glycoconjugates
TRANSCRIPT
79
Glycoconjugates
Fresenius J Anal Chem (1990) 337:79 - © Springer-Verlag 1990
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Identification and determination of individual glycosaminoglycans of human ejaculates
W. Biirgi 1, H. Ohishi 2, A. Kimura 2, and K. Schmid 2
1 Zentrallaboratorium, Kantonsspital, Aarau (Switzerland) 2 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA
The human ejaculate contains analytes such as potassium, zinc, citric acid, fructose, phosphorylcholine, spermine and pros- taglandins in higher concentration as compared with other body fluids [1]. Of the large number of proteins, including enzymes [2], some of them are believed to be specific constituents of seminal fluid [1, 3]. It is also interesting to note that Znc~2- glycoprotein, a minor plasma protein constituent, is secreted by the prostatic gland to yield a significantly higher concentration in seminal fluid than in serum [4]. However, information on glycosaminoglycans (GAGs) of human ejaculates is very scanty. The aim of the present work was to study the GAGs of human sperms and seminal plasma.
Methods
The GAGs, chondroitin sulfate C (CSC), hyaluronic acid (HA), heparan sulfate (HS), dermatan sulfate (DS), low sulfated chondroitin sulfate (LSC) and oversulfated chondroitin sulfate (CSD/E) were measured in sperm cells and in seminal plasma of 4 fertile individuals. The GAGs were assayed according to the procedure of Hata and Nasal [5]. The ejaculates were centrifuged and the sperm and seminal fluid fractions separately delipidated. After exhaustive proteolytic digestion with papain and pronase, the digests were lyophilyzed, dissolved in an appropriate volume of water and subjected to two-dimensional electrophoresis on cellulose acetate plates. The plates were stained with alcian blue and the resulting spots were analyzed photometrically for GAGs at 667 nm. Identification of GAGs was achieved by two-dimensional co-electrophoresis and de- gradation of GAGs with highly specific glycosidases (e.g. hyaluronidase, heparatinase).
Results
The results indicate that the GAG contents of sperm cells and seminal plasma were similar to each other, the average being 33.9 rag/1 ejaculate for the former and 34.1 rag/1 for the latter. In seminal fluid, the distribution of the individual GAGs, ex- pressed in relative percentages, was the following: 38% (range 23 -52) for CSC, 16% for HA (6-28) and 26% for HS (21 - 32). One specimen only contained a small amount (7%) of DS, whereas HP was absent in all of them. LSC was present in relatively high concentrations, the mean value amounting to 17% (14-23) of the total GAGs. In one seminal plasma CSD/ E was identified (4%). Sperm ceils showed a distribution pattern of GAGs significantly different from that of seminal fluid. CSC was found to account for 33% (9-56) of total GAG content, DS 20% (8-54) and HS 41% (29-57). HA was present in 2 samples in relative concentrations of 2 and 5%, respec- tively, whereas HP and CSD/E were absent in all of the specimens.
Conclusions
The occurrence and distribution of GAGs of human sperm ceils and seminal fluid are reported here for the first time. In addition CSD and CSE have not been identified until now in a normal human body fluid, i.e. seminal plasma. The lack of HP may prevent sperm cells from nuclear decondensation. The relatively high content of HA in seminal plasma may favor the migration of spermatozoa. HA and DS may prevent the sperm cells from adherence to the female genital tract wall. These findings rep- resent a significant progress in the investigation of the GAGs of seminal plasma as until now only the presence of HA in this biological fluid has been established.
