Poly(l-proline)-binding proteins from chick embryos are a profilin and a profilactin

Eur. J Biochem. 151, 291 -297 (1985) ( FEBS 1985

Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin Masato TANAKA and Hiroko SHIBATA

Laboratory of Biochemistry, Mitsubishi-Kasei Institute of Life Sciences, Tokyo

(Received June 4, 1985) - EJB 85 0613

Two poly(L-proline)-binding proteins (PBP-1 and PBP-2) were purified from chick embryos by using a poly(L- proline)-agarose column. PBP-1 was composed of two different polypeptides (molecular masses of 42 kDa and 15 kDa). The molar ratio of the two proteins in the complex was 1 : 1. The other poly(L-proline)-binding protein, PBP-2, was the 15-kDa protein itself. The 42-kDa protein was confirmed to be an actin from the amino acid composition, by immunochemical evidence and by its ability to self-polymerize. In addition, the 42 + 15-kDa protein complex (PBP-1) inhibited DNase 1, just as a monomeric actin did. The amino acid composition of the 15-kDa protein was similar to that of mammalian profilin and it inhibited the salt-induced polymerization of rabbit skeletal muscle actin. Therefore, we conclude that the two poly(L-proline)-binding proteins from the chick embryo are a profilactin and a profilin in chick embryo. The ability of profilactin to bind poly(L-proline) must bc due to profilin itself, because the profilin has a greater affinity for poly(L-proline) than does profilactin. Additionally, both the monomeric and filamentous actin from rabbit skeletal muscle have no affinity for poly(~- proline).

Poly(L-proline) is known to form unique conformations, poly(L-proline) I and I1 helices. In aqueous solution i t forms the typc I1 conformation which is a left-handed helical structure with a threefold screw axis. This secondary structure has been experimentally confirmed in a few natural proteins such as collagen, cuticlin [l, 21 and bovine pancreatic trypsin inhibitor [ 3 ] . In addition, we have already elucidated that a plant prolyl hydroxylase recognizes the poly(L-proline) I1 helix [4] and have proposed that an unhydroxylated precursor of a hydroxyproline-rich glycoprotein in plants should form the same helix. Moreover, a proline cluster has been found in the hinge region of immunoglobulin G [5] , in the gug polypeptides of rctroviruscs [6] and in proline-rich proteins secreted from the parotid gland [7, 81. Since a tetra(L-proline) cluster in peptides has been shown to form poly(L-proline) I1 helix in aqueous solution [8, 91, then immunoglobulin G, retroviral gug polypeptides and parotid gland proteins should all contain this helix. Interestingly, almost all proteins having a poly(~-proline) I1 helix are extracellular proteins.

On the other hand, a prolyl hydroxylase is known to bind to poly(L-proline) which is an effectice competitive inhibitor of this enzyme [lo, 111. Utilizing this affinity of prolyl hydroxylase for poly(L-proline), Tuderman et al. developed an affinity column procedure for purifying the enzyme from chick embryo [12]. They reported that by affinity column chromatography the enzyme was copurified with an ‘unidentified protein’ which could finally be removed by gel filtration after the enzyme was eluted from the poly(~-proline)-agarose

Correspondence to M. Tanaka, Laboratory of Biochemistry, Mitsubishi-Kasei Institute of Life Sciences, 11 -Minamiooya, Machida-shi, Tokyo, Japan 194

Ahhveviations. SDS, sodium dodecyl sulfate; PBP, poly(r,-proline)-binding protein; DNase I, deoxyribonuclease I .

Enzymes. Prolyl hydroxylase or prolyl-glycyl-peptide, 2-0x0- glutarate oxidoreductase (4-hydroxylating) (EC 1.14.1 1.2); deoxyribonuclease I (EC 3.1.21.1).

column by a poly(L-proline) solution. We have been interested in this ‘unidentified protein’, which can be regarded as a poly(L-proline)-binding protein. In addition, we can suggest that the human foetus also contains a similar poly(L-proline) binding protein based on the report by Kuutti et al. [I 31 which dealt with human prolyl hydroxylase.

This paper deals with purification and characterization of the poly(L-proline)-binding proteins in chick embryos and reports that the proteins are a profilactin and a profilin.

