Demonstration of glycoproteins which are associatedwith chromain nonhistone proteins

Int.

3.

Biochem.,

1973,

4, 345-348.

DEMONSTR,4TION ASSOCIATED

WITH

SEVALJEVI6: Institute

[Scientechnica

(Publishers)

OF GLYCOPROTEINS CHROMATIN

Ltd.]

WHICH

NONHISTONE

LJILJAiiA

AND

KRTOLICA

for Biological

Research,

(Received 12 January,

Belgrade,

ARE

PROTEINS

KOVILJKA Yugoslavia

x973)

ABSTRACT

I. Chromatin

nonhistone proteins were prepared from sea urchin embryos at different stages of development, and electrophoretically separated on acrylamide gel slab into about 20 fractions. 2. The presence of glycoproteins was revealed by the PAS reaction. 3. The carbohydrate moiety was further identified as mucopolysaccharide of hyaluranic acid type. 4. No developmental alterations of protein-bound carbohydrates have been observed.

THE class of acid-insoluble chromosomal proteins, termed ‘ nonhistone ’ (NHC), includes large varieties of the species which have been implicated as having functional or structural importance in DNA metabolism and expression. However, closer examination of the nature and functions of the constituents has been hampered by their high insolubility. Therefore most of the available information came from the electrophoretic separation of NHC proteins solubilized in sodium dodecyl sulphate. This paper reports an attempt to detect the glycoprotein components among the NHC proteins separated by this method, as well as to ascertain whether they are stage-specific. MATERIALS

AND METHODS

All studies were carried out on sea urchin embryo chromatin prepared according to the slightly modified method of Marushige and

Ozalu (1967).

Mature epes of Paracentrotus lividur were obtained by gently &ring excised gonads in natural sea water. Embryos were grown to the desired stage at 19-2 I’ C. At three developmental stages (blastula, gastrula. and pluteus) embryos were exposed to I &ml. of [Hs]amino-acid mixture (New England Nuclear) for 50 minutes. Embryos were then collected by low-speed centrifugation at 4” C. and washed three times with 50 ml. of cold sea water. Chromatin was prepared following the procedure of SIarushige and Ozaki (1967) with alterations concerning the shearing of the preparation.

Following the purification through I ‘7 M sucrose. chromatin was sonicated for I minute in IO KC Ravtheon Sonic Oscilator Model DF-101, and centrifuged at 18,000 r.p.m. for 3 hours. The supernatant was referred to as ‘ chromatin ‘. -The DNA was determined by the method of Burton ( 1gj6), the RNA by the method of Munro and Fleck (I~~cJ), and protein by the method of Lowry, Rosebrough, Farr, and Randall ( 1951). To prepare NHC protein the chromatin sample was treated as described by Elgin and Bonner (1970). Histones were extracted from chromatin with o-4 J~H?SO~. and nucleononhistone complex was pelleted by centrifugation at 10,000 r.p.m. for 30 minutes. The pellet was suspended in I per cent SDS-o*05 .M Tris,pH 8. and stirred overnight at 37’ C. After the dialyses against 0.1 per cent SDS-o.o I 1M Tris, PH 8, the DNA was pelleted by centrifugation at 36.000 r.p.m. for 36 hours at 25’ C. in a Spinco SW-50 rotor. The supematant containing NHC proteins was dialysed against 0.1 per cent SDS-~-I per cent mercaptoethanol-ro per cent glycerol in 0.01 M sodium phosphate buffer, pH 7-1, and used for acrylamide gel electrophoresis. Disc gel electrophoresis was performed in the presence of 0.1 per cent SDS (Weber and Osbom. 1969). The gels were 7 per cent acrylamide. A 200 pg. aliquot of protein containing I ~1. of tracing dye (0.05 per cent bromphenol blue in water) was submitted to the electrophoretic separation at a constant current of 8 mA per gel. The tray buffer was 0.1 M sodium phosphate, pH 7.0, containing 0.1 per cent SDS. To test the effect of chemicals on the staining for glycoproteins, the ’ blank ’ gel containing all reagents except the protein sample was prepared and submitted to electrophoresis.

