Partial characterization of five glycoprotein fractions secreted by the human parotid glands

Archr oral Bid

Vol.

19. pp. 921 to 928

Pcrgmmn

Press 1974 Prmted

in Great

Bntain

PARTIAL CHARACTERIZATION OF FIVE GLYCOPROTEIN FRACTIONS SECRETED BY THE HUMAN PAROTID GLANDS P. ARNEBERG Department of Microbiology, Dental Faculty, University of Oslo, Oslo, Norway Summary-Isoelectric focusing of human parotid saliva in gradient pH 7-10 gave a major glycoprotein fraction at pH above 10 (pl > 10 fraction), a minor pl 9.5 fraction, and low amounts of glycoproteins isoelectric around pH 9 (~19 fraction). Subsequent gel filtration (Bio-Gel P-100) separated the pl > 10 glycoproteins into three subfractions (I, II, III), whereas the pl 9 and 9.5 fractions appeared as excluded peaks. The three pl > 10 subfractions and the pl 9.5 fraction were subjected to equilibrium centrifugation in the ultracentrifuge. The pl 9.5 fraction was heterogeneous (mol. wt 30,00~70,000), whereas the three pl > 10 subfractions appeared more homogeneous. Their molecular weights were 18,000, 11,500 and less than 10,000 for I, II and III, respectively. The carbohydrate content decreased by increasing pl of the glycoproteins, and ranged from above 50 to below 5 per cent of the total glycoprotein weight. The main monosaccharides in the fractions were identified as glucosamine, glucose, galactose, mannose and fucose. Amino acid analyses showed that, in all glycoprotein fractions, proline, glycine and glutamic acid (at a ratio 2: 1: 1) accounted for 7%30 per cent of the total residues, and the basic residues (lysine and arginine) for a further l&12 per cent. The fraction of pl > 10 had a higher lysine/ arginine ratio than the fractions of lower pl. The similarity in amino acid composition indicates that all these glycoproteins are related.

INTRODUCTION The human parotid gland secretes a number of proteins characterized by a high content of proline (Armstrong, 1971). These include basic glycoproteins (Mandel, Thompson and Ellison, 1965; Levine, Weill and Ellison, 1969; Andjic, Bonte and Havez, 1970) as well as acidic proteins that contain little or no carbohydrate (Bennick and Connell, 1971; Oppenheim, Hay and Franzblau, 1971). While the acidic mucins secreted by other salivary glands have been extensively characterized in several species, as to composition, structure and biological activities (see Gottschalk, Bhargava and Murty, 1972) less is known about the function of the proline-rich proteins secreted by the parotid gland. Amsterdam et al.‘( 1971) have put forward a hypothesis that these proteins are derived from intracellular transport membranes in the parotid acinar cell. Armstrong (1971) suggested that they contribute to the formation of the acquired tooth pellicle. The present study was designed to gain more information about the glycoproteins in the human parotid secretion. Human parotid secretory proteins have previously been separated by isoelectric focusing into four main groups (Arneberg, 1972). The two basic groups had pl > 10 and pl 9910, respectively. They comprised about half of the total protein and contained most of the hexosamines of the secretion. They

were, therefore, assumed to include the principal protein fractions.

glyco-

MATERIALS AND METHODS Parotid saha. Collected from a 37-yr-old male at a flow rate of 2-2.5 ml/min/gland, as described previously (Arneberg, 1971). Isoelectric focusing. Carried out as described (Arneberg, 1972), except that carrier ampholyte of pH 7-10 (Ampholine@, LKB, Bromma, Sweden) was used in most experiments to increase the resolution in the basic part of the pH-gradient. Sucrose and carrier ampholytes were removed from the fractions by gel filtration (Arneberg, 1972). In one experiment, sucrose and carrier ampholyte were partially removed by negative pressure filtration (Sartorius apparatus 163 11 with collodium bag 132 000) and further purification was carried out by gel filtration as described below. The Sartorius apparatus was also used to concentrate protein solutions. Gel filtration. Further purification of glycoproteins was carried out at 2@22”C in 2.5 x 27-30 cm gel beds of Bio-Gel P-100 (lo&200 mesh) or Sephadex G100 (46120 mesh). The samples were eluted with 0.02 M Na-phosphate buffer pH 7, at flow rates of 0.51.0 ml/min. 921