MATERIALS AND METHODS

Matprials

Fertilized eggs of White Leghorn chickens were obtained from a local hatchery and were incubated in a moist atmos- phere at 37°C for 13 days. Poly(L-proline)s ( M , = 12000 and 6000) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). 2-O~o[l-’~C]glutarate was a product of New England Nuclear (Boston, MA, USA). (Pro-Pro-Gly), ‘ 4 H 2 0 and (Pro-Pro-Gly),, . 9 H 2 0 were purchased from Protein Research Foundation (Minoh, Osaka, Japan). Actin was ex- tracted from the acetone-dried powder of rabbit skeletal muscle and purified by the procedure of Mommaets [I41 with a slight modification in which 30 mM KC1 was used instead of 0.1 M KC1 for actin polymerization to avoid contamination by tropomyocin. Poly(L-proline)- Sepharose 4 B was prepared according to the method of Tuderman et al. [12] except using poly(L-proline) having an M , of 12000, instead of 30000.

Purification ojpoly (L-proline) -binding proteins fkom chick embryo

This procedure was essentially based on the method for purification of prolyl hydroxylase from chick embryo using poly(L-proline) - Sepharose 4 B [12]. About 600 g of frozen

292

chick embryos (1 3-days-old) stored at - 70°C were allowed to stand at room temperature. Then the half-thawed embryos were sliced and subsequently homogenized with an equal volume of 0.1% Triton X-100 and 0.01 M KC1. The homogenate was centrifuged at 12000 x g for 30 min and the solid ammonium sulfate was added to the supernatant. After the 35 - 70% saturated fraction was dialyzed against 10 mM Tris/HCl buffer, pH 7.8, containing 0.1 M NaCl, 0.1 M glycine and 10 FM dithiothreitol (buffer A), this fraction was centrifuged at 105000 x g for 90 min. The supernatant obtained was applied on a column of poly(L-proline) - Sepha- rose 4 B (2.2 x 18.5 cm) previously equilibrated with buffer A and this column was washed by seven column volumes (500 ml) of the buffer A. The unadsorbed and the first washing fractions were discarded. This affinity column was further washed by 11 of the same buffer, and this second washing fraction was concentrated to about 1 mg/ml of protein by ultrafiltration with a PM-10 membrane (Amicon). This fraction (the second washing fraction) was saved for the next step of Bio-Gel A-1.5 m purification (see below).

After the second washing, prolyl hydroxylase and poly(L- proline)-binding proteins were eluted by 20 ml of the buffer A containing 5 mg/ml of poly(L-proline) ( M , = 6000) from the affinity column as reported previously [12]. The eluted fraction was concentrated to 5 - 6 ml ultrafiltration with a PM-10 membrane. Insoluble materials were removed by centrifugation. This sample was loaded on a column (1.9 x 98,5 cm) of Bio-Gel A-1 .5 m previously equilibrated with 10 mM Tris/HCl buffer, pH 7.8, containing 0.2 M NaC1, 0.2 M glycine and 10 pM dithiothreitol (buffer B) and then eluted with the buffer B. The eluate was collected by a fraction collector and prolyl hydroxylase activity and absorbance at 280 nm in each fraction were determined. The three protein peaks were concentrated by ultrafiltration with a PM-10 membrane, and dialyzed against 10 mM Tris/HCI buffer, pH 7.4, containing 0.1 mM dithiothreitol. The samples were stored at 4'C until use. While an aliquot of the concentrated second washing fraction was also loaded on the same column of Bio- Gel A-3.5 m and eluted in the similar manner. The main protein peak was collected and concentrated in the similar manner.

Isolation of the 15-kDa protein

To isolate the 15-kDa protein without poly(L-proline) contamination, an elution with 2 M urea buffer from the poly(L-proline) - Sepharose 4 B was carried out. After the first washing of the affinity column as described above, poly(L- proline)-binding proteins were eluted with 25 mM Tris/HCl buffer, pH 7.6, containing 2 M urea, 5 mM MgCI2 and 0.2 mM dithiothreitol (urea buffer). A single protein peak was obtained and this peak was collected and dialyzed in the urea buffer overnight at 4°C. This was chromatographed at room temperature on a DEAE-cellulose column (DE-52, Whatman) previously equilibrated with the same urea buffer. The unadsorbed protein was dialyzed against 10 mM Tris/HCl buffer, pH 7.4 containing 0.1 mM dithiothreitol for 2 h, then the dialysis bag was covered with solid ammonium sulfate and stood overnight at 4-C. The precipitates present in the bag were collected by centrifugation (12000 x g for 30 min), dissolved into a sinall volume of 10 mM Tris/HCl buffer, pH 7.4, containing 0.1 mM dithiothreitol, and dialyzed against the same buffer. Using similar urea/DEAE-cellulose column chromatography, the 15-kDa protein was also isolated from

the main peak fraction (purified 42 + 15-kDa protein complex) of gel filtration with Bio-Gel A-1.5 m.

