Biochimica et Biopkysica Acta, 494 (1977) 76 91 .~':~ Elsevier/North-Holland Biomedical Press

BBA 37734 THE ISOLATION AND TUBULE PROTEINS

CHARACTERISATION

OF

PLATELET

MICRO-

ALAN G. CASTLE and NEVILLE CRAWFORD Department of Biochemistry, University ~[ Birmh~gham, P.O. Box 363, Birmingham BI5 2TT (U.K.)

(Received March 14th, 1977)

SUMMARY Tubulin, the microtubule subunit protein, has been isolated from a soluble extract of pig platelets, by an in vitro polymerisation process. Several physicochemical properties of platelet tubulin have been investigated and compared with those of mammalian brain tubulins. The molecular weight of the tubulin monomeric subunits was found to be 55 000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and the platelet protein co-migrated with rat, rabbit, guinea-pig, mouse and calf brain tubulins. The [3H]colchicine-binding dimer form of platelet tubulin sedimented in a 5 ~ to 20~o linear sucrose gradient with a sedimentation coefficient of 5.7 S, compared with that of 5.9 S for rat brain tubulin. The amino acid compositions of platelet and mammalian brain tubulins were found to be very similar, with glutamate and asparate as the predominant residues. Moreover, the one and two dimensional fingerprints of ~ZSl-labelled tryptic peptides of platelet and brain tubulins showed considerable similarity. Platelet tubulin could be separated into two nonidentical (ct and fl) subunits by Tris/glycine discontinuous alkaline sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the fl-tubulin had two or three 1251 labelled peptides not seen in the a-tubulin. Two, or perhaps three, high-molecular-weight proteins were always present in the platelet tubulin samples, prepared by in vitro polymerisation and these had molecular weights greater than 200 000 and were probably analogous to the highmolecular weight proteins reported to be present in brain microtubule preparations. Platelet tubulin with its associated high molecular weight proteins will assemble in vitro into tubular structures which in electron micrographs resemble brain microtubules and the microtubules seen in whole platelets. It is believed that this is one of the first mammalian microtubule systems other than that of nervous tissue which has been isolated and studied in detail at the subunit level. INTRODUCTION The normal, circulating mammalian blood platelet is lentiform in shape and Abbreviations: Mes, (2[N-morpholino]ethanesulphonic acid); EGTA, ethyleneglycol-bis(fiamino ethyl ether) N,N'-tetraacetic acid; SDS, sodium dodecyl sulphate; Temed, N,N,N',N'-tetramethylethylenediamine.

77 has a prominent circumferential bundle of microtubules lying just beneath the plasma membrane [1]. When the platelets are activated prior to aggregation and the release of granule stored constituents, they lose their discoid shape together with the marginal microtubule bundle. The microtubules later reappear in the pseudopodia of the activated platelet but their exact function in platelet haemostatic activities is by no means clear. Various studies with microtubule poisons, such as colchicine and the Vinca alkaloids [2] have indicated that as a labile cytoskeletal system the microtubules are at least in part responsible for platelet asymmetry and shape change. Since 1972 when Weisenberg demonstrated the in vitro assembly of brain microtubules [3] considerable knowledge has accumulated about the fundamental protein chemistry of the microtubules from nervous tissue (see reviews by Mohri [4] and Synder and Mclntosh [5]). However, because of isolation problems the microtubule subunit proteins (tubulin and the associated high molecular weight components) of tissues other than brain have not been studied in any great detail. Next to brain tissue, blood platelets are probably one of the richest sources of microtubular proteins and recently we have described a procedure for the isolation of pig platelet tubulin by a modified in vitro polymerisation procedure [6]. in this paper we report our further investigations of the platelet microtubule subunit proteins and give details of the conditions necessary for their in vitro polymerisation to microtubular forms. In addition some of the physical and chemical properties of platelet tubulin and the associated high molecular weight proteins have been described and compared with those of the analogous microtubule proteins of brain tissues. MATERIALS AND METHODS All radioisotopes (~25I for protein iodination of specific activity 80-140 mCi/ ml; [3H]colchicine, specific activity 2Ci/mmol and [)~-32P]ATP specific activity 16.0 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, Bucks, England. Mes, GTP (Type I l-S), EGTA, diphenyl carbamyl chloride treated trypsin, bovine serum albumin and the protein standards for SDS-polyacrylamide gel electrophoresis were all obtained from the Sigma Chemical Co. Ltd. All other chemicals were of Analar grade wherever possible. Pig blood and calf brains were obtained from a local abattoir. Dunkin Hartley guinea-pigs were supplied by Messrs OLAC Ltd., Bicester, Oxfordshire and random mated adult rats were obtained from the departmental animal house.

