Properties of structural proteins of bovine platelets

ARCHIVES

OF

BIOCHEMISTRY

Properties ANATOLY From

the

Biology

AiXD

86, 412-418

BIOPHYSICS

of Structural

Proteins

BEZKOROVAINY Division,

Oak

Ridge

Received

(1962)

of

Bovine

AND DAVID National August

Platelets’

G. DOHERTY

Laboratory,’

Oak

Ridge,

Tennessee

15, 1961

Bovine platelet ghosts were found to consist largely of protein with a small amount of protein-bound carbohydrate. A full spectrum of amino acids was obtained, and the N-terminal amino acid proved to be serine. Partial solubiliaation of platelet ghosts was accomplished by a mild basic hydrolysis. The bonds broken in this procedure were found to be ester-like, as shown by a positive hydroxamic acid reaction. A water-soluble DNP-fraction was isolated by mild basic hydrolysis of DNPplatelet ghosts. Ultracentrifugal analysis revealed that this fraction consisted of at least three components. INTRODUCTION

The chemical properties of blood platelet components have been studied to only a small extent compared to other cellular elements of blood. The free amino acid content of human platelet,s was investigated by several workers (l-3)) who discovered a full complement of amino acids commonly found in proteins, and a ratSherlarge quantity of taurine in the soluble portion of the cells. Woodside and Kochalaty (4) examined the carbohydrat,e composition of whole human and bovine platelet,s, and dct,ermined the presence of glucose, galactose, mannose, hcxosamines, methyl pentoses, and pentoses. Anderson and Ode11(5’) show-cdt,hc presence of chondroitin sulfatclike polysaccharide in rat blood platelets, and hyaluronic acid was found in platelets by Tropeano et al. (61. It was suggested that the acidic propert,iee of platelets were due to acid mucopolysaccharides (7). Several protein-like preparations possessing various blood-clot’ting activities have been ‘d preliminary report of this work was presented at the 45th annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City. N. J., April 10-15, 1961. “Operated by Union Carbide Corporation for the IT. S. Atomic Energy Commission.

purified (8), but homogeneity data and physical-chemical studies on most of t,hese fractions are lacking. Although histological evidence indicates that ribonucleoproteins are present in platelets (9)) no nucleic acids could be demonstrated in platelets by chemical means (10). There are significant amounts of lipids in blood platelets which are in a state of rapid turnover (11, 12). This paper is concerned with somechemical properties of structural elements of bovine platelets. MATERIALS

PREPARATION

AND

METHOD8

oF PLATELETS GHOSTS

.4xD PLATELET

Polyethylene vessels and tubing were used throughout the isolation procsdure. All centrifrrgations were performed at 2°C. Bovine blood, obtained from a slaughterhouse immrdiaicly after the animals were killed, was mixed with 0.1 vol. of 5% EDTA” in 0.9% aqueous NaCl as suggested by Marks et al. (12), and was placed on ice. The diluletl blood, pH 7.7, was rentrifuged at 5000 X g for 30 min., ant1 the plat,eletrich plasma vvvits separated and rc,centrifuged undri ‘The following abbreviations are used: EDTA, sodium ethylenediaminc tetraacrtate ; FDNB, flrrorotlinitrohenzene ; DNP, dinitrophenyl ; N, nitrogrn

PROPERTIES

OF

BOVINE

identical conditions. Platelets were obtained by centrifuging the supernatant at 20,000 X g for 30 min. The platelet button was dispersed in 0.9% aqueous NaCl and centrifuged at 300-500 X g to remove any remaining red cells and leucocytes. Platelets were reisolated by high-speed centrifuga-. tion. The platelet preparation had the expected morphological nppcarance ; it was apparently pure, since microscopic examination of platelet smears and suspensions failed to reveal presence of othri blood cells, and it. reduced the clotting time 01 rrcxlcified bovine plasma two- to fourfold. Disruption of intact, platelets was usually pcrformed by grinding the cells in :I glass homogenizer, followed by exhaustive dialysis against distilled water. The nature of the snbsequent,ly isolated structural material did not change materially if the platelets wcrc broken up by sonication, homogenation in a Waring blendor, or by freez.ing-thawing. The dialyzed material was centrifuged at 20,000 X g, and the precipitate was washed several times with water and was finally lyophilized from a water suspension. The yield of dry platelet structural material, termed platelet ghosts hcrcaftcr, was 200-400 mg. from 18 1. blood.