922

P. Arneberg

One glycoprotein fraction was tested for aggregations by rechromatography on the same G-100 column after incubation for 1 hr at 37°C in 8 M urea, or alternatively in 0.1 per cent of Tween 20@ (Koch-Light Laboratories, Colnbrook, Bucks., U.K.). The samples were applied as a 0.1 per cent glycoprotein solution. Urea or detergent was not added to the eluant. Protein. Determined by OD,,,,, using the factor OD;.;;, = 15.0 for the calculation of protein amounts (Arneberg, 1971). In amino acid analyses, an average residue weight of 110 was assumed for the calculation of protein recovery. Hydrolysis of glycoprotein. Carried out at 105°C with an excess of HCl in tubes that were evacuated, twice flushed with nitrogen and sealed. For amino acid analyses, 6 N of acid was used. For thin-layer chromatography, galactose and glucose determinations, hydrolysis in 1.5 N of acid was carried out for l-2 hr. The acid was removed in a rotating vacuum evaporator. Antino acid composition. Determined with a Technicon Autoanalyser, using a Chromobead type B resin. The standard (1%hr) procedure was followed. A Technicon type AAG integrator/calculator was used for quantitation. No correction for degradation was made. Hexosamines. Identified and quantitated on the Autoanalyser by including glucosamine and galactosamine in the standard runs and extrapolating to zero hydrolysis time. Qualitative carbohydrate analysis. Performed by TLC on cellulose plates (20 x 20 cm) (MN 300 from Macherey. Nagel & Co., Diiren, Germany). The dried residues of the hydrolysed glycoprotein samples were dissolved in pyridine. Samples of 5-10 ~1 were applied to the plate which was twice developed by the ascending technique, using a solvent of ethyl acetate, acetic acid, pyridine and water (5: 1:5:3, by vol). Reducing substances were detected with the reagents described by Trevelyan, Procter and Harrison (1950). Table 1. Molecular

weight determination

of basic parotid

-

Rotor speed (rev/min x 10e3) Protein cone (mg/ml) Temperature PSVt (ml/g) Slope (log con@*)

12 1.5 3’C 0.67 curved

Mol. wt

7o,OOt& 30,000

Zmg/ml--

PROTEIN

pH-grad,e”t I mghl--

-PI9

No10

Fracflons

-

No.20

of 5m,

Fig. 1. Isoelectric focusing of 50 ml parotid secretion on a 440-m] column with I per cent of carrier ampholyte (pH 710) for 48 hr at maximum 600 V. The protein concentratior after purification of each fracwas determined by 013~I 5nm tion on a Sephadex G-50 column. Pooling for subsequent amino acid carbohydrate analyses is indicated. Fraction No 15was lost.

Quantitaticr carholzydrate analyses. Total neutral carbohydrate was determined by the phenol/suIphuric acid method (Dubois et al., 1956), with glucose as a standard. Fucose was measured by the cysteine/sulphuric acid method (Spiro, 1966). Glucose and galactose were determined after hydrolysis with specific oxidases (Biochemica test@, GOD-Methode, Boehringer Mannheim GmbH, Mannheim, Germany, for glucose, and Galax@, Kabi, Stockholm, Sweden, for galactose). Sedimentation equilibrium runs for molecular weight determination. These were carried out in a Beckman Model E analytical centrifuge equipped with Schlieren optics. Prior to analysis, the protein solutions were concentrated 3310 times by negative pressure filtration as described above. One component was not retained secretory

glycoproteins

Glycoprotein

pl 9.5

TOTAL

I 20 2.5 20°C 0.70 straight

18,000

by equilibrium

centrifugation

fraction pl > 10 II 20 1.5 20°C 0.71 straight 11,500

III* 28 1.0 20°C 0.71 inversely curved$ < 10,000 > 5000

* This component had been concentrated 10 x by freeze drying. It was therefore analysed in 0.2 M buffer, whereas in the other experiments 0.02 M (sodium phosphate pH 7) was used. t The partial specific volumes were calculated from the amino acid and carbohydrate composition (Figs. 3 and 4, Table 2). $ Slope decreasing with distance from rotor centre.

Basic parotid secretory glycoproteins

923

Protein cone.