LJILJANA

346

AND

Gel was stained for glycoprotein by the PAS reaction as described bv Zacharius, Zeli. Morrison. and IVoodlock (~969): It was immersed in 12.3 per cent trichloroacetic acid for 30 minutes CI ). rinsed with distilled water jz), then immersed in I per cent periodic acid (made in 3 per cent acetic acid) for 50 minutes (3). The next washing was in distilled water, overnight with a few changes (4). Gel was immersed in sulphite stain in dark for 50 minutes (jj, washed with freshly prepared 0.5 per cent metabisulphite (3 Y xo mmutes), and in distilled water until excess of stain is removed. Gels were scanned at 500 nm. on Gilford Spectrophotometer Model 2400-S. Gels were stained for protein with solution containing 0.25 per cent of Coomassie brilliant blue, 50 per cent of methanol, and g per cent of acetic acid, and destained in 5 per cent methanol7 per cent acetic acid. ’ -To compare the patterns of PAS and protein staininp within single gel, the PAS-stained gel was scanned at 500 r&r., ‘then re-stained for protein with Goomassie blue and re-scanned at 6oo nm. The relative mobilities of stained fractions were calculated as : ssobilitv_distance

of protein migration

x

length after staining length before staining distance of dye migration’ Determination and electrophoretic separation of acidic mucopolysaccharide (AMPS) was carried out as described by Stefanovich and Gore (1967). Protein was digested with activated papain solution and fractionation of isolated AMPS was achieved on an Ecteola cellulose column. Elution was done with a solution of 3 LzI NaGl in 0.1 j\r HCI. Uranic acid in eluate was determined bv the orcinol method. Electrophoretic separation of mucopolysaccharides was performed on cellulose polyacetate strip (Sepraphore III), using a buffer ofcopper acetate and glacial aceticacid,pH 3-6, at a constant current of 0.5 mA/cm. for 2 hours. The strips were stained with alcian blue (0.1 per cent of dye in 5 per cent acetic acid and IO per cent ethanol) and scanned on Universal Densitometer Elektrofor, using red colour filter. The radioactivities of glycoprotein fractions were expressed as the percentage of total c.p.m. of proteins which entered the gel. The gel was cut into r-mm, slices and each slice was dissolved in 0.5 ml. of 30 per cent hydrogen peroxide. To the clear sample solution, 12 ml. of the mixture containing 8 ml. of toluene scintillation liqxid and 4 ml. of methylcellosolve were added. Counting was done by a Beckman Scintillation Counter.

RESCLTS

iWD

DISCUSSION

Electrophoresis of chromatin SHC on SDS-acryl-amide gel resulted

protein in the

KOVILJGA

hf.

3. Bmhem.

appearance of 20-24 bands (Fig. xi. Two fast-moving major bands, I 8 and I 9, involved about one-half of total protein, while the slow-moving protein species appeared as a set of close and very weakly represented bands. We have shown previously that this pattern did not alter significantly during the development of sea urchin embryos from blastula to pluteus stage. However, the pattern of the relative rate of biosynthesis of protein fractions was found to exhibit developmental alterations (SevaljeviC, manuscript submitted for publication). Staining of the ge1 for carbohydrate by the PAS procedure resulted in the appearance of two very weak bands and one intense band in the region of the fastest moving protein fractions (Fig. I). They were eluted from the

gel by proiongation of the electrophoresis until the fastest moving fractions were eluted into a dialysing bag fastened around the lower part of the tube and immersed into a tray buffer. The presence of acidic mucopolysaccharides in this eluate was detected by the method described under Materials and Methods. They were then isolated from the whole NHC protein sample and submitted to electrophoretic separation on a cellulose polyacetate strip. The predominant fraction on the electrophoretogram was identified as hyaluronic acid (Fig. 2). No detectable peaks in the region where heparitin sulphate and chondroitin sulphate would run have been observed. The more intense staining reaction was always seen just ahead of the tracking dye. The relative mobility of this rather broad band was about 1.17. It coincided with the fastest moving band stained with Coomassie blue. Although protein forming this band was minor with respect to protein entering the gel, it was the major glycoprotein with respect to the protein-bound carbohydrate. Its appearance as a broad, sometimes spiit, diffuse band, reveated the heterogeneity of the material. The stage-related alterations were not observed either in the carbohydrate or in the protein moiety of this fraction. .%t all investigated stages of development about 6 per cent of the total radioactivity was found in this fraction.

CHROMATIN

NONHISTONE

PROTEINS

FIG. I .-Electrophoretogram and the pattern ofdistribution of radioactivity (-O---O_) of chromatin protein from embryos at gastrula stage. Continuous line, Protein stained with Coomassie blue; Broken line, Carbohydrate stained by the P.4S procedure. Abscissa: relative mobilitia of protein fractions.

