X G A < T C > G G X G C X C C X C A < A G > G A < A G > G A
<AG>GG-3' ( X represents inosine) (peptide sequence of TGAAD-G A P Q E E G ) . T h e l a s t o n e ( 5 ' -G A < T C > G C X G C X C C X -(32-mer)) corresponds to another peptide sequence (DAAPAA-SDSKP). These oligonucleotides were 32P-labeled using T4 polynucleotide kinase and used as probes for screening the library. About 8 X IO6 clones were screened, and five positive clones were picked up.

GCX<TA><CG>XGA<TC><TA><CG>XAA<AG>CC-3'
All of these clones were reacted with three probes. The clone having the longest insert was selected, and the insert was subcloned into Bluescript KS I1 (Stratagene, La Jolla, Ca). DNA sequencing was performed using the dideoxy chain termination method using the Sequenase I1 kit (United States Biochemical Corp.).
Expression of NAP-22 cDNA in Escherichia coli-To express NAP-22 cDNA in E. coli, the EcoRI fragment of the clone was subcloned into the EcoRI site of the vector pUC119. Since there was no appropriate restriction enzyme site in the upstream region of the open reading frame, 5"untranslated regions were removed using a deletion kit (TaKaRa, Co., Japan) to construct expression vectors after digestion with SphI and BamHI. From the DNA sequencing of obtained clones, one recombinant NAP-22 (rNAP-2) is expected to have an additional 13 amino acids (MITPSLHPKPNSK) in its NH2-terminal and the other recombinant NAP-22 (rNAP-1) is expected to lack 22 amino acids from its original amino-terminal and to have additional 6 amino acids (MITPSL). After induction of proteins with 0.5 mM isopropyl-/3-D-thio-galactopyranoside for 3 h, cells were collected and soluble fractions were recovered after sonication at 0 "C in 10 mM Tris-HC1, pH 7.8, containing 1 mM each of EGTA, EDTA, and PMSF. Solid ammonium sulfate was added to bring 40% saturation, and the precipitates were removed by centrifugation. The supernatant was applied to a phenyl-Sepharose column. Proteins eluted with 1.5 M NaCl solution were recovered and dialyzed against 10 mM MES-KOH, and 0.2 mM EGTA, pH 6.8.
Production of a Monoclonal Antibody-Partially purified NAP-22 fraction (the sample after phenyl-Sepharose column chromatography) was treated with performic acid according to the method of Hirs (37). Each 0.1 mg of protein was injected intraperitoneally after mixing with the Ribi adjuvant system (RIB1 ImmunoChemical Research, Inc. Hamilton, MT). After four injections, untreated NAP-22 fraction was injected, and 4 days later a spleen was dissected and used for cell fusion with myeloma cells. The fusion of cells and screening of hybridomas were performed as described previously (38). One clone was finally obtained and used for immunological studies.
Zmmunostaining of Neural Cells-Cortices from brains of E18 and of P3 were dissected and dispersed using papain and DNase I. Cells were spread on polylysine-coated cover glass and cultured in a CO, incubator using a mixed medium of Ham's F-12 and Dulbecco's modified Eagle's medium (1:l) supplemented with 5% horse serum, 5% fetal calf serum, and 1% rat serum. Cells were fixed at 37 "C with 3.7% formaldehyde in PBS (20 mM potassium phosphate, 140 mM NaCl, pH 7.4) and permeabilized with acetone at -20 "C after washing with PBS. Immunostaining of cells was performed as described previously (39). Subcellular Fractionation-Brains from 10-day-old rats were homogenized in 0.32 M sucrose, 5 mM HEPES-KOH, 1 mM PMSF, pH 7.2. After centrifugation at 3,000 X g for 10 min, the pellet (nuclear) fraction was separated from the supernatant. The supernatant was centrifuged at 200,000 X g for 60 min, and the supernatant (soluble fraction) was recovered. The pellet fraction was homogenized in a solution containing 10 mM Tris-HC1, 1 mM EGTA, 0.8 M NaCl, and I mM PMSF, pH 7.8, and recentrifuged. The supernatant (NaCl extract) was recovered, and the pellet fraction was extracted with a solution containing 10 mM Tris-HC1, 1 mM EGTA, 1 mM PMSF, and 1% Triton X-100. After centrifugation the supernatant (Triton extract) and the pellet (skeletal) fraction were recovered.
