Exp. Eye Res. (1975) 20,541-548

Further Studies on the Polypeptide Chains of 8-Crystallin* P. HERBRINK,

HANSKE

Depwtment

VAN

WESTREENEX

of Biochemistry, University The Netherlands

AND

H. BLOEMENDAL

of ,Vijm,eyen,

S$megen,

(Received 28 October 1974, Lordor,) The isolation and partial characterization described. Three of them can be obtained fi-crystallin substructure is proposed.

of four polypeptide chains of p-crystallin is both from j3~ and 6~. A nomenclature for the

1. Introduction In comparison with a-crystallin the subunit structure of bovine fi-crystallin has been lessextensively studied (Herbrink and Bloemendal, 1974; Bloemendal and Herbrink. 1974; Zigler and Sidbury, 1973). In fact the separation of a purified fl-polypeptide chain has, for the first time, been reported only recently (Herbrink and Bloemendal, 1974; Bloemendal and Herbrink, 1974). We have proposed to designate this polypeptide. which is shared both by /?u and &,, PB,. The fact that two proteins have a major polypeptide in common possibly indicates a closerelationship between the two species.In the present paper we provide further evidence for this interrelationship of the fl-crystallins by demonstrating that, in addition to the /3B, chain, three other chains are shared by flH and flL. Th ese chains have been isolated

from both p fractions.

Furthermore the acidic polypeptide from /2n has been purified. A partial characterization

of the chains is also given.

2. Materials and Methods Preparatiorb

of lens extract

The eyes of 2%s-month-old calves were obtained fresh from the slaughter-house and the lenses removed. After dissection of the epithelial cells the lens co&ices were dissolved in distilled water by gentle stirring. After removal of the nuclei the turbid solution was centrifuged at 15 OOOxg for 20 min. The clarified solution was dialyzed against water and lyophilized. The dry material, referred to as lens extract, was stored at 4°C. PuYi$cntl,on

of /3-orystallin

Purification of j?-crystallin was achieved by gel-filtration on Sephadex G-200 (Van Dam. 1967). The swollen material was equilibrated with O-1 M-Tris-HCl, pH ‘7.5, containing 0.05 v-NaCl and 0.001 M-EDTA. Samples containing 500 mg of lens extract in a volume of 5 ml were applied to the top of the column (100 x 4 cm). The elution rate was 20 ml/hr. The fractions were desalted by gel filtration on Sephadex G-25 in 0.1 N-ammonia and lyophilized. Polyacrylamide gel electrophoresis Polyacrylamide gels at pH 8.5 containing 6 M-urea were prepared according to Hloemeudal (1967). The final concentration of the acrylamide was 7.5%. The buffer used * Part (19741.

of this paper

has been presented

at the International

Congress

of Eye

Research,

Capri,

Italy

.X2

I’. HEltBl?IXlI-urea, pH 7.7, 0.02°/0 dithiothreitol. Elution was carried out at 4°C at. an average tlow r;tt,e of 24 trll/hr. The effluent was monitored at, 280 nm with an LKB Fvicord absorptiometer. Protein fractions were pooled, desaketl on Sephades G-Z and lyophiliaed. Amino

acid analysis

Amino acid analysis was conducted on a Beckman Multichrom automatic amino acid analyser using a single column. Samples of protein (0.2 mg) were hydrolysed under vacuum at 110°C with 6 M-HCl for 24, 48 and 72 hr. Cysteine was determined as cysteic acid after performic acid oxidation (Hirs, 1956). Tryptophan was measured according to Benzce-Schmid (1957). Peptide mapping Of the proteins 2.5 mg was dissolved in lo/i NH,HCO,, pH 8.9. Trypsin (U426 mg) was added and digestion wasallowed to proceedfor 2 hr at 37°C.At the end of this period the digestion mixture was frozen and lyophilized. The peptide mixture was dissolvedin

50 ~1 of water and subjected to high-voltage electrophoresis,which was performed on Whatman 3 MM at 47 V/em and 35 mA in pyridine-acetic acid-water (25 : 1: 225. by vol), pH 6.5, for 75 min. Thereafter descendingchromatography in n-butanolLacetic acidwater-pyridine (75 : 15 : 60 : 50. by vol) was carried out for 16 hr. After drying the peptides were detected with a 0.2% (w/v) ninhydrin solution in acetone containing l”h (v/v)

pyridine and l”/;, (v/v) acetic acid. 3. Results Nomenclature Previously

of the /I-chains we proposed

to subdivide

the P-crystallin

chains

into

a basic and an

acidic group indicated by the capitals B and A behind the symbol/3 (Bloemendal and Herbrink, 1974) (Fig. 1). This is in accordance with the generally accepted nomenclature of the cc-crystallin chains aA and cxB(Bloemendal, 1972). A further subdivision of u-chains was based on their electrophoretic behaviour in alkaline polyacrylamide urea gels. At this moment a similar approach for the /3-crystallin chains seemsto be less meaningful since t,he interrelationship between the individual chains is not yet fully understood. One should also keep in mind that a few polypeptides occur only in minor quantities which even may vary in different experiments. For these reasons we suggest designating the bands, with exception of PA-, in correspondencewith their position in the elution profile from DEAE columns (compare Fig. 3).

