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The isolation of heparin from mast cells of the normal rat
278
PRELIMINARY NOTES
VOL. 31 (1959)
Based on the preceding observations, it is possible to propose for Compound A the formula of a 4-acetamido-2-amino-2,4,6-tfideoxyhexose and for Compound B the formula of a 2,4-diacetamido-2,4,6-trideoxyhexose. Thus, it appears to be the first isolation of a diaminohexose from natural sources. This work was supported b y grants from the American Cancer Society and from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service (Grant F-I965). NATHAN SHARON* The Robert W. Lovett Foundation, Massachusetts ROGER W. JEANLOZ** General Hospital and the Department o[ Medicine, Harvard Medical School, Boston, Mass, (U.S.A.) N. SI~ARON,Nature, 179 (1957) 919. 2 S. GARDELL,Acta Chem. Stand., 7 (1953) 207. 8 M. SOMOGYI,J. Biol. Chem., 195 (1952) 19; N. NBLSON,J. Biol. Chem., 153 (1944) 375. E. C. COCKINGAND E. W. Y~MM,Biochem. J., 58 (1954) xii. 6 M. V. TRACEY,Biochem. J., 52 (1952) 265. * R. J. BLOCKAND D. BOLLING,The Amino Acid Composition o] Proteins and Foods, 2rid ed., C.C. Thomas, Publ., Springfield, 1951, p. 261. J. F. O'DEA AND R. A. GIBBONS,Biochem. J., 55 (1953) 58o. s A. S. PERLIN,J. Am. Chem. Soc., 76 (1954) 41Ol. z
Received September 29th, 1958 * On leave of absence from the Weizmann Institute, Rehovoth, Israel. ** Special Investigator of the Arthritis and Rheumatism Foundation.
The isolation of heparin from mast cells of the normal rat Evidence for the production of heparin b y m a s t cells has been obtained by a variety of indirect techniques. These include the demonstration of (I) metachromasia in cells stained with toluidine blueZ; (2) a parallelism between the concentration of mast cells and apparent heparin content of tissues 1; (3) mS uptake b y mast cells of rats * and b y heparin of mast-cell tumors both in vivo 8 and in vitro*,~; and (4) the isolation of heparin from mast-cell tumors s. Although synthesis of heparin has been demonstrated in mastocytomas 8-5, there is no direct chemical evidence for the presence of heparin in the normal mast cell. In view of the hypothesis that hyaluronic acid is formed by mast cells s, it seemed desirable to obtain direct chemical evidence regarding the nature of the acid mucopolysaccharides present in m a s t cells. Mast cells were obtained from the peritoneal fluid of 5o0 normal, male SpragueDawley rats by the method of PADAWAR AND GORDON~, using a 0. 9 % NaCI solution containing o.15 % disodium ethylenediaminetetraacetate for flushing the peritoneal cavity. Approximately I. IOs m a s t cells were isolated from the peritoneal fluid of each rat. Differential counts indicated that these cells comprised 9 ° to 98 % of the total cell population of each preparation.
VOL. 31 (I959)
PRELIMINARY NOTES
279
The cells were washed with acetone, dissolved in a 2 % NaOH and dialyzed against distilled water for 72 h. The dialyzed material was digested at 37 ° with 5 mg crystalline trypsin in o.I M phosphate buffer, pH 7.8, for 5 days in a manner similar to that described previouslys. M t e r proteolysis, the extract was dialyzed against running tap water for 24h. (NH,),SO, was added to 0.6 satn. and the small precipitate that formed was removed by filtration. The filtrate was dialyzed thoroughly against distilled water and concentrated to a volume of 25 ml. Approximately one-half of the material was passed through a small column of Dowex-5 o (H+). Nitrogen, hexosamine and uronic acid (by the carbazole method) were determined on the effluent; ester sutfate, uronic acid and the specific rotation were measured prior to treatment with the ion-exchange resin. The analyses are shown in Table I. The amino sugar was identified as glucosamine by the method of STOFFYN AND JEANLOZTM. Paper chromatography, using a 40 % aqueous solution of tert.-butanol as the solvent system TM, showed the presence of a single substance with a mobility TABLE I ANALYSES OF HEPARIN OF RAT MAST CELLS Values expressed as m o l a r ratios w i t h h e x o s a m i n e t a k e n as I .oo Pr~aration Mast cell Na h e p a r i n * (I45 U / m g )
Hexosamin@*
N***
Uronic a~id~
S04~§
[¢1]D
I.OO i.oo
i. 78 1.42
1.38 1.39
3.13
-~- 54.0
* A c o m m e r c i a l p r e p a r a t i o n obtained f r o m General Biochemicals, Inc. ** A modification of t h e ELSO~-MORGAN m e t h o d ~ following hydrolysis in 4 N HC1 for 2 4 h t 100 °. *** B y the micro-Kjeldahl method. § B y t h e carbazole reaction l°. §| B y a colorimetric reaction with b a r i u m chloranilate n following h y d r o l y s i s in N HC1 for 2 h a t IO0 °.