FIG. a.-Electrophoretic pattern of acidic mucopolysaccharides isolated from chromatin NHC protein and separated on cellulose polyacetate strip. Single arrow indicates origin; double arrow where hyaluronic acid would run in this system. Two other PAS-positive bands were very weakly represented. They coincided with two major protein fractions, 18 and 18.

Developmental alterations were exhibited by protein moieties only. At the early blastula stage the band 19 involved about 30 per cent of total radioactivity, while at the gastrula stage this value dropped to about IO per cent. The, radioactivity of protein forming band I 8 was found to increase from 21 to 28 per cent in the period between the early and the late blastula stage, while at the later stages a drop to the value of 18 per cent was observed. The PAS staining was very discrete at all investigated stages of development. This argues against the significant stage-related quantitative alterations of the carbohydrate moieties, but still does not prove the absence of the minor changes. The polyanionic nature of mucopolysuggests that the H,SO,saccharides insolubility of the glycoproteins was due rather to the carbohydrate than to the protein moiety of the complex. Therefore these proteins, at least those forming band 20, cannot be considered, on the basis of the acid-insolubility of the complex, as acidic

348

LJILJANAAND KOVILJKA

ones. two

Xmino-acid major

of rat liver ?;HC bands contained (Elgin finding

that

proteins, showed that these primarily acidic proteins

the did

alterations

forming

on the elec~rOphoretogram

and Bonner,

these fractions specific

analysis of protein

peaks

I g7oj. This, as well as our carbohydrate not respond

of protein

moieties

of

to the stage-

moieties,

suggest

that glycoproteins and two major NHC fractions are different species which exhibited the same electrophoretic mobiiities. Among the number of roles in which the mucopolysaccharides are implicated is that of mediator of the specific cellular interaction. Hyaluronic acid was found to be one of the constituents of the ground substance. It is a large random coil polymer, able to exhibit the rapid transition from viscous to elastic behaviour. The main glycoprotein forming band 20 was found to be stage-unspecific. If its presence was not an artifact due to the contamination of chromatin preparation by matrix material, then what kind of properties may it confer upon chromatin function and structure? While this question remains to be answered by some more amenable experimental approach, one may speculate that mucopo~ysacch~d~ play some role in the phenomenon of chromatin condensation and extension, as well as that they might, owing to their polvanionic nature, compete with DSA for basic proteins.

ACXNOwLEDGEMENTS \Se gratefuli>acknowledge tifiss Slilica Levental for the helpful discussion concerning the mucopolysaccharide determination and ,Lliss Borka 112 for her technical assistance. REFERENCES

BURTON, K. (x956), ’ A study of the conditions

and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid ‘, B&hem. 3.. 6z, 3Ij-322. ELGIN, S., and BONNER, J, (1g7o), ‘ Limited heterogeneity of the major non-histone chromosomal proteins ‘, Biochemistry, s 4440-4.446. LOWRY, O., ROSEBROUGH,N. J., FARR, k L., and RANDALL, R. J. ( rg5r ), ‘ Protein measurement with the Folin phenol reagent ‘, 3. biol. Chem., 193,265-27j. MARUSHIGE, K., and OZAKI, H. (1967)) ‘ Properties of isolated chromatin from sea urchin embryos ‘, Deal B~oL’.~ 16, 47&38. MUNRO, N. H.. and FLECK. rl. i1g6g), in .Vefhod in B~ochemi~o~ Analysis (ed. Glick), vol. XIV. p. rjg. New York: 1Viley. STEFANOVICH, V.. and GORE, I. i 196jj, ’A micromethod for the determination of acid muconoivsaccharides in vascular tissue ‘. _7. ~h~o~~o~., 3x,473-478. WEBER. K.. and O~BORN. M. i r&c+). ‘ The reliability of’ molecular weight’ deter&nation by dodccyl sulfate-polyacrylamide gel electrophoresis ‘? 3. bicl. Chem., w ++06-44x2. ZACHARIGS, R., ZELL, T., MORRISOX, J., and WOODLOCK, J. (196+), ’ Glycoprotein staining

following electrophoresis on acrylarnide gels ‘, Anal. Biochem.. 30, 148-152.

Kq CtTord Index: Glycoproteins. chromatin, ~arace~iro~~ iiuidw, nonhistone proteins.