Isolation of platelets Platelets were isolated from flesh pig blood by the differential centrifugation procedure described by Harris & Crawford [7].

Isolation of platelet tubulin by & vitro polymerisation Pig blood platelets, freshly prepared at room temperature, were resuspended in ice-cold polymerisation buffer (1.5 ml for every gram wet weight of platelets). The polymerisation buffer consisted of 1 mM EGTA; 1 mM EDTA, 0.1 M Mes; 0.3 M sucrose, adjusted to pH 6.8 at 22 °C with 8 M NaOH, 1 mM GTP. The G T P was added just before use. The platelet suspension was placed on ice for 10 min and then

78 homogenised in an ice-cooled vortex flask, using a MSE top drive, blender-type homogeniser operated at maximum speed for a total of 5 rain, with 4 one rain intervals for recooling. The platelet homogenate was centrifuged at 100 000 ~ .%~ for 60 rain at 4 ~C and the supernatant or "platelet soluble phase" was then carefully decanted to avoid contamination by particulate material. An equal volume of 8 M glycerol was added and the soluble phase in glycerol was incubated for 20 rain at 30 'C, during which time thin fibrous strands of varying length were observed to form in the solution. The incubated material was layered onto 6 ml of 6 M glycerol, (buffered at pH 6.8) in 40 ml polycarbonate tubes and centrifuged at 50 000 ~, ,~,,~for I h at 20 C in a MSE 3 i~ 43 ml swing-out rotor in a MSE Superspeed 50 centrifuge. The supernatant was decanted and the sides of the tube washed and dried with filter paper before the pellet was taken up in the desired buffer to a concentration of about 5 mg/ml protein. Occasionally the preparation was given gentle hand homogenisation (one or two passes) in a glass Potter-Elvehjem type homogeniser using a teflon pestle.

Isolation of brain tubulins by #7 vitro polymerisation Rat, rabbit, calf, guinea-pig and mice brain tubulins were all prepared by the procedure of Shelanski et al. [8].

Isolation of muscle and platelet act& Acetone-dried powders of muscle and platelets were prepared by the method of Carsten and Mommaerts [9] and actin was extracted from these powders by the method of Adelstein and Kuehl [10] and polymerised after the method of Carsten and Mommaerts [9].

SDS-polyacrylamide gel electrophoresis This was performed using two different buffer systems (A and B). A Tris/borate buffer system, pH 7.0, was used for most of the analytical work (System A), whilst a Tris/glycine discontinuous buffer system, pH 8.6, was used to split the two subunits of platelet tubulin (System B). The protein samples for electrophoresis were denatured by making to a final concentration of 4 ~,, SDS, 8 M urea and 0.1 M /~-mercaptoethanol and placing in a boiling water bath for 5 min. For molecular weight determinations standard curves were constructed from the log molecular weight/mobility relationships determined for the following standard proteins: myosin heavy chain 200 000, phosphorylase 94 000, catalase 60 000, ovalbumin 43 000, trypsin 23 800 and ribonuclease 13 000. With the Tris/borate buffer gel system the relationship was linear throughout this range of molecular weights. A. Tris/borate continuous buffer system. Both 7.5 ~ polyacrylamide slab gels (dimensions 20 ;~. 6 ~ 0.2 cm) and polyacrylamide stick-type gels (9 × 0.5 cm) were used. The latter were mainly used for molecular weight determinations. A stock solution (12 ml) consisting of 30~o (w/v) acrylamide, 0.8)/o (w/v) bisacrylamide was mixed with 24 ml of a stock solution consisting of 5 ~o (w/v) boric acid; 20~o Tris; 0.2~o SDS and the total made up to 47.35 ml with distilled water. Temed (50/~1) followed by 0.6 ml of 1 ~ ammonium persulphate were used to catalyse the polymerisation. The running buffer for the electrophoresis was 0.4 M boric acid adjusted with Tris (approx. 0.1 M) to pH 7.0, containing 0.1 o/jo SDS. Denatured