1

0.5 mglml

Fractions

10

20

t C&40

30

40

50

ml)

t (V, 137 ml)

Fig. 2. Glycoproteins of pl > 10 fractionated on a Bio-Gel P-100 column. A fraction (5 ml) of pH 10.6 was obtained by isoelectric focusing (pH 7-10). Sucrose and carrier ampholytes were removed by negative pressure filtration. During this procedure 5 ml of phosphate buffer (0.02 M, pH 7) was added twice. A final sample of 4 ml was applied to the P-100 column.

by the bag and was therefore concentrated ten times by freeze drying. The molecular weights were calculated according to Lamm (1929). RESULTS

Isoelectric

jbcusiny.

For 48 hr at maximum 600 V in the pH range 7-10 gave one major (pl > 10 fraction) and one minor protein peak (pZ9.5 fraction). Low Table 2. Characteristics

amounts of protein were found at 8.69.2 (pl 9 fraction) (Fig. 1). Gel jiltration. The pl > 10 fraction on Bio-Gel P100 gave one excluded (I) and two retarded subfractions (II and III) (Fig. 2). With the pl 9 and 9.5 fractions, no further separation was obtained. Both fractions appeared as narrow excluded peaks. Similar results were obtained on a Sephadex G-100 column, although for the pl > 10 fractions slightly

of basic parotid secretory glycoproteins. pl-values, and carbohydrate composition Glycoprotein

Gel filtration K,fi, Bio-Gel P- 100 K,, Sephadex G-100 Ultracentrifugation Mol. wt Hexosamine residues per 100 amino acids Glucosamine Galactosamine Neutral carbohydrates? Total (y<,) Galactose (y/i) Fucose (‘;3

gel filtration

data, molecular

weights

fraction

PI 9

pl 915

I

pl > 10 II

III

0.0 0.0

0.0 0.0

0.0 01

0.2 0.4

0.4 0.6

70,00@ 30,000

18,000

11,500

< 10,000 >5Oc0

11.5* 0.2

1o.o* 0.2

0.5 0.0

0.0 0.0

0.0 0.0

54

32, 38$ 4.6, 5.5 6.0, 5.6

16 trace trace

2.5 trace trace

2.5 trace trace

* Values corrected for degradation (uncorrected values of the pl 9 glycoprotein: 24-hr hydrolysate 8.3, 4%hr 5.0; of the pl 9.5 glycoprotein: 24-hr hydrolysate 7.4, 4%hr 4.7, 72-hr 2.7). t Carbohydrates (percentage of Cbh by weight) calculated by: (Cbh x lOO)/(protein + neutr. Cbh). Protein was determined by ODz , 5nm. $ Purified from different samples of parotid saliva.

P. Arneberg

924

40 E2 SUBFRACTION

I I&,

18000)

H

SUBFRACTION

II IM,

0

SUBFRACTION

111 (10000~M,>5000)

*

TRACES

I

B

11500)

E

i

$0 3 e %

_

r

secretory glycoprot&s. Samples from three glycoprotein components after isoelectric focusing were hydrolyzed. Two hydrolysates (24 and 48 hr) of the ~19 component and three hydrolysates (24, 48 and 72 hr) of the pl 9.5 and of the pl > 10 components were analysed. The range of values is indicated (vertical bar).

Fig. 4. Amino acids in basic parotid secretory glycoproteins. The glycoprotein component of pI > 10 was separated into three subfractions by gel filtration (Fig. 2). One sample from each subfraction was hydrolysed for 24 hr and analysed. For comparison the range of values from analyses of an unfractionated pl > 10 component (Fig. 3) is indicated by vertical bars to the right.

higher K,,-values were found (Table 2). On this column, subfraction I had an elution position similar to that of human serum albumin. Samples of subfraction I gave a single peak of the same &-value after treatment with disaggregating agents. Equilibrium centr@gation. The pl 9.5 component revealed size heterogeneity with molecular weights ranging between 70,000 and 30,000. The molecular weights of subfractions I and II of pl > 10 were 18,000 and 11,500, respectively, and both were homogeneous in this system. For subfraction III, the molecular weight was lower than 10,000 and higher than 5000, but, as the slope of the curve decreased towards the bottom of the cell, its definite molecular weight could not be established (Table 1). Amino ucid compositiorz. Glycoproteins from the three different levels of the pH-gradient were similar