References
1. Coffey DS (1986) Campbell's urology, 5th edn. W. B. Saunders Company, Philadelphia, USA, p 255
2. Edwards JJ, Tollaksen SL, Anderson NG (1981) Clin Chem 27:1335-1340
3. Schaller J, Akiyama K, Tsuda R, Hara M, Marti T, Rickli EE (1987) Eur J Biochem 170:111 - 120
4. Biirgi W, Simonen S, Baudner S, Schmid K (1989) Clin Chem 35:1649-1650
5. Hata R, Nagai Y (1973) Anal Biochem 52:652-656
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Fresenius J Anal Chem (1990) 337:80-81 - © Springer-Verlag 1990
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Glycosaminoglycan patterns in the skin of the rat: Changes after wound-healing
P. Mailiinder 1, N. Giissler 2, A. Delbriiek 2, and A. Berger 1
1 Clinic of Plastic, Hand and Reconstructive Surgery, Medical School Hannover 2 Institute of Clinical Chemistry (II), Medical School, Podbielskistrasse 380, D-3000 Hannover 51, Federal Republic of Germany
During the past decade the skin expander technique has become more and more important in Plastic Surgery, It is often used for breast reconstruction and for covering skin defects. Simulta- neously to the clinical use some authors examined the reaction of cutis and subcutis on biomechanical stretching. Prolonged obesity produces an increase of collagen fibers while the relation between skin diameter and collagen content is constant. In 1982 Austad [1] histomorphologically examined Guinea pig skin after controlled expansion. He found an atrophy of the pann{culus carnosus without any thinning of the dermis. Studies by electronmicroscopy showed larger groups of tonofilaments in the epidermis and an increase of compact as well as the thin collagen fibers in the dermis. In this study the distribution patterns of glycosaminoglycans (GAGs) are investigated using a stretching model to gain more insight in the biochemical changes during wound-healing.
\
Material and methods
11 male Spray-Dawley rats weighing 300 g were divided into two groups with 7 animals in the treated skin group and 4 animals in the control group. 7 rats got a undermining operation under the back skin at the midthoracic level beneath the panniculus carnosus. 60 days after the operation a 2 x 2 cm skin area was taken from the point of the undermined skin area and from the back of the control animals.
The skin biopsies were frozen and kept by -80°C. The GAGs have been isolated according to the method of GURR [5]. In this method specific GAG degrading enzymes and high performance liquid chromatography (HPLC) are employed for the specific and quantitative determination of the individual GAG-fraction. The amounts of GAG are calculated from the amounts of respective disaccharide metabolites using disaccha- ride calibrators. The uronic-acid content of the GAGs have been assayed by the carbazole reaction [3].
Results and discussion
The results are shown in Fig. 1. The fractions of GAGs yielded from skin of rats were:
hyaluronic acid: 3.30 gmol/g (70.3%) in healthy and 2.65 gmol/g (77.2%) in treated rat skin chondroi t in : 0.23 gmol/g (4.8%) in healthy and 0.46 gmol/g (13.4%) in treated rat skin chondroi t in-6-sul fa te : 0.02 gmol/g (0.5%) in healthy and 0.00 lamol/g (0.0%) in treated rat skin chondroi t in-4-sul fa te : 0.12 lamol/g (2.5%) in healthy and 0.21 gmol/g (6.1%) in treated rat skin dermatan." 0.13 gmol/g (2.7%) in healthy and 0.09 gmol/g (2.6%) in treated rat skin dermatan-6-su l fa te : 0.02 lamol/g (0.4%) in healthy and 0.00 Ixmol/g (0.0%) in treated rat skin derma tan -4 - su l f ate : 0.88 gmol/g (18.8%) in healthy and 0.02 gmol/g (0.7%) in treated rat skin to tal G A G : 4.70 gmol/g (100%) in healthy and 3.43 gmol/g (100%) in treated rat skin.
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[~]
8O
70
2o
I0
Hyaluronat Chondroitin Chondr.- Chondr,- Dermatan 6-sulfat 4-sulfat
(p < 0.10) (p < 0.05) (p < 0.01) (p < 0.03) (n. S,)
Dermatan- Dermatau- 6-sulfat 4-sulfat
(p < 0.05) (p < O.Ol)
I0
5
Fig. 1 Glycosaminoglycan pattern in healthy rat skin connective tissue (n = 4) and treated autografts (n = 7). The differences in each GAG-fraction are calculated with the U-Test of Wilcoxon, Mann and Whitney ([~ glycosamino- glycan fraction from treated connective tissue of rat skin, [] glycosaminoglycan fraction from healthy connective tissue of rat skin)
All figures are given in relation to the weight of the specimens.