Others-Tissue extracts were prepared from an 8-week-old rat as described previously (39). Calmodulin was purified and coupled to CNBr-Sepharose 4B (Pharmacia) as described previously (40). About 1 mg of protein was coupled to 1 ml of gel.
GAP-43 was purified according to the method of Baudier et al. (31) from rat and bovine brains. Microtubule proteins and purified tubulin was prepared and kindly supplied by Dr. H. Murofushi (Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo) as described (41). Rabbit muscle actin was purified as described previously (42). Monoclonal mouse anti-tubulin antibody and monoclonal rat antitubulin antibody (YL1/2) were purchased from Amersham (United Kingdom) and Sera Labs (U.K.), respectively. SDS-PAGE, twodimensional electrophoresis, and protein determination were performed as described previously (39).

RESULTS
Purification and Biochemical Characterization of NAP-22-To characterize proteins in the growth cone fraction, successive extractions with low salt, high salt, and detergent soh-tions were carried out. A fraction resistant to these solubilization procedures was first analyzed with SDS-PAGE. The major components were electrophoresed with estimated molecular masses of about 55 kDa. Two-dimensional acrylamide gel electrophoresis of this fraction revealed that these 55 kDa bands represented three proteins, two major components with similar isoelectric points, and one minor one, all in the acidic region (Fig. 1). The major spots were identified as tubulin from their positions in two-dimensional gel and their immunoreactivity with anti-tubulin antibody (data not shown). The minor spot having a much more acidic isoelectric point than tubulin was further characterized. For this purpose, biochemical purification of this protein from whole brain was attempted, since only small protein amount was recovered in the growth cone fraction.
Among several solubilization methods tried, alkaline solubilization was found to be fairly effective (see "Materials and Methods"). Following this treatment, several conventional column procedures were used to purify this protein (Fig. 2B). Although this protein was hardly extractable using low and high salt, the protein, once solubilized, was found very hydrophilic because more than 2 M NaCl was needed to bind the protein to a phenyl column and 1.5 M NaCl was effective to elute this protein from the column (Fig. 2 A ) . About 0.4 mg of protein was purified from 30 g of brain, and purified protein showed the same mobility in two-dimensional gel electrophoresis with the acidic protein detected in the growth cone-insoluble fraction. In several experiments, heat or PCA treatment was attempted, and this protein was found to be resistant to these treatments. NAP-22 has, hence, common physicochemical features with GAP-43 and MARCKS (31).
Low hydrophobicity and rod-shaped structures of the proteins are thought to cause anomalous behaviors in SDS-PAGE in cases of GAP-43 and MARCKS, namely different molecular masses were estimated under different acrylamide concentrations (24, 34). Fig. 3 shows the mobility of NAP-22 compared to those of molecular mass standard and purified GAP-43. Using a 7.5% acrylamide gel, the molecular mass of this protein was estimated to be about 58 kDa. On the other hand, using 12.5% acrylamide, the value was about 45 kDa. The estimated molecular masses of GAP-43 also changed under these conditions, but its mobility was clearly different from that of NAP-22.
The interaction of NAP-22 with calmodulin was studied because both MARCKS and GAP-43 are known to have calmodulin binding activities. NAP-22 showed an ability to bind calmodulin in a Ca2+-dependent manner. Fig. 4 shows its binding to a calmodulin affinity column and its elution in the absence of Ca2+ ions. In the absence of Ca2+ ions, NAP-22 does not bind to calmodulin at all. NAP-22 does not bind to a control column without calmodulin. Ca2+-dependent calmodulin binding of NAP-22 was also confirmed with a gel filtration experiment (data not shown). Since this protein was first identified in a fraction enriched with tubulin, the binding of this protein to microtubules was examined with a cosedimentation assay. This protein showed no binding activity to polymerized tubulin in the presence or absence of microtubule-associated proteins. Little binding of this protein to actin filaments was also detected with a cosedimentation assay.