AmLmIcAL

METHODS

Dinitrophenylation Thirty to 50 mg. of platelet ghosts were incubated with 0.1 ml. FDNB at 40°C. and pH 8.0 in 3-4 ml. water. The uptake of FDNB by the suspension was followed titrimetrically, as described by Fraenkel-Conrat el al. (13). Hydrolysis of DNPplatelet ghosts was performed with peroxide-free constant-boiling HCl in a sealed tube at 105°C. for 12 hr. DNP-amino acids were separated and identified by the two-dimensionai paper chromatography system of Biserte and Osteus (14). DSP-serme,4 treated in a manner identical to the DNP-platelet ghosts, was used as a standard in al1 N-terminal group detenninatione.

Constituent

-Analysis

Analysis for the various components of platelrt ghosts was done on its peptic digest (pH 2.~ HCl; 2 hr.; 40°C.; enzyme to substrate ratio, 1:50) and on thr soluble portion of its 2 LY HC!l 2-hr. hydrolyzate. 1\Jo such treatment vvas performed on the water-soluble materials derived from 1)latelet ghosts. Hexose, hexosamine, and methylpentose were tletermined by the methods summarized by FVinzler (15), sialic acid by the method of Warren (16), uranic acid by the method of Dische (17)

41:3

PLATELETS

using galacturonic acid as standard, and pento.se according to Albaum and Umbreit (18). Nitrogen was determined by digestion with HBOA for 45 min. and nesslerization, phosphorus by digestion with perchloric acid for 30 min. and the method of Fiske and SubbaR.ow (19), and moisture by drying at 1lO’C. to constant weight. “Protein” was determined to the method of Lowry et nl. (20), using crvstalline human serum albumin as standard. Ester, bonds were estimated by the method of \Veissmann and MLlr.ver (21) using peptic digests of plntelol ghosts. Ac~ucous ethyl acetate, prepared immr&ttely I)efore the :~~a\-, was used :IJ the ~t:lll(I~l.d.

Periodate

Oxidations

Oxidation of platelet ghosts with periodate was carried out in the dark with shaking at pH 7.7 (0.02 J1 phosphate), and its uptake by the platelet material was measured spectrophotometrically accortling to Dixon and Lipkin (22). Since platelet ghosts were insoluble in the reaction medium, an aliquot of the reaction mixture was centrifuged before determining thr optical density. Stock solutions of periodate were standardized spcctrophotometrically by oxidation of a standard solution of galactose. The portion of the periodate III'take vs. time plot having a constant slope was assumed to liare been caused by o\-eroxidation, and n-a,5 cstrapolatcd to zero time to obtain the actual uptake of pcriodate l)y the I)latelct ghosts. By this method orodomucoitl took up 2 moles periodate! mole sialic acifl, s:: 1Ii,c)\-iou.ly foimrl by f’opcnoe (23).

Paper Chroma toyraphy Qualitative identification of carbohydrates and peptitles was carried out by descending paper chromatography using the ethyl acetate-pyridinewater (10:4:3) system of Partridge (24), and the butnno-acetic acid-watrr (4: 1:5) system of Bradfield and Flood (25). Reducing carbohydrates were identified by aniline osalate spray (24), and peptides were identified by spraying with a 0.39; solution of ninhytlrin in acetone.

Electrophoresis Moving-boundary electrophoresis was plished in an .Intweiler microelectrophoresis paratus at 15°C. and 65 v.

accomap-

Ultracentrifzigation Ultracentrifugal analysis was performed with the Spinco model E instrument at 20°C. and 59,780 r.p.m. Sedimentation constants were calculated by the method of least squares.