The data on healthy rat skin subcutaneous connective tissue are in agreement with the observations by yon Figura [4] on human skin specimens. In human skin 90% of the total GAG accounted for hyaluronate and dermatan sulfate while less than 10% were identified as chondroitin sulfates without further differentiation as to the subfractions.
Further the GAG pattern in this study are in general agree- ment with the results by Barker et al. [2] and Schiller [6] using ion-exchange chromatography followed by detection of the indi- vidual fractions with paper chromatography or electrophoresis. These authors found hyaluronic acid and dermatan sulfate as the major GAG-fraction in healthy rat skin. In addition small amounts of heparin, chondroitin-4-sulfate, chondroitin-6- sulfate and keratosulfate have been detected. The differences in the concentrations of GAG in comparison to the results of the method applied here are explained with the more specific and quantitative detection with the combined enzymatic and t-IPLC method. The observed increase in the concentrations of chondroitin and chondroitin-4-sulfate and the simultaneous decrease in dermatan-4-sulfate in tissue after undermining rat skin could be explained by a possible epimerization of the C 5 carbon in the uronic acid moiety of the repeating disaccharides in the glycan side chains of proteoglycans [7].
The pattern of GAGs from tissue of the treated rat skin is similar to the distribution pattern from skin of human foetus
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[4]. Therefore we suppose that the analyzed tissues of treated rat skin are in an early phase of repair. This interpretation is supported by the increase in chondroitin-4-sulfate in the specimens.
Beside the changes in the GAG patterns in wound healing the decrease in the total amount of GAG compared with the untreated skin portions is remarkable. One may assume that in early stages of wound healing the tissue contains other components like proteins in higher concentrations than the healthy control skin. These findings needs further investiga- tion.
References
1. Austadt ED, Pasyk KA, McClatchey KD, Cherry GW (1982) Plast Reconstr Surg 70: 7 0 4 - 719
2. Barker CN, Cruickshank D, Webb T (1965) Carbohydrate Res 1 : 5 2 - 6 1
3. Bitter A, Muir H (1962) Anal Biochem 4 :330 -334 4. von Figura K (1976) Der Hausarzt 27 :206-213 5. Gurr E, Pallasch G, Tunn S, Tamm C, Delbriick A (1985) J
Clin Chem Clin Biochem 23 : 77 - 87 6. Schiller S (1966) Biochim Biophys Acta 124:215-217 7. Scott JE (1988) Biochem J 252: 313 - 323
Fresenius J Anal Chem (1990) 3 3 7 : 8 1 - 82 - © Springer-Verlag 1990
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Characterization of basement membrane associated heparan sulfate proteoglycans from human tissues
G. StOeker, H.-D. Haubeck, C. Wagener*, and H. Greiling
Institute for Clinical Chemistry and Pathobi0chemistry , Medical Faculty, Aachen University of Technology, Pauwelsstrasse 30, D-5100 Aachen, Federal Republic of Germany
Heparan sulfate proteoglycans (HS-PG) represent a family of molecules which are involved in fundamental biological pro- cesses like cell-cell interaction, proliferation and differentiation [1]. Different types of HS-PG were found on cell surfaces, in basement membranes and in extracellular matrix. In basement membranes HS-PG create ionic charge barriers, participate in cell adhesion and bind other basement membrane components. Here we report on the isolation and characterization of basement membrane associated HS-PG from various human tissues.
Materials and methods
HS-PG were isolated and purified from human aorta as de- scribed [4]. Briefly, proteoglycans were extracted from the tissues
* Presen t address: Department of Clinical Chemistry, Medical Clinic II, University of Hamburg, Martinistrasse 52, D-2000 Hamburg 20, Federal Republic of Germany
M r 1 2
A
205,000 -,.
116,000 -,. 97,000 .--.,.
66,000 -,.
45,000 --,.
29,000 -,.
20,100 -,. .....