identical to that of a commercial preparation of heparin. The rate of hydrolysis x4,16 was similar to that found for heparin and much slower than that of either the chondroitinsulfuric or hyaluronic acids. When the effect of the mucopolysacchafide on coagulation of blood was compared with that of a commercial preparation of beef heparin, it was found to possess only IO % of the activity of heparin. That rat heparin has a lower anticoagulant activity than beef or dog heparin has been noted by others TM. Although the anticoagulant activity of the substance is lower than that found in a preparation of beef heparin, the compound isolated from the mast cells behaved like heparin in all other respects. There was no indication for the presence of any other mucopolysaccharide. This is in marked disagreement with the view of ASBOEHANSENe regarding the mast cell as a source of hyaluronic acid. KORN4 has reported the presence of a chondroitinsulfuric acid fraction as well as heparin in mast-cell tumors of mice. MAGNUSSON AND LARSSON3 alSO obtained evidence of a sulfated mucopolysaccharide which contained galactosamine as the amino sugar in a preparation from a dog mastocytoma. These findings do not necessarily disagree with the present observation since the mast cells in tumors are held together by connective
280
PRELIMINARY
NOTES
V0L. 31 (1959)
tissue which undoubtedly contains chondroitinsulfuric acid. On the other hand, the mast cells harvested from the peritoneal fluid of rats as described in the present paper, were free of all other connective tissue components. The authors are indebted to Mrs. KATHRYN F. DEWEY for valuable technical assistance. This work was supported by grants from the National Heart Institute, U.S. Public Health Service (No. H-3II) and the Chicago Heart Association.
LaRabida-University o] Chicago Institute and the Departments o] Biochemistry and Pediatrics, University o] Chicago, Chicago, Ill. (U.S.A.)
SARA SCHILLER ALBERT DORFMAN
j . E. JORPES, Heparin, 2 n d ed., O x f o r d U n i v e r s i t y Press, L o n d o n , 1946. 2 j . E. JoRPES, E. ODEBLAD AND H. BOSTROM, Acta Haematol., 9 (1953) 273. 3 S. MAGNUSSON AND B. LARSSON, Acta Chem. Seand., 9 (1955) 534. * E. D. KORN, J. Am. Chem. Sos., 8o (1958) 152o. s L. SPOLTER AND W. MARK, Federation Pros., 17 (1958) 314. 6 G. ASBOE-HANSEN, Ann. Rheumatic Diseases, 9 (195 o) 1497 j . PADAWAR AND A. S. GORDON, Proc. Soc. Exptl. Biol. Med., 88 (1955) 29. 8 S. SCHILLER, M. B. MATHEWS, H. JEFFERSON, J. LUDOWlEG AND A. DORFMAN, J. Biol. Chem., 211 (1954) 717 . 9 L. A. ELSON AND W. T. J. MORGAN, Biochem. J., 27 (1933) 1824. i0 Z. DlSCHE, J. Biol. Chem., 167 (1947) 189. 11 R. J. BERTOLACINI AND J. E. BARNEY, Anal. Chem., 29 (I957) 281. 12 p. j . STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. xa j . E. SCOTT, Thesis, M a n c h e s t e r U n i v e r s i t y (1956). la j . E. JORPES AND S. GARDELL, J. Biol. Chem., 176 (1948) 267. is j . A. CIFONELLI AND A. DORFMAN, J. Biol. Chem., 231 (1958) i i . is F. C. MONKHOUSE, R. G. MAcKNESON AND G. BAMBERS, Proc. Soc. Exptl. Biol. Med., 95 (I957) 489 • 1
Received August 29th, 1958