To know its molecular structure, three amino acid sequences (SDAAPAASDSKPS, AGEASAESTGAADGAPQ-EEG, and ETPAASEAPSSAAKAPAPAA, single letter code) of isolated trypsin-derived peptides of this protein were de- termined, while the NH2-terminal end was blocked. A computer homology analysis showed no identification of this protein with known proteins.
Using oligonucleotides corresponding to these peptide sequences as probes, rat cDNA library was screened and five positive clones were isolated. Because all of these clones were positively and evenly reacted with three probes, the clone having the longest insert was sequenced (Fig. 5). Sequencing revealed an insert of 1037 base pairs, including an open reading frame of 660 base pairs, and the deduced amino acid sequence contained the partial sequences determined from direct analysis of the purified protein (underlined residues in Fig. 5). Because only one ATG codon was present in the reading frame and there were two termination codons in the 5' upstream region of this frame, this ATG was assumed to be the initiation site. The protein coded by this gene was fairly interesting in its amino acid content; it does not contain cysteine, histidine, arginine, phenylalanine, isoleucine, or tryptophan. Instead, it was rich in alanine, glutamine, lysine, and proline. The isoelectric point was calculated to be 4.3 which coincided well with its acidic behavior in two-dimensional PAGE. This protein has, however, a cluster of basic residues in its NH2-terminal 25 residues (PI = 10.4) which contrasted to the acidic nature of the remaining part. The estimated molecular mass was 21,790, which was much smaller than the value obtained from mobility in SDS-PAGE.
Two recombinant NAP-22 were, hence, expressed in E. coli, and their mobilities in SDS-PAGE were compared with that of purified brain NAP-22 (Fig. 6). The mobilities of these proteins were fairly well coincided under different acrylamide concentrations and exhibited same retardation behavior in gels of decreasing acrylamide concentration. We therefore concluded that this clone coded for NAP-22.
A hydropathy plot (Fig. 7) showed its very hydrophilic character having no hydrophobic region in the molecule, coinciding with its behavior in a hydrophobic column (Fig. 2). The Chou-Fasman plot of NAP-22 suggested that NAP-22 contains extensive cy-helical domains linked with turns, but not p-sheets. A homology search using the Genetic Computer Group (University of Wisconsin) system in the EMBL and Genbank Databases picked up several interesting proteins. One was CAP-23 (chicken cortical-associated protein (44)) with 65% similarity and 52% identity, and the others were MARCKS (also called p87 or p80 (35)) with 53% similarity and 31% identity, and GAP-43 (also called neuromodulin, pp47, p57, B50, F1) (25) with 43% similarity and 30% identity. The homology between NAP-22 and CAP-23 was especially high in their NH2-terminal 50 amino acids; almost identical sequences were found (Fig. 8). In the following 50 amino acid sequences, homology is still high. In the COOH-terminal half, although there are some homologous regions, these regions were separated with fairly long gaps present in each side of the proteins. Because the sequence of the rat homologue of CAP- 23 has not yet been reported, it is unclear at present whether NAP-22 is a rat homologue of CAP-23 or whether this is a different protein composing a family. This protein also showed moderately high similarity with MARCKS and GAP-43, but the identity was not as high compared with CAP-23. In these cases, homologous regions were detected throughout the sequence. Interestingly, the predicted calmodulin-binding site (and kinase C phosphorylation sites) in the MARCKS sequence is not present in NAP-22 sequence (Fig.  9A) (33). The calmodulin-binding site and the NH2-terminal 25 amino acid sequence of GAP-43, which is important for interacting with Go, are also not found in the sequence of NAP-22 (Fig. 9B) (32,45).
Since this protein was noticed in the insoluble materials of the growth cone fraction, it is interesting to know the intracellular localization of this protein. A monoclonal antibody was produced for this purpose. Fig. 1OA shows the result of an immunoblotting of this antibody with the purified protein and several tissue extracts. This protein is highly expressed in brain, and little immunoreactivity was observed in the other tissues. In the liver extract, one protein band was reacted with the antibody. This band, however, shows a little slower mobility than NAP-22 in this system and in other systems, using different acrylamide concentrations, showed no change in the estimated molecular mass (data not shown). This protein, thus, was judged not to be NAP-22. Extracts of established rat cell lines such as 3Y1, NRK, and PC12 cells showed little reactivity with this antibody, suggesting its specific expression in the nervous tissue.