(Figs. 1 and 3). Proline, glycine and glutamic acid, accounting for 7&80 per cent of total residues, appeared at a ratio of 2: 1: 1. All three samples contained also 11 per cent of the basic amino acids lysine and arginine, as well as 4-6 per cent of aspartic acid and of serine. Compared with the pl 9 and 9.5 fractions, the pl > 10 fraction had a higher lysine/arginine ratio and a higher alanine content. Furthermore, this most basic fraction contained only the eight amino acids mentioned, whereas the pl 9 and 9.5 fractions had low amounts of five additional amino acids, i.e. threonine, valine, iso-leucine and histidine (Fig. 3, Table 5). Trace amounts of some or all the last-mentioned amino acids were detected in separate analyses of the three pl > 10 subfractions, purified from another parotid saliva sample. In all other respects, the amino acid

Fig. 3. Amino acids in basic parotid

Table 3. Protein

load and amino acid recovery in amino acid analyses

PI 9

pl 9.5

“pooled”

pl > 10 I

pl > 10 II

pl > 10 III

0.40

0.54

0.75

0.61

0.38

046

6.3 (1667;) 4.7 (130%)

5.8 (118%) 2.7t (110%) 26t (105%)

3.W (117%) 3.0t (89%) 2.2.t (65%)

5.5.t (99%)

1.5.t (87%)

2.1t (85%)

pl>

Fraction Protein amounts* per hydrolysate (mg) Recovery 24-hr Hydrolysis (,umole) 48-hr Hydrolysis

(pmole)

72-hr Hydrolysis

(pmole)

10

Percentage recovery from the Autoanalyser calculated by an average included. * Based on OD, 1snmvalues. t Only half of total hydrolysate was applied to Autoanalyser.

residue

of 110; carbohydrates

not

925

Basic parotid secretory glycoproteins Table 4. Amino acid composition of pl > 10 subfractions (residues per 100) Subfraction* Pro GUY Glx Asx Ser Lys Arg His Leu Ala Val Be Thr

I

II

III

44.1 17-I 18.3 4.4 4.1 6.7 3.1 0.2t traces 18 traces traces traces

39.0 19.9 20.1 38 44 6.6 3.2 traces traces 3.0 0.0 traces 0.0

42.0 189 19.2 3.6 4.1 I.0 3.2 traces traces 2.2 traces traces traces

* Separated on Bio-Gel P-100 (Fig. 2). t Based on recorded value of 0.012 pmole. Traces are values recorded as < 0.005 pmole. profiles of the three subfractions were highly similar and in excellent agreement with the previous analyses of the “pooled” pl > 10 fraction (Fig. 4, Table 4). The recovery of amino acids from the 24-hr hydrolysates were, with one exception, within 85-l 18 per cent of protein amounts (Table 3). In spite of a decrease in total recovery by prolonged hydrolyses, the amino acid profiles after 48 or 72 hr of hydrolysis showed very small variations from the 24-hr values (Fig. 3). Carbohydrate analyses. The qualitative analysis gave satisfactory resolution only for the samples with

Table 5. Amino acid composition

Pro G’Y Glx Asx Ser Lys Arg His Leu Ala Val Be Thr

of basic parotid

a high carbohydrate/protein ratio (pl 9 and 9.5 fractions). Reducing substances with Rr-values corresponding to glucosamine, galactose, inannose and fucose were detected (Fig. 5). A substance with the Rrvalue of glucose was also found, but the sucrose density gradient and the Sephadex bed material are possible sources of glucose contamination. The quantitative analyses gave low carbohydrate values for the pl > 10 fractions. The lowest amounts were found in subfractions II and III, where the neutral carbohydrates were only a few per cent of total weight and hexosamines below detection limits of the Autoanalyser. The ~19.5 fraction contained one hexosamine (glucosamine) residue per every 10 amino acids and the pI9 fraction slightly more. Similarly, the neutral carbohydrate accounted for about one-third of total glycoprotein weight in the pl 9.5 fraction, and about one-half in the pl 9 fraction. suggesting an inverse relationship between pl-values and the proportion of carbohydrates in the glycoprotein fractions (Table 2). Specific analyses for fucose and galactose gave only trace amounts in the pl > 10 fractions, and about 5 per cent in the pl 9.5 fraction, in accordance with the differences in total carbohydrate (Table 2).

DISCUSSION

Isoelectric focusing in a pH 7710 gradient gave an improved resolution and essentially the same pl-distribution of basic parotid secretory glycoproteins as a pH 3-10 gradient (Arneberg, 1972). The high pl-values must be related to the 11 per cent of lysine and arginine residues in all fractions, as these two residues carry a

secretory glycoproteins. ferent workers

Residues per

100,

as reported

by dif-

Levine et al. (1969) (DEAE and Sephadex G-200)?