Polyacrylumide gel electrophoresis

Electrophoresis on a 7.5% polyacrybamide gel was carried out at pH 8.3 according to Davis [15]. Gel electrophoresis with sodium dodecyl sulfate (SDS) was performed according to Laemmli [16]. The gels were stained with Coomassie brilliant blue R in 7% acetic acid and 50% methanol. To analyze the subunit structure of the poly(L-proline)-binding protein, we used a combined system of two disc electrophoresis with native polyacrylamide and SDS/polyacrylamide gels, according to Kanda et al. [17] with minor modification as follows. After the gel was stained for 1 - 2 min, a faintly stained band was cut off, rinced once in 62.5 mM Tris/HCI, pH 6.8, containing 1 ?'n SDS and 1 % 2-mercaptoethanol, and incubated in the same fresh solution for 30 min at 45°C. Then, the gel was applied on the top of a SDS/polyacrylamide gel directly. Affinity electrophoresis [18] in the presence of poly(~- proline) ( M , = 6000) was performed using 6% polyacrylamide gels in Tris/glycine, pH 8.3 [l 51 at room temperature.

Enyzme ussays

The activity of prolyl hydroxylase was assayed in a final volume of 0.3 ml by measuring the I4CO2 evolution from 2- 0x0-[I -14C]glutarate using 0.15 mg of (Pro-Pro-Gly), . 4H20 as a substrate according to the modified method [I91 of the procedure of Rhoads and Udenfriend [20]. The assay mixture contained the same concentration of other compounds as indicated by Tuderman et al. [12].

The DNase I inhibition assay was performed as described by Lindberg [21].

Visrometry

Actin polymerization was followed by measuring an increase of viscosity. The viscocity was measured with an Ostwald-type viscometer requiring a volume of about 0.7 ml.

Other methods

Protein concentration was determined by the method of Bradford [22] using rabbit skeletal muscle actin as a standard. Actin concentration was determined spectrophotometrically using the value of A:&, 11 .O.

Amino acid compositions were analyzed with Irica model A-5550 automatic amino acid analyzer (Irica Instrument Inc. Kyoto, Japan), after the samples were hydrolyzed at 110 'C for 20 h with 6 M HCl containing 0.2% phenol in evacuated sealed glass tubes.

RESULTS

Isolar ion ojpoly (L-proline) -binding proteins

When we prepared a prolyl hydroxylase from chick embryos according to Tuderman et al. [I21 to compare with a plant prolyl hydroxylase [4, 191, a large amount of proteins were co-purified together with the chick enzyme by poly(L- proline)- Sepharose 4 B column Chromatography. Fig. 1 shows a typical elution profile of this affinity chromatography. Proteins which could be eluted by 5 mg/ml poly(~- proline) from this column were separated into a prolyl hy-

293

Column effluent (Ilter) Column eluate (mu)

Fig. 1. Affinit,v chromatography qfpoly (L-proline)-binding proteins on polL(iL-proline)-agarose column. Horizontal bars labelled lst, 2nd and E show fractions of the first washing, the second washing and the elution with poly(L-proline) solution, respectively. Vertical arrow indicates the start of the elution with poly(L-proline) solution

Fraction Number

Fig. 2. Separation of poly(L-proline)-binding proteins, PBP-I and PBP-2, f rom prolyl hydroxylase and poly (L-proline) by gel filtration with Bio-Gel A-1.5 m. The eluate (indicated by E in Fig. 1) with poly(r- proline) solution from the column of poly(L-proline) - agarose was loaded on a column (1.9 x 98.5 cm) of Bio-Gel A-1.5 m, and eluted with 10 mM Tris/HCI buffer, pH 7.8, containing 0.2 M NaCI, 0.2 M glycine and 10 pM dithiothreitol. The fractions of 4.7 ml werc collected. Prolyl hydroxylase activity (0-0) and absorbance at 280 nm (0-0) were measured. The peak of protein eluted just after prolyl hydroxylase and the last peak were designated as PBP-1 and PBP-2, respectively

droxylase and two poly(L-proline)-binding proteins by gel filtration with Bio-Gel A-1.5 m as shown in Fig. 2. The protein from each peak was examined by SDS/polyacrylamide slab gel electrophoresis as shown in Fig. 3, lanes c, d. The bands of lane c clearly indicates that the first peak in Fig. 2 contains a prolyl hydroxylase composed of a and ,8 subunits.