POLYPEPTIDE

CHAIKS

OF

B43

,B-CRYSTALLIN

Fro. 1. Polyacrylamide gel electrophoresis of PH and FL. Acrylamide gels, 7.5%, were described elsewhere (Bloemendal, 1967). Electrophoresis was performed at pH 8.5 in a buffer 6 ~-urea and 100 mg dithiothreitol per liter. After a pre-electrophoretic run for 1 hr samples 100 ue of protein were layered on ton of the uel and subjected to electrouhoresis for 5 hr at 2 Gels with Cfoomrtssie bkliant Ike accordi”ng to Mslik and Berrie (1972).

0

20

40

60

80

100

120

140

160

277-----

(b)

Sot0

/& 20

I

\/

40 Fraction

60

80

100

number

FIG. 2. Chromatography of /la (a) and &, (b) on DE-52. /3= and &, were applied to a (40.0 y 1.6 cm), equilibrated with 0.005 hl-Tris-HCI, pH 7. 7, containing 6 ix-urea and t,hreitol. A linear gradient was applied ranging from 0.006 to 0.030 M-Tris-BCI, pH 7.7, 0.02% dithiothreitol. Elution was accomplished at 4°C at 24 ml,‘ht,. The fraction volume

pzcrijicntion

of

the

prepared as containing containing mA per gel.

DE-52 column 0.02% dithio6 M-urea and was 5 ml.

PB2,/3BI,/3B5md /IA chins

Fractionation of these chains has been achieved by chromatography of /lH and #JL on DE-52. The separation of the polppeptides has heen improved as compared to the

FIG. 3. Basic polyacrylamide gels of the purified legend of Fig. 1. Twenty pg of the purified chains * The symbol

j3B, stands

for the highly

chains of flrr and &,. Conditions were layrred on top of the gel.

basic polypeptide

chains

as indicated

in the

of PH.

fractionation reported previously (Herbrink and Bloemendal, 1974) by using a longer column (40-O x 1.6 cm) and a less steep gradient (O-005-O-030 M-Tris-HCl, pH 7.7. containing 6 M-urea and O*O2o/0dithiothreitol). Typical elution diagrams are shown in Fig. 2. The numbered fractions were analysed on basic urea gels. The third peak

FIG. 4. Sodium dodecyl sulphate gel electrophoresis of the purified chains of fl-crystallin. Thirteen per cent aorylamide gels were prepared according to Laemmli (1970). Eleotrophoresis was performed at 5 mA per gel. Gels were stained with Coomassie brilliant blue. The numbers 1, 2, 3 correspond to the fractions obtained after DEAE chromatography [compare (a) and (b) of Fig. 21. In SDS gels )BB5 migrates as j3B, and /~BP as ,3B4.

POLYPEPTII)E

(‘HAINS

OF

/J-(‘RY

646

STALLIS

usually consists of pure flB, chains but occasionally flB, is eluted together with the /3B, chains. If this is th e case /3B, can be isolated by rechromatography using a linear gradient ranging from O-005 to 0.020 M-Tris-Hcl, pH 7.7, containing 6 M-urea and 042~/, dithiothreitol. The PB, chain is obtained pure by rechromatography of the TARLE Amino

Amino

acid

of the /3 ch~ains

(nld

7.7 -‘.(j* 7.1* 13.6 3.0 9.1 A.0 14t 6.21 0.9 2-s: 7.3 3.8 4.2 6.1s 4.2 1.6 84

peptides

ft,om the j?B,

I’(,)

X.3 3-t* Y-S* 1.i.h 6.8 9.3 4.2

Extrapolated to zero t.ime hydrolysis. Estimated as cyst&c acid. Values for 72 hr hy’drolysis. Determined according to Benzce-Schmid

5. Map of thr: tryptic one map is shown.

FIG.

only

analysis

(nlr,l “J

Asparke acid Threonine Serine Glutamic acid Proline Glycim: Alanine Cyst&e Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lgsine Histidine Arginine * T $ s

mid

I

PA (mol sb) ti.3 :l.i* s.4* 15.4 34 IO.1 t5.8

1 of

I .9?

7.5f 1.0 3.11 54 44 441 :i.og 6.0 4.1 5.1)

tie3~ lb8 L’3f 6.4 3.6 645 :1.6g 3.2 3.1 7.0

(19%).

and /3B, chain.

Since both tingerprints

are identica,

Po~~/f/~r~~ztrltl~it~f~

qd

f’lf’cfrol/l/ot’f’sr:.~.