79 protein samples (5-100/A) were applied to the top of the gel under the running buffer and gels were run at 2-3 mA/gel using bromophenol blue as tracking dye. The gels were stained overnight in 0.25 ~% Coomassie Brillant Blue R in methanol/acetic acid/ water (45:10:45, by volume) and destained by diffusion in methanol/acetic acid/water (1:1:8, by volume). B. Tris/glycine discontinuous buffer system. The gel solution was prepared by mixing together 10 ml of 30~,, (w/v) "Cyanogum 41"; 20 ml of 3 M Tris. HC1, pH 8.9, 0.5 ~,, Temed, and 10 ml of water. 40 mg of SDS was dissolved in this solution and then 0.5 ml of 10~o ammonium persulphate was added to initiate polymerisation. Slab gels were used. A stock buffer of 0.38 M glycine, 0.05 M Tris, I ~o (w/v) SDS, pH 8.6 was prepared and the running buffer was made from this by dilution with water (1 volume stock buffer + 9 volumes water). Denatured protein samples were applied to the gels and were run at 50 mA for about 6 hours until the "refraction line" was at the bottom of the gel. Gels were stained and destained as in procedure A. Densitometer traces of gels were obtained at a wavelength of 570 nm using a Gilford Model 2000 spectrophotometer with a Gilford gel scanning attachment.

Colchicine-binding assay The DEAL-cellulose filter-disc assay of Borisy (1972) was used under the optimum conditions established by Castle [11]. The [3H]colchicine-binding protein was adsorbed onto a stack of four DEAL-cellulose filter discs and free [3H]colchicine was removed by extensive washing. This procedure was rapid and reproducible and for example in an assay of the [3H]colchicine-binding activity of a rat brain tubulin preparation a value of 56 580 ~ 1110 dpm (mean ~ standard deviation) was obtained for 12 replicate determinations. Estimation of the sedimentation coeaficients of prote#~s by sucrose densiO,-gradient centrifugation The method used was essentially that of Martin and Ames [12]. The sedimentation of the protein species under investigation (in this case tubulin as identified by [3H]colchicine-binding activity) in a sucrose density gradient after ultracentrifugation was compared with that of marker enzymes of known sedimentation coefficients (catalase and alcohol dehydrogenase). Linear sucrose gradients of approximately 5 ~o to 20°//o (w/v) sucrose were used and after high speed centrifugation, fractions were taken and assayed for [3H]colchicine-binding activity (by the DEAL-cellulose filter disc method), for marker enzyme activity and for sucrose concentration (using an Abbe refractometer). Catalase was determined by the method of Baudhuin et al. [13] and alcohol dehydrogenase by the method of Martin and Ames [12]. Amino acid analyses These were performed using the system of Spackman et al. [14], modified for elution from a single column with three buffers as used by Wilkinson et al. [15]. Duplicate samples were hydrolysed in evacuated sealed tubes in 6 N HCI at 110 ':'C for 24 and 72 h. After hydrolysis HCI was rapidly removed in a rotary evaporator, and the dried sample was resuspended in buffer and applied to the column. A Beckman model 120B amino acid analyser was used fitted with a locarte automatic loader. Amino acids were separated on the one column by sequential elution with sodium citrate buffers, pH 3.25 and 4.25 (0.2 M) and pH 6.65 (1.0 M).