414

BEZKOROVAINY R.ESULTS SOLVBILIZATIOS

OF PLATELET

GHOSTS

Lyophilized platelet ghosts appcnrcd as white flakes and were quite insoluble in water and neutral salt solutions. Wlicn the platelet ghosts were trcatcd with alkaline salt solutions or buffers, they became partly soluble. Dialysis of platclct ghosts against 0.05 M Verona1 buffer at pH 8.6 for 16 hr. at room temperature or at, 4°C’. resulted in the solubilization of 35’;; of the original material. The base-soluble material prccipitated n-lien the solution wax acidified or dialyzed against water. Solubilization al)pearcd t,o be due to a mild basic hydrolysis, and the presence of base-labilta bonds such as ester bonds was. therefore, suggested. Peptic digest’ of a platclct ghost l)rcparation was rcactcd with hydroxylamine before and after the mild alkaline llydrolysis in order to cst,imatc the number of “r&Y’ bonds broken. The base-treated material had 1.83 pequiv. of “est~cr!’ bontls,‘lng. X:. while the control had 2.86 ,quiv. of “ester” bonds/ 60 1

3’

/a~A’

A-A

HA-~*BLANK

0" 0

I

20

40

REACTION

/ , , 60 80 100 TIME

, 120

(m(n)

Fm. 1. Dinitrophenylation of platelet ghosts (40.8 mg.), human serum albumin (15.5 mg.), and the blank reaction. The reaction wssel contained the protein in 4 ml. water at pH 8.0 and 0.1 ml. PDSB. The temperature was maintained at 40°C. by circulating warm water through a jacket surrounding the reaction vessel. The pH was held constant by titration with 0.05 12’ NaOH from a mirroburet with a Beckman model CT pH-meter. Constant agitation w:w ~wrform~d with a magnetic stirrer.

APJD DOHERTY

mg. N. Thus, 367; of the “ester” bonds present were broken in solubilization of 35s’:, of ptatelc$ ghost material.

Although ttic base-solubilized platelet ghost material prowd to be water-insoluble, a water-soluble fraction could be isolated when they were first rcactcd with FDNB, and the DEP-platclct ghosts hydrolyzed as described. Figure 1 shows the progress of dinitrophenylation of platelet ghosts resulting in an uptake of 0.7 +~olc FDSB/mg. of platclct ghosts. Bftw removal of excess FDNB and tlinitrophenol by cthcr extraction, the DNP-platrlet ghosts wcw isolated frown the ucidificd reaction mixture by ccntrifugation, wasliecl ae~~~ralt,inics with 0.1 .\- HCl, suspcntlctl in tlic T’cronal buffer, dialyzed against the saiw buffer for 16 hr., and finally diulyzcd against distilled water. From I/$, to l/l of the original DSP-platelet ghost material provc~l to tw water-soluble as a result of t(his trcatlncnt. The soluble frwction will bc wfcrrctl to as the wtublr: DNPfraction and tliv insoluble one as the insotublc DNP-fract.ion. Treatnwnt~ of the insolublr DNP-fraction n-it11 the basic buffer resulted in it,b further solllbilization, honever! at a much sloww rate. The sotublc DSP-fraction was prccipitablt~ at pH vaturs below 5.25 (acctatc buffer, ionic stwngth 0.02)) and \vas l)rccil)itatctl with sodium sulfate at, a concentration of 0.16 g.-ml. \\‘trcn the soluble DSP-fraction was subjcctcd to c~lcctroptlol.~~si~ at w~wl alkatint~ l)H values, it lnovcd to thr l)ositiw pole :IS essentially one leak (Fig. 2,. In ttw ultracentrifuge, honcwr. tlwc pwks with sctlimentation constants of 5.8. 23.2 (major peak 1, and 29.9 at a total concentration of 8.3 mg./ml. W~I’Colwrvctl (Fig. 31.

N-terminal groups were identified in DNP-platclrt ghosts, tlw soluble-, and the in~olubtc DNP-fractions. The lat,ter two were sub,iwtcd to analy& n-it,hout further dinitrophenylation. 111 all thtw cases tt1c major DNP-amino acid found in the ether l)hase of t,trc acitl trydrolyzatc was DNP-

b

wrinc, :trcounting for 755; of total Stcrnlinal DNP-anho aci(L4 wcowwd in case of DNP-plntrlct ghosts and the ~olu~~h~ DNP-fraction, and 1005; in tmirc case of the insolulnlc DNP-fraction. T11c contnnlinating yellow iuat~erial was DSP-glutamir :wid. The nrcr:qg molrcular weight of ill<&

c

solul)lc DSP-flnction, calculatctl on tllc hsis of DKP-swine rccorered, was 2-3 x IO;‘, and 1 X 10” in tlw case of the insolul)le DNP-fraction. Aqueous phases of arid Iiycll’ol;vzatc~s of DNP-p1ate1ct g11osts, the solu~~l~~ DNP-fraction, and t,lic insolul)lc DNP-fraction were dinitrophcIl~lntct1 and