Andjic et al. (1970) (Sephadex G-200 and prep. electrophoresis)t

Present work* pI9.5 fraction

Present work* pl > 10 fraction

33-6 20.5 20.9 6.7 5.2 5.0 4.9 0.9 0.1 0.5 0.6 0.3 0.4

34.3 20.1 20.5 5.2 5.1 5.1 5.1 1.5 0.4 o-7 0.3 0.2 0.9

39.9 17.6 18.6 4.6 4.5 5.2 5% 1.2 0.1 0.5 0.7 0.31 0.3$

396 19.4 18.7 4.8 4.4 7.2 3.3 0.0 0.0 2.5 0.0 0.0 0.0

* Data presented in Fig. 3. t Purification procedure. 2 Amounts insufficient for accurate

quantitation.

926

P. Arneberg

positive charge at neutral pH. The pl-values of 911 are compatible with neutralization through titration of lysine e-amino groups. These groups have p&-values of 9.4 10.6, whereas the arginine guanidinium groups are still charged at pH 11 (Edsall, 1943). The pl-variations within this group of basic glycoproteins, however, cannot be explained by the higher p&-values of the guanidinium group, since the highest arginine content was found in the pl9 and 95 fractions (Figs. 1 and 3). Sialic acids have been detected in similar glycoproteins (Levine et al., 1969; Andjic et a/., 1970) and may have contributed to the pl-differences. A lower proportion of this negatively charged carbohydrate residue by increasing pl-values, would be in agreement with the present observations on total carbohydrate (Table 2). Sialic acid and fucose are terminal residues on the carbohydrate side chain. whereas the linkage to the polypeptide is most commonly through a hexosamine residue (see Clauser et al., 1972). The hexosamine values presently observed in the pl > 10 fractions may have been underestimated as a result of degradation. Hydrolysis in 1.5 N HCl gave higher hexosamine values in comparable fractions (Arneberg, 1972). The amino acid profiles reported here are in accordance with previous analyses on parotid secretory protein, of comparable pl by Armstrong (1971). Close similarity in carbohydrate composition is also apparent between the present pl 95 fraction and the homogeneous parotid secretory glycoproteins reported by Levine et al. (1969) and Andjic et a[. (1970) (Table 5). In comparison, the pl > 10 fractions characterized in the present experiments had lower molecular weights, less carbohydrate, and contained a restricted variety of amino acids. Only eight different residues were detected in analyses of the “pooled’ pl > 10 fraction (Fig. 3). The detection limit on the Autoanalyser by our procedure was below 0.01 ,nmole, as 0.03-0.04 pmole was required for quantitation. Based on molecular weight and total recovery (Tables 2 and 3) a single amino acid residue in subfractions I and III would correspond to amounts sufficient for quantitation. whereas only traces were observed (Fig. 4, Table 4). In subfraction II analysis, the total load was too low to exclude the possibility that other amino acids were inherent in its polypeptide chain. The slight differences in amino acid profiles between glycoproteins of different pl-values (Fig. 3) are too small to exclude a common polypeptide for all. The carbohydrate is the less constant part of glycoproteins (see Clauser et ul.. 1972) and fractions analysed here showed a decreasing content of carbohydrate by decreasing molecular weight (Table 2). However, since the differences in molecular weights were that large, the heterogeneity may not have been confined to the carbohydrate moiety. This problem could be studied by “fingerprinting” techniques. Subunit structures within these glycoproteins were considered. Similar or identical polypeptide chains, with one alanine residue out of a total of about 50,

might appear as mono-, di- and trimers in subfractions III, II and I, respectively. Rechromatography of subfraction I after urea or Tween 20 treatment did not, however, indicate a subunit structure. Oppenheim et al. (1971) proposed that acidic proline-rich parotid secretory proteins might be aggregates. This was based on the remarkably low K,,values in gel filtration compared to their sedimentation equilibria in the ultracentrifuge. The basic proline-rich glycoproteins showed the same discrepancy. Subfraction I ofpl > 10 had a K,, on Sephadex G-100 similar to that of human serum albumin (M, 69,000) whereas equilibrium centrifugation gave a molecular weight of only 18,000. As the present analyses were performed in the same buffer and at similar protein concentrations, aggregate formation during gel filtration and not during ultracentrifugation seems unlikely. It appears more probable that the proline-rich proteins have nonglobular forms, This would lead to low &,-values without obscuring the results from ultracentrifugation (Edwards and Shooter, 1970). The present investigation shows that the basic proline-rich protein fraction secreted by the human parotid gland constitutes a group of closely related glycoproteins, but the exact nature of this relationship is unknown. Further studies concerning their biosynthesis, with special reference to an intracellular membrane origin (Amsterdam et al., 1971) have been started in this laboratory. Acknowledgements-Terje Christensen, Institute of Biochemistry, University of Oslo, performed the ultracentrifugation. Tone De Best carried out the amino acid analyses. Anette Melsom