The second and the third peak in Fig. 2 are shown to be composed of the 42-kDa and 15-kDa proteins (lane d) and the 15-kDa protein itself (lane e), respectively, though Tuderman et al. [I21 pointed out that the second and the third peaks were an unidentified protein and poly(L-proline), respectively. Although the third peak of our preparation was confirmed to contain poly(L-proline) judging from the A230/ Azso ratio and the amino acid composition, the following results indicate that the 15-kDa band in Fig. 3 was not due to poly(L-proline) contamination. (a) Poly(L-proline) solution

Fig. 3. SDS/polyacrylamide gel electrophoresis of poly(L-prolinej- binding proteins. The electrophoresis was done by using 12% polyacrylamide slab gel in the presence of SDS. Lanes (a - f) as follows: (a) the concentrated first washing fraction in Fig. 1 ; (b) the concentrated sccond washing fraction in Fig. 1 ; (c) the first peak fraction in Fig. 2 (prolyl hydroxylase); (d) the second peak fraction in Fig. 2 (PBP-1); (e) the third peak fraction in Fig. 2 (PBP-2); (f) the purified 15-kDa protein described in Materials and Methods. Number on the sides represent molecular mass in kDa

used for this purification showed no absorbance at 280 nm. (b) This poly(L-proline) was not stained with Coomassie brilliant blue R on the gel electrophoresis. (c) Proteins eluted from poly(L-proline) - Sepharose 4 B by a 2 M urea buffer instead of poly(L-proline) solution also gave the 15-kDa band in addition to the 42-kDa band and a$ bands of prolyl hydroxylase (described below). Thus, we could purify the two poly(L- proline)-binding proteins, one of which was composed of the 42-kDa and 15-kDa proteins and the other was the 15-kDa protein itself. In this paper, we refer to the two poly(L-proline)- binding proteins, the 42 + 15-kDa complex and the 15-kDa protein, as PBP-1 and PBP-2 respectively.

We found that the yield of PBP-1 remarkably changed depending on the volume of the buffer used to wash the affinity column. If the step of the second washing (see Ma- terials and Methods) was omitted, the second peak (PBP-1) in the final gel filtration with Bio-Gel A-1.5 m was remarkably increased. Therefore, PBP-1 seemed to have a weak affinity for poly(L-proline) and was eluted from the affinity column continuously. Then, we tried to isolate the PBP-1 also from the second washing buffer, because this preparation should be free from poly(L-proline) essentially. Fig. 3, lane b, shows that the second washing solution contains PBP-1. In fact, this PBP-1 could be isolated as a main peak in the gel filtration with Bio-Gel A-1.5 m (Fig. 4B). Although a concentrated sample of the first washing solution shows a similar staining pattern to that of the second washing solution (Fig. 3, lane a), the PBP-1 could never be separated by gel filtration with Bio-Gel A-1.5 m (Fig. 4A). The preparation from the first washing solution gave many minor bands other than 42-kDa and 15-kDa bands on the SDS gel electrophoresis, if it was loaded at about threefold increase in concentration (data not shown).

According to the procedure described here, using 600 g of chick embryo as starting materials, we could obtain 17.7 mg and 8.45 mg of pure PBP-1 from the second washing and the

294

E 0 0.4 co 01 H H

0.1

N

0 10 20 30 40 50 60

0) 0

m 0.8 e 0

2 0.6

0.4

0.2

0 10 20 30 40 50 60

m .

0 . 4 1

0 10 20 30 40 50 60

Fraction Number

Fig. 4. Separation ojproteins contained in the washing fractions f rom the poly (L-proline) - agarose column by gel filtration with Rio-Gel A-1.5 m. (A) An aliquot of the concentrated first washing fraction (indicated in Fig. 1) was loaded on a column of Bio-Gel A-1.5 m. The fractions of 4.3 ml were collected. Other conditions were the same as in Fig. 2. (B) An aliquot of the concentrated second washing fraction (indicated in Fig. 1) was loaded on the same column and fractionated under the same condition

eluate with poly(L-proline), respectively. In this purification, 5.02mg of prolyl hydroxylase and 2.19mg of PBP-2 were also obtained. Total purified poly(L-proline)-binding proteins, therefore, was calculated at 28.4 mg. Since we could estimate the concentration of these proteins to be at least 75 pg/g wet tissue of 13-days-old chicken embryo judging from the staining intensity of 42-kDa and 15-kDa bands on SDS gel electrophoresis (data not shown), the yield of the total poly(L- proline)-binding proteins (PBP-1 + PBP-2) during the purification procedure could be estimated to be about 63%. Most of the losses were caused by precipitation of the proteins at the ultrafiltration step.