14,400 - - , .
B
Mr
205,000
116,000 97,000
66,000
45,000
29,000
20,100
14,400
2 ~ii I ~ / ~ i
Fig. 1 A, B. Diffusion blot of native and enzymatically digested heparan sulfate proteoglycans (HS-PG) from human aorta and kidney. HS-PG's have been submitted to SDS-PAGE on a 8 - 25% gradient gel and subsequently blotted onto nitrocellulose membranes. Immunochemical detection of untreated HS-PG's and heparinase/heparitinase detection of HS-PG's was done using the HS-PG specific mAb 1F10/B8. HS-PG from kidney (Fig. IA, lane 1), HS-PG from aorta (Fig. IB, lane 1); heparinase/heparitinase digested HS-PG's from kidney (Fig. 1 A, lane 2) and aorta (Fig. 1 B, lane 2)
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by 4 mol/1 guanidine hydrochloride in the presence of protease inhibitors and CHAPS. HS-PG were purified by several chroma- tographic steps including anion-exchange chromatography, size exclusion chromatography and hydroxylapatite chromatog- raphy. Biochemical characterization of the isolated HS-PG was done by amino acid analysis, electrophoresis and blot onto nylon membranes prior and after enzymatic digestion.
Monoclonal antibodies (mAB) were raised in mice against HS-PG. These HS-PG-specific mAB have been used for immu- nohistochemical analysis of the distribution and localization of HS-PG in different human tissues. Tissues with positive staining reactions in immunohistochemistry were selected as sources for the isolation of HS-PG. For these preparations a short isolation procedure was used including only one anion-exchange chroma- tography and subsequent affinity chromatography using the mAB 1F10/B8. Characterization of the isolated HS-PG was performed by electrophoresis, blot and subsequent immuno- chemical detection.
Results and discussion
A series of monoclonal antibodies were raised against HS-PG isolated and purified from human aorta. The specificity of mAB 1F10/B8 was shown by immunochemical analysis of HS-PG after SDS-PAGE and subsequent transfer to membranes. This HS-PG-specific mAB was used for immunohistochemical analy- sis of the distribution and localization of HS-PG in different human tissues. These analysis demonstrated that the mAB 1F10/ B8 predominantly recognizes basement membrane related structures. For comparison of basement membrane associated HS-PG from different tissues, HS-PG were isolated by affinity chromatography. Results of immunochemical analysis on blots
of HS-PG from human aorta and kidney are shown in Fig. 1. Molecular weights after SDS-PAGE have been determined to Mr 80,000-200,000 for HS-PG from aorta (Fig. 1 B, lane 1) and to Mr 25,000-160,000 for HS-PG kidney (Fig. 1 A, lane 1). Molecular weights of the core protein moieties were determined after heparinase/heparitinase digestion of the respective HS- PG's (aorta: Fig. 1 B, lane 2, kidney Fig. 1 A, lane 2). Whereas for aorta only one band at Mr 24,000 could be detected, for kidney two bands at Mr 22,000 and Mr 15,000 were found. Whether the second smaller band reflects a degradation product of the core protein or indicates two different types of core proteins has to be determined. However core proteins of dif- ferent sizes (Mr 21,000-34,000) have been described by Kato for HS-PG from Engelbreth-Holm-Swarm mouse tumor [2]. Different sizes of the native HS-PG's as well as differences for the core protein moieties indicate that different basement membrane associated HS-PG's are present in human aorta and kidney. These findings are supported by results obtained by Klein [3]. Whether the different sizes of the core proteins are due to protein "tailoring" [3] or reflect different gene products has to be clarified by sequence analysis.