80 The amounts of threonine and serine were corrected for losses during hydrolysis by extrapolation to zero time of" hydrolysis. Valine and isoleucine were determined after 72 h of hydrolysis. Other amino acids were determined from the mean of duplicate analyses of the 24 and 72 h hydrolysates. Tryptophan was determined by ~'Procedure K" of Spies and Chambers [16].

Peptide mapping of 1251-1abelledprotein from polyacrylamide gels Subunit proteins eluted from polyacrylamide gels were labelled with 12Sl in the presence of chloramine T (Hunter and Greenwood [17]) and then digested with diphenyl carbamyl chloride treated trypsin. The resulting peptides were separated in either 1 or 2 dimensions according to the method of Bray and Brownlee [18].

Protein estimation Protein was determined by the method of Lowry et al. [19] as modiiied by Eggstein and Kreutz [20]. This modification replaces sodium tartrate by sodium citrate in Reagent B to give a more stable reagent. Bovine serum albumin was used as the standard.

Electron microscopy Samples for electron microscopy were mixed with an equal volume of "microtubule stabilising buffer" [21] (i.e. 50~o glycerol, 10~o dimethylsulphoxide, 0.02 M Mes, 0.4 mM EGTA, 0.2 mM MgCI2, 0.4 mM GTP (pH 6.5) and applied to a carbon coated specimen grid. These grids were stained with 1 ~/o uranyl acetate and examined in a Philips 301 electron microscope. RESULTS

Isolation of plateIet microtubule proteins Platelet microtubule proteins were isolated by the procedure outlined in the Methods section and the fractions obtained during this isolation were analysed by SDS-polyacrylamide gel electrophoresis (Fig. 1). The soluble phase of platelets (Fig. 1 gel A) contained a large number of polypeptides ranging in molecular weight from 300 000 or above, down to about 15 000. The pellet (Fig. 1 gel B) obtained by subjecting this soluble phase to polymerising conditions, contained a major protein band which had a molecular weight estimated to be 55 000 by SDS-polyacrylamide gel electrophoresis. This band was often seen as a closely spaced doublet. From the experimental evidence presented in this paper and elsewhere [11] we feel that this component can be referred to as platelet tubulin. The pelleted material also contained 2 or possibly 3 proteins of higher molecular weight ( ~ 200 000) and we refer to these as high-molecular-weight proteins, the term used by brain tubulin investigators for similar components in their microtubule preparations. The supernatant fraction remaining after the removal of the pelleted material was shown to have a very similar protein profile on gels to that of the original platelet soluble phase, except that the content of the 55 000 tubulin component was greatly reduced (Fig. 1, gel C). The pelleted material represented 5 ~o to 6 ~o of the total soluble phase protein and the specific [3H]colchicine-binding activity of the pellet protein was 35 times greater than that of the supernatant measured after removal of the pellet.

81

A

13

Fig. I. SDS-polyacrylamide gel electrophoresis (Tris/borate buffer system; see Methods). Gel A, platelet soluble phase; Gel B, platelet pellet containing the microtubule proteins; Gel C, remaining supernatant after the removal of the microtubule proteins by centrifugation.

C~;-electrophoresis experiments The tubulin isolated from pig b l o o d platelets was co-electrophoresed with n e u r o t u b u l i n s p r e p a r e d from various m a m m a l i a n brains and also with actins from rabbit skeletal muscle and from pig platelets. The results o f one such experiment are shown in Fig. 2. The tubulin from platelets c o - m i g r a t e d with t u b u l i n isolated from rat, rabbit, guinea-pig, mouse and calf brains, whereas b o t h platelet and r a b b i t skeletal muscle actins migrated well a h e a d o f the platelet and b r a i n tubulins with a mobility c o r r e s p o n d i n g to a molecular weight o f 43 000.