assisted

in preparing

the manuscript.

REFERENCES

Amsterdam A., Schramm M., Ohad I., Salomon Y. and Selinger Z. 1971. Concomitant synthesis of membrane protein and exportable protein of the secretory granule in rat parotid gland. J. Cell Biol. 50. 187-200. Andjic J., Bonte M. and Havez R. 1970. Purification et etude dune glycoproteine basique de la salive parotidienne humaine. C. r. hehd. S&WC. Acad. Sci., Paris, Ser. D, 270, 564566. Armstrong W. G. 1971. Characterisation studies on the specific human salivary proteins_adsorbed in vitro by hydroxypatite. Caries Rrs. 5, 215-221. Arneberg P. 1971. Quantitative determination of protein in saliva. A comparison of analytical methods. &and. J. dent. Res. 79, 60-64. Arneberg P. 1972. Fractionation of parotid salivary proteins by isoelectric focusing in a wide pH range. Stand. J. dent. Res. 80, 134138. Bennick A. and Connell G. E. 1971. Purification and partial characterization of four proteins from human parotid saliva. Biochem. J. 123, 455-464. Clauser H., Herman G., Rossignol B. and Harbon S. 1972. Biosvnthesis at the cellular and subcellular level. In: Glvcoproteins. Their Composition, Structure and Function (Edited by Gottschalk A.), 2nd Rev. Edn, Vol. 5, Part B, Chap. 10, pp. 1151L1169. B.B.A. Library, Elsevier, New York.

Basic parotid

secretory

Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A. and Smith F. 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 35&356. Edsall J. T. 1943. Proteins as acids and bases, In: Proteins, Amino Acrds and Peptides as Ions and Dipolar Ions (Edited by Cohn E. F. and Edsall J. T.), p. 445. Monograph Series No. 90. Reinhold. New York. Edwards P. A. and Shooter K. V. 1970. A study of calf thymus histone fraction F2(b) by gel filtration. Biochem. J. 120, 61-66. Gottschalk A., Bhargava A. S. and Murty V. L. N. 1972. Submaxillary gland glycoproteins. In: Gl~coproteins. Their Compositiotl, Structure and Function. 2nd Edn, (Edited by Gottschalk A). Vol. 5. Part B. Chap. 7. pp. 811% 829. B.B.A. Library. Elsevier, New York. Lamm 0. 1929. Die Differentialgleichung der Ultrazentrifugierung. Ark. Mat. Astr. Fys. 21B (2). l-4.

glycoproteins

921

Levine M. J., Weill J. C. and Ellison S. A. 1969. The isolation and analysis of a glycoprotein from parotid saliva. Biochim. hiophys. Acta 188, 16>167. Mandel I. D., Thompson R. H., Jr. and Ellison S. A. 1965. Studies on the mucoproteins of human parotid saliva. Archs oral Biol. 10, 4999507. Oppenheim F. G., Hay D. I. and Franzblau C. 1971. Proline-rich proteins from human parotid saliva-l: Isolation and partial characterization. Biochemistry, Iv’.Y 10, 4233-4238. Spiro R. 1966. Analysis of sugars found in glycoproteins. In: Methods in Enzymology (Edited by Neufeld E. F. and Ginsburg V.). Vol. 8, pp. 3-26. Academic Press, New York. Trevelyan W. E., Procter D. P. and Harrison J. S. 1950. Detection of sugars on paper chromatograms. Nature. Lond. 166, 444445.