Molecular masses vf the poly (L-proline) -binding proteins

The apparent molecular mass of PBP-1 was estimated to be 56 kDa by gel filtration with Bio-Gel A-1.5 m (data not shown). The molecular mass of PBP-1 was also estimated to be 57 kDa by native polyacrylamide gel electrophoresis according to Hedrick and Smith [23] (data not shown). When the band sliced out was applied again on SDS gel electrophoresis according to Kanda et al. [17], both the 42-kDa and 15-kDa proteins were observed. Consequently, PBP-1 is a complex composed of the 42-kDa and 15-kDa proteins; the molar ratio of these proteins is 1 : 1. Since the apparent molecular mass of PBP-2 was estimated as below 15-kDa by gel filtration with Bio-Gel A-I .5 m, PBP-2 may exist as a mono- mer, at least in 10 mM Tris/HCl, pH 7.8, containing 0.2 M NaC1, 0.2 M glycine and 10 pM dithiothreitol.

IdentiJl'cation of the 42-kDa and 15-kDa proteins

To analyze the 42-kDa and 15-kDa proteins chemically, we attempted to isolate each subunit from PBP-1 under

0 .3 -

0) 0 c 0.2 - $ u) 0.1 - 9

Fract ion Number

Fig. 5. Isolation of the 42-kDa and 15-kDa proteins,from PBP-1 under denuturing conditions. PBP-1 dissolved in 10 mM Tris/HCl buffer, pH 7.4, containing 6 M guanidine- HCI and 10 mM dithiothreitol was stood for 30 min at room temperature. The sample was loaded on a column (0.65 x 60 cm) of Bio-Gel A-1.5 m previously equilibrated with 10 mM Tris/HCI buffer, pH 7.4, containing 6 M guanidineHC1 and 0.1 mM dithiothreitol, and eluted by the same buffer. Fractions of 0.4 ml were collected. The gel filtration was performed at room temperature

Table 1. The amino acid compositions o j the 42-kDa and 15-kDa proteins. The values for actin and profilin were calculated from the sequences of rabbit skeletal muscle actin [33, 341 and calf spleen profilin [35], rcspectlvely. n.d. indicates not determined

Amino acid Amount in ~~

42-kDa actin 15-kDa profilin protein protein

mol/lOO mol

Asx Thr Ser Glx Pro GlY Ala CYS Val Met Ile Leu Tyr Phe

His LYS

Arg TrP

9.43 9.09 10.07 10.56 6.79 7.49 9.65 8.45 1.11 5.88 5.78 6.34

11.28 10.43 4.37 6.34 5.60 5.08 5.51 2.82 9.10 7.49 9.69 11.97 8.30 7.75 10.05 1.75

n.d. 1.43 0.74 2.11 5.44 5.61 8.76 1.15 3.39 4.28 2.88 4.23 7.35 7.75 4.19 4.93 7.39 6.95 7.91 7.75 3.39 4.28 3.88 2.82 3.45 3.21 3.71 3.52 5.36 5.08 6.53 6.34 1.92 2.41 2.29 1.41 4.10 4.81 3.91 3.52

n.d. 1.09 n.d. 1.41

denaturing conditions. Lyophilized PBP-1 was dissolved in 6 M guanidine . HCI and 10 mM dithiothreitol, and then the sample was loaded on a column of Bio-Gel A-1.5 m. Two protein peaks were observed as shown in Fig. 5. The first and the second peaks were confirmed to be the 42-kDa and 15- kDa proteins, respectively, on SDS/polyacrylamide gel electrophoresis. The last peak eluted at the column volume was derived from dithiothreitol. The amino acid compositions of the 42-kDa protein was closely similar to that of actin, and the composition of the 15-kDa protein was similar to that of mammalian profilin (Table 1). The amino acid composition of PBP-2 was similar to that of the 15-kDa protein preparation