References
1. Hassell JR, Kimura JH, Hascall VC (1986) Ann Rev Biochem 55: 539- 567
2. Kato M, Koike Y, Suzuki S, Kimata K (1988) J Celt Biol 106: 2203 - 22] 0
3. Klein D J, Brown DM, Oegema TR, Brenchley PE, Anderson JC, Dickinson AJ, Horigan EA, Hassell JR (1988) J Cell Biol 106: 963 - 970
4. St6cker G, Liickge J, Greiling H, Wagener C (1989) Anal Biochem 179 : 245 - 250
Fresenius J Anal Chem (1990) 337:82-84 - © Springer-Verlag 1990
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Determination of the position of the keratan sulphate chain at the branched mannose
D.-Ch. Fischer, H. W. Stuhlsatz, and H. Greiling
Institute for Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology, Pauwelsstrasse 30, D-5100 Aachen, Federal Republic of Germany
Previous research on the molecular structure of pig corneal keratan sulphate proteoglycan showed that to the core protein two keratan sulphate chains and two N-glycosidic oligo- saccharides are attached [1, 3]. Methylation studies indicated the branched mannose being substituted in 1, 3 and 6-position [4] (Fig. 1 d). We used GC/MS analysis to determine whether the 3- or 6-position of the biantennary ]~-mannose is linked to the keratan sulphate chain.
Material and methods Peptidokeratan sulphate from porcine cornea was prepared as described previously [5]. To liberate either the 3- or 6-position prior to derivatisation for GC/MS-investigations, two different sequential enzymatic digestions (A and S) were performed. In preparation A the keratan sulphate chain was digested with keratanase followed by incubation with /%N-acetylhexos- aminidase, /~-galactosidase and c~-mannosidase. Separation of
the linkage region and the liberated mono- and oligosaccharides was performed by ion-exchange and gel-permeation chromatog- raphy. In preparation S the oligosaccharide chain was sequentially digested with neuraminidase, ]~-galactosidase and ]%N-acetylhexosaminidase. Before removing the ~-mannose enzymatically, incubation with keratanase was done. The re- sulting degradation product containing the linkage region was purified by chromatography. These two peptidokeratan sulphate molecules, modified in different ways, and the trisac- charide Man ~1 ~ 3Man/31 ~ 4GlcNAc as reference, were subjected to further structural analysis. Prior to GC/MS exam- ination the molecules were transformed to the corresponding permethylated alditol acetates by the procedure of Levery and Hakomori [2], GC/MS examination was performed on a Finnigan MAT 1020 equipped with an OV 101 capillary column (50 m, 0.32 mm ID); ionisation was done by electron impact (70 eV).
Results and discussion Two different enzymatic treatments were applied to corneal peptidokeratan sulphate to obtain oligosaccharides containing a 1,3- or a 1,6-substituted/%mannose. The oligosaccharides were further processed to a mixture of permethylated alditol acetates separated by gas chromatography and analysed by mass spectrometry. To decide whether preparation A or S results in a 1,3- disubstituted /~-mannose, that means to determine the former substituent of the 6-position, a trisaccharide of known structure was subjected to the same derivatisation procedure
83
Sel l
e i ~ $PECT~U~ e i .Oo /~ 10, ~a : t ) e * 16 ,25 ~ t " lP l .E t REFEREHZ-T I |~ .d I~CF I~ | I~ [ , PERPI{TICr~|ERT [TC .
O~TRJ R~4 01571
61
°'lit ,3 .i I ? I~I
" 1t, I ~,',l 1,1!. i[...ll . ...... I 141,1, I . e
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I'tAS$ ,~0t~CTm.Jn IO~TA! l~02 029~J~1
I , ] , ~ - t r l -O-&cety1-2 ,4 ,6 - t r i -O-~m ~hyl - I Lnm l ~ol
14S
S l
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"' ~
84
before GC/MS investigation. This procedure resulted in a mixture of three clearly different alditol acetates, one derived from a monosubstituted saccharide, one from a disubstituted saccharide and one from an aminosugar. Spectrum and reten- tion time of the interesting 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl- mannitol (Fig. la) coding for a 1,3-disubstituted mannose was determined from the analysis of the reference mixture and used to identify the same product in preparation A or S. Thereby we found that only preparation A leads to a 1,3-disubstituted p- mannose resulting in a 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl- mannitol (Fig. 1 b) after further derivatisation. Since only in preparation A the keratan sulphate chain was completely re- moved, this chain has to be linked to the 6-position of the branched/~-mannose. In agreement with this we were able to identify the mass spectrum of a former 1,6-disubstituted mannose in preparation S (Fig. 1 c).