A -B ...... C ......... '~ ...... ~Fig. 2. SDS-polyacrylamide gel electrophoresis (Tris/borate buffer system; see Methods). Coelectrophoresis studies of platelet and brain tubulins and mixtures of these tubulins with platelet and rabbit skeletal muscle actins. A, rat brain tubulin; B, platelet tubulin F platelet actin; C, rat brain tubulin t platelet tubulin - platelet actin; D, platelet actin ~ muscle actin; E, muscle actin.

82

Estimation q/ the sedimentation coqfficient of platelet tuhulin and its comparison with brain tubulin A sample (130#1; 0.35 nag) of platelet tubulin in I mM E G T A , I mM E D T A , 0.1 M Mes, pH 6.8, 0.1 M sodium glutamate, 1 mM GTP, was mixed with catalase (10ffl; 0,1 nag) and then layered onto a 12 ml linear sucrose gradient (5 to 20 °/ / o ~V,/V sucrose). After centrifugation (196000 > g;,v; 20 h: 4 C ) 36 equal fractions were collected from the gradient by aspiration and these were assayed for colchicine binding activity and catalase activity. The results are shown in Fig. 3. A peak of [3H]colchicine-binding activity was found about one-third of the way down the gradient, and a peak of catalase activity occurred about two thirds of the way down the gradient. Using the catalase as the reference protein (S~o.w 11.3) the peak of [~H]colchicine-binding activity was determined to have a sedimentation coefficient of 5.3 S. Five separate determinations of the sedimentation coefficient of [3H]colchicine binding platelet tubulin were performed using different preparations. The values obtained ranged from 5.3 S to 6.0 S (mean 5.7 S).

to



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The sedimentation coefficient of [3H]colchicine-binding rat brain tubulin was also determined by the sucrose gradient procedure and the results from two experiments were 5.8 S and 6.0 S.

Amino acid composition of platelet tubulin and a coml)arison with brain tubul#ls The platelet tubulin samples chosen for these analyses were the highest purity preparations obtained throughout this study. Although an actin band was faintly visible on overloaded polyacrylamide gels, it represented less than 2~o o f the total protein present in the sample as determined by semiquantitative analysis of the gel scans. The brain tubulins were all o f high purity and free from actin. Both the platelet and the brain tubulin preparations contained some high molecular weight protein (about 9 % to 15 % of the total protein in the sample as determined by gel scanning). N o other protein bands were visible on the gels.

83 The a m i n o acid compositions of platelet and b r a i n tubulins, expressed as residues per 55 000 × g, are given in Table I. Two different preparations of pig platelet t u b u l i n were analysed, together with five preparations of b r a i n t u b u l i n , isolated from three different m a m m a l i a n species, viz. guinea-pig, rat and calf. The a m i n o acid compositions of the t u b u l i n s were all very similar with the acidic residues consistently o u t n u m b e r i n g the basic residues. The tubulins from the two cell sources (viz. platelets a n d brain) have considerable similarities. Only two a m i n o acids (serine and threonine) showed significant, t h r o u g h small differences, but the reliability of serine and threonine d e t e r m i n a t i o n is possibly n o t as great as with the other a m i n o acids, due to losses during hydrolysis.

TABLE I AMINO ACID COMPOSITION OF PLATELET AND BRAIN TUBUL1NS Figures are moles per mole, 55 000. N.D., not determined. Pig platelet tubulin prepn. 1

Pig platelet tubulin prepn. 2

49.0 28.5 32.0 60.0 29.0 41.0 40.0 39.0 11.5 24.5 41.0 17.0 20.5 13.0 28.0 25.5 N.D.

54.0 27.7 33.2 60.9 31.7 37.7 39.6 34.2 8.9 22.8 38.6 14.9 19.8 15.9 28.7 25.3 2.5

Guinea-pig brain tubulin prepn, l

Guinea-pig brain tubulin prepn. 2

Rat brain tubulin prepn. I

Rat brain tubulin prepn. 2

Bovine brain tubulin

49.3 32.4 35.3 63.7 26.9 38.3 38.8 34.8 12.4 23.4 39.8 15.9 20.9 12.9 29.4 23.9 N.D.