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I none

1 . 5 1 actin PEP-1 I

1.0 0 1 2 3

Time (hours) Fig. 6. Polymerization of the 42-kDa prolein conluined in PBP-I. PBP-1 and rabbit skeletal muscle actin were dialyzed against 5 mM potassium phosphate buffer, pH 7.6, containing 0.1 mM CaCI,, 0.2 mM ATP and 0.1 mM dithiothreitol at 4°C. The PBP-1 and the actin were diluted to 0.61 mg/ml and 0.45 mg/ml, respectively, by the same buffer. Polymerization of PBP-1 (A-A) and actin (0-0) were initiated by the addition of 2 mM MgCIZ and 10 mM KCI, and monitored by viscometry at 22.5 “C. After polymerization, the viscous solution obtained from PBP-1 (combining two experi- ments) was centrifuged at 105000 x g for 2 h. The resultant pellet was dialyzed against 1 mM Tris/HCl, pH 8.0, containing 0.1 mM CaCI2, 0.2 mM ATP and 0.1 mM dithiothreitol overnight. Insoluble materials were centrifuged at 105000 x g for 1 h, and the supernatant was obtained. The protein concentration of the supernatant was diluted to 0.45 mg/ml by the same phosphate buffer as described above and polymerizability of this sample ( 0 , 4 2 K-rich fraction) was examined under the same conditions described above

except large amounts of proline which might be due to poly(L- proline) contamination (data not shown). These data suggest that PBP-1 and PBP-2 are a profilactin and profilin [24], respectively, in chick embryo.

Acriidike propertios of the 42-kDa protein

To confirm further that the 42-kDa protein in PBP-1 is an actin, some properties of PBP-1 were examined, because the purified 42-kDa protein could not be isolated without denaturation. First, by measuring the changes of viscosity, we examined whether or not the 42-kDa protein component of PBP-1 had the ability of salt-induced polymerization. As shown in Fig. 6, PBP-1 could increase its viscosity after a long lag time. This result is analogous to the profile of profilactin polymerization. The resultant viscous solution of PBP-1 was centrifuged and the pellet obtained was dialyzed against a buffer of low ionic strength just as G-actin is prepared. This sample, which contained at least 90% of the 42-kDa protein, could polymerize without lag time in a manner similar to G-actin. Furthermore, we confirmed that PBP-1 inhibited DNase I, as monomeric actin does [25] (Fig. 7), and that PBP- 1 can react with an antibody against the actin prepared from chick gizzard (Fig. 8). Since the 15-kDa protein did not react with the antibody, the 42-kDa protein in PBP-1 might react with the anti-actin antibody. These properties of PBP-1 strongly support the idea that the 42-kDa protein is an actin.

Isolation and properties of’ the 15-kDa protein

To study the properties of the 15-kDa protein, we prepared it again by an altered method as described in Materials and

0.1

r > 0

.I- .- .- - a 0.05 - 0 u) m 2 D

‘0 1 2 3 4 5 6 7 8

Time ( m i d Fig. I . ,5ffects gf PBP-1 on DNase I activity. DNase I (2 pg) and various amounts of PBP-1 were preincubated at room temperature for 5 min in a final volume of 20 pl, and subsequently 0.5 ml of substrate solution (calf thymus DNA) was added to the mixture. Immediately, increase of absorbance at 260 nm was monitored by an automatic spectrophotometer (Hitachi model 220). The figure besides each curve indicates the amount of added PBP-1

Fig. 8. immunochemical clzaracterizalion of PBP-1 by immunodif .fitsion. The central well (Ah) contained antiserum raised against actin from chick gizzard. Peripheral wells contained PBP-1 (PBP-l), 15-kDa protein (1 5K), actin from chick gizzard (A) and sodium-phosphate- buffered saline (PBS)

Methods, because the former preparation of PBP-2 contained a large amount of poly(L-proline). Part of this method is based on the procedure for profilin isolation from profilactin. Proteins which could bind to poly(L-proline) - Sepharose 4 B were eluted directly by a buffer containing 2 M urea and this eluate was passed through a DEAE-cellulose column previously equilibrated with the same ‘urea buffer’. Prolyl hydroxylase and actin were adsorbed to the column and only the 15-kDa protein was recovered in the pass-through fraction. The purity of the protein was examined by SDS/polyacrylamide gel electrophoresis (Fig. 3, lane f). This preparation of the 15-kDa protein was not contaminated by poly(L- proline), but was unstable in a buffer of low ionic strength. For instance, freezing and thawing of this preparation in 1 mM Tris/HCl, pH. 7.4, containing 2 mM ATP, 0.1 mM CaC12 and 0.1 mM dithiothreitol resulted in precipitation of the protein. In addition, when the 15-kDa protein was concentrated by ultrafiltration with a PM-10 membrane in the same buffer, much of the protein was lost due to adsorption onto