.??I1

prifietl

chains

were

huhjecked

t(J

gd

dect~ro-

phorwis on ljasic ~~olyawylm~ide gels containing 6 wurea. The result is shown irL Fig. 3. The separated chains have also been analysed by SDS gel electrophorttG\ (Pip. 4). It, is clear that the first peak elutecl after chromatography of /3,%on DE-Z!. consist’s of the two high-molecular weight chains of PH. The /3B,, chain as ~11 as BP, has :L lrwlccubr weight of approximately r 47 000 Daltons whereas PR, and flB5 reveal i\. n~olccrd:~r weight of approximately 30 000 Daltons. The /IA chain has a molecular weight of 25 000 Daltons. These molecular weights were obtained irrcspcti\-e of whether the chains wcrc &rived cit,her frorrl /3,, Jr p,,.

FIG. 0. Map of the tryptic

peptides

from the ,!?BP- (a) and the fiB,-chain

(111.

I’OLYPEP?‘II~~

(.‘HAlXS

OF

ST

/3-~~‘r,\-siraLL1S

A rr/ir~ crcici!crr~lysis. Hydrolysis of the p chains was carried out for 24, 48 and 72 hr. respect i\-rl\-. The amino acid composition of the different polypeptides is depicted in Table I. No difference was fount1 in the amino acid composition of the corresponding chains of PH :tnd /$,. Moreover ,LIB, am1 ,i3B4. as well as /3B, and ,f3B, $elcl also the same aluino acid composition. 7’~!/+: pptirlv w~~#~ag. The tryptic peptide map of PB,, is shown in Fig. 5. Again no tliR’&~~nce was found -Then this map was compared to t’hat, of /3B4. The peptidr tniips

of /3B, and /3B, (F ig. 6) differ

mainly

in. t’wo spots

(dashed

arrows).

The

tliffitrenct: in electrophoretic behaviour betKeen these spots might well be due t,o :I tli~~~rencein two acidic groups. The tryptic peptide nlap of /3$is shown in Fig. 5.

t

4. Discussion ()ur finding that /In and /IL share, in addition to PB,,. three other chains favours the idea that the P-cry&all& might be less complex than thought previously. A close of the r&t,ion between a number of chains is also suggestedby the st.riking similarity corresponding peptide maps. Moreover it would not be surprising if a certain degree of homology existed between the different chains. As much as 55% of homology has recently been found in comparative sequencestudies between GA, and crB, chain5 (Van der Ouderaa, De Jong, Hilderink and Bloemendal. 19i4). but further investigations, especially sequencestudies. are neededin order to enable definite statement,s conwrning

the homology

of the difFerent

/3-crwtsllin

chains.

The authors wish to thank Miss M. Verst,ec,n and JIr G. Groenewoud for performing the anli~bo acid analyses and Dr W. de Jong for raluable Iliswssiou. The present investigation, 11aw pnrtlp been carried out under auspices of the Set,herlands Foundation for Chemical Research (SO?;) and with financial aid from the Netherlands Organization for the Adwncement of Pure Research (ZWO).

Jienzre. IV. L. and Schmid, K. (1957). Determination of tyrosine and tryptophan in proteins. Anal. Chem. 29, 1193. Hloemendal, H. (1967). Electrophoresi.~. Theory, Nethodn’ and Application (Ed. Bier. >I.). \‘ol. 2. pp. 379422. Academic Press, View Stork. Bloemendal. H. (1972). Alpha-crystallin. Sructural asperts and biosynthesis. dcfrr Morphal. iVeerl.-Scand. 10, 197. Bloemendal, H. and Herbrink. P. (1974). Growing insight into the structure of ,8 c*rystallin. Ophthal. Res. 6, 81. Herbrink, P. and Bloemendal, H. (1974). Studies on /3 crystallin. I. Isolation and partial characterization of the principal polypeptide chain. Biochim. Biophys. Acta 336, 370. Hirs. C. H. W. (1956). The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219, till. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature (Lo~~don) 227, 680. Malik. K. and Berrie, A. (1972). IGw stain fisa,tive for proteins separated by gel isoelectric, focusing based on Coomassie brilliant blue. r-ltw/Z. Biochem. 49, 173. Van Dam, A. F. (1967). Ooglenseiwitt.en. lsolering en Karakterisering. Thesis, University of Xijmegen, Kijmegen, The Netherlands. Van der Ouderaa, F. J., De Jong, 11:‘. IV., Hilderink, A. and Bloemendal. H. (1974). The amino arid sequence of the aB, chain of bovine sc-crystallin. Eur. J. Biochem. 49, 157. Zigler, .J. S. and Sidbury, .J. B. (1973). Structure of calf lens fi-crystallin. Esp. Eye Res. 16, 207.

Note Added in Proof Recent sequence studies on /IBp revealed that the C-terminal sequence is not Arg-Ser, as published earlier (Herbrink ;tnd Bloemendal, 1974) but Ser-Ser.