R&urn&La focalisation isoelectrique de la salive parotidienne humaine dans le gradient pH 7-10 a donne une fraction glycoprottinique majeure a un pH au-dessus de 10 (fraction pl > lo), une fraction mineure pl 95 et des petites quantites de glycoprottines isoelectriques autour du pH 9 (fraction pl 9). La filtration subsequente du gel (B&Gel P-100) separa les glycoproteines pl > 10 en trois subfractions (I, II, III) tandis que les fractions pl9 et 95 se montraient comme des sommets exclus. Les trois subfractions pl > 10 et la fraction pl 9,5 ont Cte sujettes a la centrifugation en equilibre dans l’ultracentrifugeuse. La fraction pl 9,5 Ctait heterogbne (poids mol. 3O.OOt&70.000) tandis que les trois subfractions pl > 10 paraissaient plus homogenes. Leur poids moleculaires Ctaient de 18.000. 11.500 et moins que 10.000 pour I, II et III respectivement. Le contenu en hydrate de carbone diminua, en augmentant le pl des glycoproteines et variait den haut de 50 jusqu’a 5 pour cent au-dessous du poids total des glycoproteines. Les monosaccharides principales dans les fractions furent identifiees comme dextrosamine, glucose, galactose, mannose et fucose. Les analyses d’aminoacides montrerent que dans toutes les fractions de glycoproteines la proline, la glycine et l’acide glutamique (dans une proportion de 2: I : 1) comptaient pour 7&X0 pour cent des residus totaux et les residus de base (lysine et arginine) pour encore Is-12 pour cent. La fraction du pl > 10 avait une proportion plus forte de lysine/arginine que les fractions dun pl inferieur. La similarite dans la composition d’aminoacides indique que toutes ces glycoproteines sont apparentees.

Zusammenfassung-Isoelektrisches Zentrieren von menschlichem Ohrspeichel im Gradient pH 7-10 et-gab ein griiI3eres Glykoproteinfragment in pH iiber (pl > 10 Fragment), ein kleineres pl 9.5 Fragment und schwache Mengen von Glykoproteinen, isoelektrisch urn pH 9 (pl 9 Fragment). Nachfolgende GellFiltrierung (B&Gel P-100) trennte die pl > 10 Glykoproteine in drei Subfragmente (I, II, III), wahrend die pl 9 und 9,5 Fragmente als ausgeschlossene Scheitelwerte erschienen. Die drei pl > IO Subfragmente und das pl9,5 Fragment wurden in der Ultrazentrifuge einer Gleichgcwichtsschleuderung ausgesetzt. Das pl9,5 Fragment war heterogen (Mol. Gew. 30.00@ 70.000). wghrend die drei pl > 10 Subfragmente mehr homogen erschienen. Ihre Molekulargewichte waren 18.000, beziehungsweise 11.500 und weniger als 10.000 fur I, II und III. Ihr Kohlenwasserstoffgehalt nahm mit steigendem pl der Glykoproteine ab und bewegte sich zwischen iiber 50 bis auf unter 5 Prozent des gesamten Glykoproteingewichts. Glukosamin, Glukose, Galaktose, Mannose und Fukose wurden als Hauptmonosaccharide in den Fragmenten festgestellt.

928

P. Arneberg Aminsaureandlysenzeigten, daf3 Prolin, Glycin, Glutaminsaure (in einem Verhaltnis von 2: 1: 1) 7680 Prozent des gesamten Riickstandes in allen Glykoproteinfragmenten darstellten, und die basischen Riickstande (Lysin und Arginin) weitere l(212 Prozent. Das Fragment von pl > 10 hatte ein gr6Beres Lysin/Arginin Verhaltnis als die Fragmente von niedrigerem pl. Die Ahnlichkeit der Aminsaureverbindung zeigt an, dab all diese Glykoproteine mit einander verwandt sind.

Basic parotid secretory glycoproteins

Galactosamine Glucosamine Galactose G I ycoprotein Glycoprotein Glycoprotein

of pl >I0 of

pl >9-5 of

plY3

Glucose Mannose Fucose

Fig. 5. Qualitative carbohydrate analysis of basic parotid secretory glycoproteins by TLC. Glycoproteins of different pIvalues were hydrolysed for 2 hr for carbohydrate liberation. The dried residues were dissolved in pyridine and 5-10 ~1 was applied to the plate. The amount of neutral carbohydrates in the samples applied was 5.8 pg (pZ > 10 glycoprotein), 19 pg (pZ 9.5 glycoprotein), 16.5 pg (pZ 9 glycoprotein). Of each standard 5 pg was applied.

A.O.B. f.p. 928