To discover the distribution of this protein within the cell, a tissue fractionation experiment was done and each fraction was immunoblotted. Fig. 10B shows its concentration in the Triton-extractable membrane fraction in addition to the Triton-insoluble fraction from which the protein was purified. The presence of NAP-22 in the Triton extract was confirmed by the purification of this protein from this fraction after treatment with 2.5% PCA (data not shown). This protein was recovered in the soluble fraction after the treatment with PCA as were GAP-43 and MARCKS (31).
The primary culture of rat brain cells was for the intracellular distribution of this protein by an immunostaining method (Fig. 11). In addition to growth cones, dendritic protrusions and cell bodies were well-stained. Double staining using anti-tubulin antibody showed that NAP-22 was more widely distributed than tubulin in some growth cone regions and in some fine protrusions having fibrous structures.

FIG. 5.
Nucleotide sequence and alignment of deduced amino acid sequence of NAP-22 coding clone. The potential myristoylation site is indicated by an arrowhead. All obtained amino acid sequences in the trypsin-digested peptides were found in the deduced sequence (underlined).

S T E P A P S S K E T P A A S E A P S S
-790 8 10 830 In this paper we showed the biochemical purification of a calmodulin-binding protein from rat brain and its cDNA cloning. This protein was originally noticed in an insoluble protein mixture of a growth cone-enriched fraction, although the presence of this protein in the Triton-soluble fraction was also shown by an immunological method and purification. The molecular mechanism that enables tubulin and NAP-22 to become resistant to such solubilization procedures is unknown at present. This protein might be identical to a protein called MP1, noticed in rat brain plasma membrane fraction, because these proteins show strikingly similar behavior on two-dimensional gels (46). The result of cDNA cloning showed mass of 22 kDa. This protein has a possible myristoylation site at its amino terminus. This modification could explain the membrane localization of this protein shown in the cell fractionation experiment (Fig. loll). A homology search using the GCGS databases showed that this protein is fairly homologous with CAP-23 (65% similarity and 52% identity), a protein detected in chick brain. CAP-23 was noticed electrophoretically from its anomalous behavior at different acrylamide concentrations and is known to be phosphorylated by protein kinase C, but little biochemical characterization has been done. Our preliminary result shows that NAP-22 is also a substrate of protein kinase C (data not shown). The homology between CAP-23 and NAP-22 is, however, relatively low, since the homology of MARCKS of these species is calculated to be 83% similarity and 67% identity (35, 36). CAP-23 has less residues of alanine and proline than NAP-22, and the presence of arginine, phenylalanine, tryptophan, and histidine in CAP-23 is also different from NAP-22. NAP-22 does not have a basic region in its COOH-terminal that is present in CAP-23. Whether NAP-22 is a rat homologue of CAP-23 or these proteins are the members of a family is, hence, unknown at present. The biochemical characters of NAP-22 such as acidic isoelectric point, low hydrophobicity, heat stability, anomalous behavior in SDS-PAGE, and calmodulin binding activity, are common to MARCKS, GAP-43, and neurogranin, a recently found GAP-43-like molecule having much smaller molecular mass, although GAP-43 and neurogranin bind calmodulin in the absence of the Ca2+ ions (24, 45, 47). These proteins  showed sequence similarity, but the conserved regions of the MARCKS family such as the effector domain and the MH2 region and the calmodulin-binding site of GAP-43 are clearly lacking in the sequence of NAP-22 (Fig. 8) (45,47-50). GAP-43 is known to be present in two states; one is extractable with a detergent solution and the other is resistant to this treatment, suggesting its tight interaction with membrane skeletal structures (51, 52). Similarly, some part of NAP-22 was also recovered in the Triton-extracted fraction. Further treatment of this Triton-extracted fraction with perchloric acid showed its recovery in the soluble fraction with GAP-43 and MARCKS. GAP-43 was also detected during the purification of NAP-22 from the Triton-insoluble fraction, an indication of their association with proteins in the Tritoninsoluble fraction coincided with other reports (47, 50-53).