50.2 33.3 40.7 64. I 24.3 36.8 36.8 33.8 12.9 22.9 38.8 15.9 20.4 ~2.9 28.8 24.8 N.D.

49.2 33.3 33.8 68.1 26.4 38.3 37.3 35.8 12.4 22.9 40.3 14.9 18.4 12.4 28.8 25.4 N.D.

54.6 31.6 37.6 59.7 27.6 43.1 39.6 35.6 8.5 21.6 35.1 12.5 19. I 22.6 28.6 25.1 N.D.

53.0 36.5 37.5 64.0 35.5 37.0 38.5 33.0 8.0 20.0 34.5 15.0 19.5 15.5 29.0 24.0 N.D.

_

Asp Thr Ser Glu Pro Gly Ala Val Met lle Leu Tyr Phe His Lys Arg Trp

Subunit heterogeneiO, of platelet tubulin O n electrophoresis in the c o n t i n u o u s Tris/borate buffer system at pH 7.0, platelet t u b u l i n occasionally migrated as a very closely spaced doublet. However, when the platelet t u b u l i n was run on gels in a discontinuous Tris/glycine buffer system at pH 8.6 there was a clear separation into two subunits (Fig. 4a). We have designated these subunits as a-platelet t u b u l i n (slower migrating c o m p o n e n t ) and fl-platelet t u b u l i n (faster migrating c o m p o n e n t ) according to the c o n v e n t i o n applied to brain tubulins. Densitometer traces of the a and fl-platelet t u b u l i n s were made (Fig. 4b) and these showed that the ratio of the two subunits was approximately equal. By careful excision of the ~t and /3-platelet t u b u l i n bands from gels it was possible to elute sufficient protein for a study of the tryptic peptides after ~ZSllabelling.

~4 0

Tubul n Tubulin

J Fig. 4. Discontinuous Tris/glycine SDS-polyacrylamide gel electrophoresis. (a) Gel showing the separation of platelet tubulin into two subunits (tt and/3 tubulin). (b) Densitometer traces of the a and [~ subunits of platelet tubulin (HMW refers to the high molecular weight proteins present in tubulin preparations).

85

Tryptic peptide mapping of lZSl-labelled platelet tubulin and its comparison with rat brain tubulin One-dimensional tryptic peptide maps of platelet and brain tubulin 125I-labelled peptides were made by high voltage paper electrophoresis at pH 2.0. Comparisons were made between rat brain and platelet tubulins which migrated as single 55 000 molecular weight bands in the Tris borate gel system and preparations subjected to Tris glycine discontinuous gel electrophoresis in which the c~ and [4 tubulin components could be separately excised. The maps were very similar for all the tubulins analysed, with 6 or 7 radioactive spots. Fig. 5 shows one-dimensional tryptic peptide maps of platelet and brain tubulin ~25I-labelled peptides separated by high voltage paper electrophoresis at pH 6.5.

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86 Again the maps of the tubulins were similar, the most prominent radioactive peptides being those designated "2" and "6". However, one peptide designated "'5"' was observed in fl-platelet tubulin but was absent in (t-platelet tubulin. This peptide was seen as a faint spot in whole platelet tubulin and in rat brain tubulin. Another possible difference between {~ and #-platelet tubulin was in the faint spots designated peptide "4". The peptides from the (~- and #-platelet tubulins separated by high voltage electrophoresis at pH 6.5 were further resolved by chromatography with n-butanol/ acetic acid/water (120:30:50, by volume) at right angles to the electrophoretic separation. These two-dimensional fingerprints are shown in Fig. 6. Comparison of the fingerprints of c~- and #-platelet tubulins showed one striking difference. Peptide "5" of fl-platelet tubulin was now resolved into 2 or possibly 3 peptides (shaded in Fig. 6) all of which were absent in (~-platelet tubulin. A

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