The keratan sulphate is thought to guarantee the transpar- ency of the cornea by controlling collagen fibrogenesis. This function requires the possibility of free rotation which is for sterical reasons only possible, if the keratan sulphate chain is linked to the 6-position of the branched fl-mannose. Our analy- sis of the molecular structure of corneal keratan sulphate
showed that the keratan sulphate chain is attached to the 6-position of the branched mannose confirming the biological function supposed [6] (Fig. 1 d).
References
I . Lennartz L (1987) Dissertation RWTH Aachen 2. Levery SB, Hakomori S (1986) Methods Enzymol 121 : ~ 3 -
25 3. Oeben M, Keller R, Stuhlsatz HW, Greiling H (1987) Bio-
chem J 248:85-93 4. Stein T, Keller R, Stuhlsatz HW, Greiling H, Ohst E, Miiller
E, Scharf HD (1982) Hoppe-Seyler's Z Physiol Chem 363 : 8 2 5 - 833
5. Stuhlsatz HW, Hirtzel F, Keller R, Cosma S, Greiling H (1981) Hoppe-Seyler's Z Physiol Chem 362:841- 852
6. Stuhlsatz HW, Keller R, Becker G, Oeben M, Lennartz L, Fischer DC, Greiling H (1989) In: Greiling H, Scott JE (eds) Keratan sulphate - chemistry, biology, chemical pathology. The Biochemical Society, London
Fresenius J Anal Chem (1990) 337:84-85 - © Springer-Verlag 1990
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Determination of UDP-sugars from cultured human chondrocytes by high-performance liquid chromatography
G. Fedders, R. Kock, E. Van de Leur, and H. Greiling
Institute for Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology, Pauwelsstrasse 30, D-5~00 Aachen, Federal Republic of Germany
The aim of the present study was to develop a sensitive method for qualitative and quantitative determination of UDP- activated sugars from cultured human chondrocytes. Previously synthesis-rate of proteoglycans, measured as 3 ~S_incorporation, and other extracellular matrix molecules have been used as marker for cell activity [1]. Quantitative determination of syn- thesized proteoglycans is rather tedious and requires the use of 3H-labeled serine as well as 14C-labeled N-acetylglucosamine and 35S-labeled sulfate. UDP-activited sugars are precursor molecules for glycosaminoglycan synthesis [2]. We report here on the establishment of a highly sensitive HPLC-method for detection and quantification of these molecules in extracts of in-vitro cultured human chondrocytes. By this method we have determined the amount of UDP-N-acetylglucosamine, UDP-N- acetylgalactosamine, UDP-glucose and UDP-glucuronic acid.
Material and methods
HPLC analysis was done on a Bruker Model 31 Chromatograph equipped with a Waters 990 Photo-Diode-Array-UV/VIS Detec- tor. The separation-procedure was established by using defined solutions of different nucleotides (Sigma) and UDP-activated sugars (Sigma) as well as mixtures of them. Different columns and eluents have been tested. A Partisil 10 SAX column
(250 mm x 4.6 mm i.d.) (Chromatographie Service) eluted with a concave gradient performed from 0.015 tool/1 phosphoric acid, pH 3.8 to 0.75 mol/1 phosphoric acid, pH 4.8 (pH fixed with NH3, both eluents containing 3% methanol) gave the best re- sults [3].
UDP-N-acetylglucosamine and UDP-N-acetylgalactos- amine as well as UDP-glucose and UDP-galactose coelute and were further separated by rechromatography on the same column by the sequential use of water and a borate buffer (0.37 tool/1 boric acid, 0.15 mol/1 disodium tetraborate and 2.0 mol/l glycerol, pH 6.45) as mobile phases [41. Quantification was done by correlation peak area (absorbance at 262 nm) and amount of standard-substance applied to the column. By this method the amount of UDP-activated sugars in in-vitro cultured human chondrocytes was quantitatively determined.