296

0 30 60

Time ( m i d

Fig. 9. EjJects of 15-kDa protein on actin polyrnerizability. The 3 5- kDa protein was dissolved in 0.5 M KCI to a final concentration of 0.65 mg/ml and sonicated, previously. Actin from rabbit skeletal muscle (0.36 mg/ml) was preincubated with this protein (0.13 mg/ml) in 5 mM potassium phosphate buffer, pH 7.6, containing 0.1 mM CaCI,, 0.2 mM ATP, 0.1 mM dithiothreitol and 0.1 M KCI at 0°C for 10 min. Polymerization of this sample (0-0) was initiated by addition of 2 mM MgClz and incubation at 22.5"C. Control exper- iment (0-0) was performed in a similar manner except there was no addition of 15-kDa protein

the membrane. The effects of the 15-kDa protein on actin polymerizability was examined. The protein, when dissolved in the buffer of low ionic strength (see above), did not affect either polymerization of actin or depolymerization of actin fiber. However, the protein stored in a buffer containing 0.5 M KCl could decrease the rate of polymerization of actin in a final concentration of 0.1 M KCl, 1 mM Tris/HCl, pH 7.4, 2 mM ATP, 0.1 mM CaCI, and 0.1 mM dithiothreitol (Fig. 9). This suggests that the 15-kDa protein, that is PBP- 2, can interact with rabbit skeletal muscle actin in vitro. Therefore, it must be a profilin in chick embryo.

Affinities ? fPRP-I und PBP-2 towardpoly(L-proline)

To confirm that PBP-1 and PBP-2 have proper affinities toward poly(L-proline), purified PBP-1 and PBP-2 were both separately rechromatographed on columns of poly(L-proline) - Sepharose 4 B. Both proteins were adsorbed to the column and could be eluted with the poly(L-proline) solution in a similar manner as described in Materials and Methods. This indicates that the binding abilities to poly(L-proline) are specific characteristics of the proteins themselyes. Addi- tionally, PBP-1 could be eluted also with an oligo(L-proline) solution, t-butyloxycarbonyl-octa-(L-proly1)-benzyl ester- (Boc-Pro8-OBzl, 5mg/ml), but PBP-2 could not be eluted with this solution. Accordingly, PBP-2 may have a greater affinity toward poly(L-proline) than PBP-1.

We attempted to measure Kd values for PBP-1 and PBP-2 toward poly(L-proline) by affinity electrophoresis according to the method of Takeo and Nakamura [18]. This method is based on quantitative determination of the strength of interac- tion between an entrapped ligand in polyacrylamide gel and a protein. In other words, the decrease in the mobility was measured after the electrophoresis of a sample on gels with various concentrations of a ligand. The decrease in the mobility of both PBP-1 and PBP-2 were observed to be depen- dent on the concentration of ligand (Fig. 10). However, accu-

Poly(L-proline) mg/ml

F i g . 10. Ajfinify electrophoresis of' PBP-I on gels containing p o l y ( ~ - proline). The electrophoresis was carricd out at pH 8.3 (electrode buffer) by using 60/0 polyacrylamide gels containing various concentrations of poly(L-proline) indicated in this figure. 5 pg of PBP-I was loaded on each of the gels

rate Kd values for PBP-1 and PBP-2 could not be obtained. Then, we calculated their ratios of mobilities on gels with a fixed concentration of poly(L-proline) (2 mg/ml) to that without poly(L-proline), assuming the mobility in the absence of poly(L-proline) as 1.0. The values for PBP-1 and PBP-2 were 0.61 and 0.27, respectively. This also suggests that PBP- 2 has a greater affinity to poly(L-proline) than PBP-1.