Soluble DNPfraction

8 .2 0.6 1.4 1.1 0.7 0.1

0.8-

2. 8 0.0 0 6 1.4 0 .3 0.2 0.8

4.1 0.0 1.0

1.1 0.5 0.2 0.7

39% of the original nit#rogen and 30% Of hexose; the controls gave 7.1 and 7.970, rcspectively.” The dialyzable fraction consisted of a rather heterogeneous mixture of peptidcs that were revealed in the ethyl acet,ate-pyridine-water and the butanol-acetic acid-water chromatography systems. The mixture contained peptide-bound carbohydrate that could be released by hydrolysis with the Dowex 50 cation-exchange resin opcrntcd at 100°C. for 1 hr. Hcxosaminc, gulactose, glucose, mannose, and methylpentosc were identified chromntographitally. Platelet ghost material was also susrcptiblc to digestion wit,11 pepsin at pH 2.0 and 4O”C., as shown by its partial xolubilization and the nppcarancc of dialyzable pcptidc material, which was identified by leaper chromatography. Both the soluble and the insoluble DKP-fractions were not susceptible to peptic digestion.

The elimination of charged amino groups from plat,elet ghosts by dinitrophenylation appears to stabilize the tertiary structure of the soluble components, probably by inhibiting random coil formation and conscquent lack of solubility in neutral solutions. It is probable, therefore, that the prccipitation of base-soluble platelet ghost components upon neutralization is due to a rcformation of salt linkages.

FVe wish to thank Dr. X. G, Anderson aud R. Canning for their nsistumcc in opwating ult wventrifuge.

1. 2. 3.

DISCUSSION

Structural elements of platelets appear to be composed largely of protein, as shown by direct nitrogen analysis, amino acid analysis, and digestibility by proteolytic enzymes. The carbohydrates, which account for 7.15 of the platelet ghosts, appear to be tightly bound to the protein, and the hexoses, hexosamines, and pentose are unavailable for pcriodate oxidation. The partial oxidation of sialic acid and uranic acid is certainly not sufficient to account for the total uptake of periodate by the platelet ghost material; and, therefore, it is necessary to postulate the presence of an unknown, periodatc-oxidizable structure in t#lic platelet ghosts. Important links in maintaining the structural integrity of platelet ghosts arc basolsbilc acyl bonds, which give a positirc hydroxamic acid test. Comparable treatment, of numerous esters rcsult,s in their liytlrolysis; it, is likely, therefore, that t,hc basclabile bonds in platelet, ghosts arc tstcr bonds. It appears that the degree of solubilization of platelet ghosts is directly connected with the rupt’ure of these base-labile bonds.

4. 5. 6. 7. 8. 9. 10.

11. 12 13

14 15 16 17 18

’ T’isking

casings,

1 cm. diamet,er.

Mr. tllc

418

BEZKOROVAINY

19. I”ISKE, c. H., AM) SUBBAROW, y., J. Bid. Chem. 66,375 (1925). 20. LOWHY, 0. H.. R~SEKBROGGH, S. J.. FARR, A. L., .~SD RAKI)ALL, R. J., J. Biol. Chenz. 193, 265 (1951). 21. \~EISSMAAT, B., AKD MEYER, K., J. Am. Chem. Sot. 76, 1753 (1954).

AND

DOHERTY

22. DIXOK, J. S., ARI) LIPKIS, I).. A//t/l. Clrrw. 26, 1092 ( 1954 ) 23. POPEA-OE, E. A., Biochim. c/ Hio$r!/~. Aclo 32, 584 (1959). 24. PARTRIDGF,, S. &I.. LVulwx 164, 443 (1949). 25. BRADFIELD. A. E., ASD Fr,oon. A. E.. I\‘rtlurc 166, 264 (1950).