Chondrocytes from hyalinic cartilage of the knee joint of a 25 years old patient were isolated by collagenase treatment (two steps). Chondrocytes were cultured in LGTA (low-gelling- temperature-agarose, Sigma) in Opti-MEM (Gibco) supple- mented with 10% fetal calf serum, 1% Ultroser G (Gibco) 1 gg/ ml insulin and 50 gg/ml ascorbic acid. For the experiments described chondrocytes were first cultured as mouolayers and passed in agarose at the 6th passage. For estimating the UDP- sugar pool cells were extracted according to the method de- scribed by Wice et al. [3], incorporation of 35S-sulfate in pro- teoglycans of cultured human chondrocytes was determinated according to Griesmacher et al. [5].
ResuLts and discussion
A two-step procedure for quantitative determination (pmol range) of UDP-activated sugars was established. Molecules were separated by HPLC on a Partisil 10 SAX colmnn and detected by a highly sensitive photo-diode-array-UV/VIS detector. The coeluting 4'-epimeric UDP-aminosugars as well as the 4'- epimeric UDP-sugars were further separated by rechromatog- raphy on the same column using a borate buffer instead of the former used phosphate-buffers. The method was established by the use of pure UDP-sugars and nucleotides and mixtures of
85
Table 1. Estimation of UDP-sugar concentrations and incorporation of 3SS-sulphate in proteoglycans of human chondrocytes cultured for different times
Culture time
Concentration of UDP-Sugars/10 6 cells in pmol
UDP-GlcNAc UDP-Glc UDP-GlcUA + UDP-GalNAc
35S-Sulfate incorporation in PG (24 h)/ 10 6 cells in Bq
agarose-fraction medium-fraction
6 days 3094 + 155 (5.0%) 1390 _+ 160 (11.5%) 672 _+ 64 (9.5%) 291 + 15 (5.1%) 86 _+ 6 (7.3%) 12 days 1417 _+ 139 (9.8%) 766 _+ 55 (7.2%) 305 _+ 18 (5.8%) 255 _+ 4 (1.5%) 53 _ 4 (8.2%) 20 days 756 _+ 100 (13.2%) 364 + 35 (9,6%) 280 _+ 26 (9.3%) 246 _+ 10 (4.2%) 89 _+ 2 (2.4%) 28 days 1026 _+ 37 (3.6%) 591 _ 24 (4.7%) 574 _+ 30 (5.2%) 307 _+ 12 (3.8%) 91 + 2 (1.8%)
(UDP-GlcNAc = UDP-N-acetylglucosamine, UDP-GalNAc = UDP-N-acetylgalactosamine, UDP-Glc = UDP-glucose, UDP- GlcUA = UDP-glucuronic acid, PG = proteogtycans)
them and then applied to extracts of cultured human chondrocytes. The pool of UDP-activated sugars was estimated and compared with the amount of proteoglycans found in culture-supernatants and cell-fractions. Determination of pro- teoglycan-synthesis by using 3SS-sulfate incorporation allows no decision whether chondroitinsulfate- or keratansulfate-pro- teoglycan is produced. Nevertheless our data indicate that the amount of synthesized proteoglycans correlates with the mea- sured UDP-sugar pool. Therefore this method seems to be useful for a rapid and sensitive monitoring of the biosynthesis of pro- teogtycans in in-vitro cultured chondrocytes (see Table 1).
References
1. von der Mark K (1986) Rheumatology 10: 272- 315 2. Silbert JE (1982) J Invest Derm 79:31s-37s 3. Wice BM, Trugnan G, Pinto M, Rousset M, Chevalier G,
Dussaulx E, Lacroix B, Zweibaum A (1985) J Biol Chem 260:139-146
4. Weckbecker G, Keppler DOR (1983) Anal Biochem 132:405-412
5. Griesmacher A, Hennes R, Keller R, Greiling H (1987) Eur J Biochem 168:95-101