DISCUSSION

In the present study, two poly(L-proline)-binding proteins (PBP-1 and PBP-2 ) were purified from chick embryos. Ini- tially, we considered that these proteins might have a role in collagen biosynthesis, because individual chains of collagen triple helix form poly(L-proline) I1 helix. We did not expect that these proteins could have any other function, because the poly(L-proline) helix is much less abundant in natural proteins. Then, we tested the effect of the PBP-1 and PBP-2 on denaturation and renaturation of the collagen model peptide, (Pro-Pro-Gly),, and on the fibril formation [26] of type I collagen (rat tendon) in vitro. No effects were observed. In addition, PBP-1 and PBP-2 could not bind to immobilized native collagen prepared by the procedure of Nagai and Hori [27]. Next, we tested the effect of PBP-1 and PBP-2 on prolyl hydroxylase activity using (Pro-Pro-Gly), as a substrate. No effects were observed.

Surprisingly, the amino acid composition of the 42-kDa protein component in PBP-1 showed close similarity to that of actin from rabbit skeletal muscle. And we show here that the 42-kDa protein has an ability of polymerizing (Fig. 5), inhibits DNase I (Fig. 6) and reacts with anti-actin antibody

297

(Fig. 7). From this evidence, the 42-kDa protein must be an actin in chick embryo. Accordingly, the other component in PBP-1, the 15-kDa protein, should be regarded as an actin- binding protein. On the other hand, many actin-binding proteins are known in non-muscle cells [28]. Among the actin- binding proteins, profilins which stabilize monomeric actin [29] in many eukaryotic cell [30 - 321 have molecular masses 12 - 16 kDa. The amino acid composition of our 15-kDa protein preparation was similar to that of mammalian profilin. Considering that the chick 15-kDa protein inhibits actin polymerization, we can conclude that the 15-kDa protein is a profilin in chick embryo. However, its inhibiting ability against actin polymerization was lower than that of mammalian profilin. In addition, this chick profilin migrated a little slower than calf thymus profilin on SDS/polyacrylamide gel electrophoresis, indicating that the chick protein has a higher molecular mass than the mammalian one (data not shown). These results may be responsible for a slight difference between avian and mammal proteins. Alternatively, these results may be caused by differences between the embryo and the adult, as in the case of r-fetoprotein and albumin.

Since the molecular size and amino acid composition could not distinguish PBP-2 from the 15-kDa protein component in PBP-I, PBP-2 also may be a profilin in chick embryo. Consequently, we can say that the poly(L-proline)-binding proteins (PBP-1 and PBP-2) from chick embryo are a profilactin and a profilin, as named by Carlsson et al. [24]. We can further consider that the binding ability of the profilactin from chick embryo to poly(L-proline) is due to its profilin moiety, because the profilin itself, PBP-2, can bind to poly(L- proline). This idea is supported by their strength of affinity for poly(~-proline), that is, profilin could bind to poly(~- proline) more strongly than the profilactin. Of course, this also explains the reason why PBP-1 (profilactin) could be eluted continuously from the column of poly(L-proline) - Sepharose 4 B by a large volume of the washing buffer. I n addition, actin from rabbit skeletal muscle could not bind to poly(L-proline) - Sepharose, regardless of its monomeric or filamentous state (G- or F-actin). Therefore, the profilin from chick embryo has a poly(L-proline)-binding site in addition to an actin-binding site.

If a profilin generally has this affinity to poly(L-proline), the procedure of the affinity chromatography in the present work will be the most effective method for isolation of profilin and profilactin. Our preliminary work shows that the poly(L- proline)-binding proteins exist at least in calf thymus, rat liver and rat embryo. These proteins may also be profilins and profilactins. Thus, this binding ability of profilin to poly(~- proline) seems to be conserved in evolution. We therefore consider that this ability of profilin has an important role in the formation of a cytoskeleton in a living cell. We also speculate that there is a protein having poly(L-proline) I1 helix in a cell and that the hypothetical protein can regulate the profilin function. This discovery of a profilin property will provide a new approach to research on the cytoskeleton.

We wish to thank Dr T. Uchida, chief of our laboratory, for her constant interest, encouragement and critical reading of this manu- script. Helpful discussions with Drs M. Takahashi, J. Masai, M. Miki and H. Sugino of our Institute are gratefully acknowledged. We are indebtcd to Dr S. Tanaka for providing chick embryo, Dr K . Kitamura for his immunological assays and D r M. Miki for providing

dried muscle from rabbit. We are also grateful to Prof. D. Fujimoto of Hamamatsu University School of Medicine and Prof. H. Sakai and D r E. Nishida of Tokyo University for their valuable discussions.

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Poly(l-proline)-binding proteins from chick embryos are a profilin and a profilactin