Biosynthesis of mucopolysaccharides in bovine cornea Mucopolysaccharides are present in high concentration in the cornea, where together with the collagen fibers they are intimately involved in maintaining the transparency and structure of this specialized tissue. MEYER and coworkers z, ~ have separated crude corneal polysacchafide material into three acid mucopolysacchafide fractions, which together comprise about 2 % of the dry weight of this tissue: (I) keratosulfate, a polymer containing N-acetylglucosamine, galactose, and sulfate in equimolar proportions, (2) a chondroitin sulfate, and (3) a chondroitin. In the present communication, evidence is presented for the biosynthesis of these compounds in isolated adult corneas from 14C-labeled glucose. The general procedure employed was as foUows. Individual adult-bovine corneas were incubated with shaking in air at 38 ° in 3 ml of Krebs-Ringer carbonate buffer plus L-glutamine and uniformly labeled D-glucose. After thorough washing with distilled water, each cornea was hydrolyzed in I ml 4 N HC1 at 96-1oo ° in sealed tubes for 16 h. The hexosamines were isolated in pure form by adsorption and elution
PRELIMINARY NOTES
VOL. 31 (1959)
Based on the preceding observations, it is possible to propose for Compound A the formula of a 4-acetamido-2-amino-2,4,6-tfideoxyhexose and for Compound B the formula of a 2,4-diacetamido-2,4,6-trideoxyhexose. Thus, it appears to be the first isolation of a diaminohexose from natural sources. This work was supported b y grants from the American Cancer Society and from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service (Grant F-I965). NATHAN SHARON* The Robert W. Lovett Foundation, Massachusetts ROGER W. JEANLOZ** General Hospital and the Department o[ Medicine, Harvard Medical School, Boston, Mass, (U.S.A.) N. SI~ARON,Nature, 179 (1957) 919. 2 S. GARDELL,Acta Chem. Stand., 7 (1953) 207. 8 M. SOMOGYI,J. Biol. Chem., 195 (1952) 19; N. NBLSON,J. Biol. Chem., 153 (1944) 375. E. C. COCKINGAND E. W. Y~MM,Biochem. J., 58 (1954) xii. 6 M. V. TRACEY,Biochem. J., 52 (1952) 265. * R. J. BLOCKAND D. BOLLING,The Amino Acid Composition o] Proteins and Foods, 2rid ed., C.C. Thomas, Publ., Springfield, 1951, p. 261. J. F. O'DEA AND R. A. GIBBONS,Biochem. J., 55 (1953) 58o. s A. S. PERLIN,J. Am. Chem. Soc., 76 (1954) 41Ol. z
Received September 29th, 1958 * On leave of absence from the Weizmann Institute, Rehovoth, Israel. ** Special Investigator of the Arthritis and Rheumatism Foundation.
The isolation of heparin from mast cells of the normal rat Evidence for the production of heparin b y m a s t cells has been obtained by a variety of indirect techniques. These include the demonstration of (I) metachromasia in cells stained with toluidine blueZ; (2) a parallelism between the concentration of mast cells and apparent heparin content of tissues 1; (3) mS uptake b y mast cells of rats * and b y heparin of mast-cell tumors both in vivo 8 and in vitro*,~; and (4) the isolation of heparin from mast-cell tumors s. Although synthesis of heparin has been demonstrated in mastocytomas 8-5, there is no direct chemical evidence for the presence of heparin in the normal mast cell. In view of the hypothesis that hyaluronic acid is formed by mast cells s, it seemed desirable to obtain direct chemical evidence regarding the nature of the acid mucopolysaccharides present in m a s t cells. Mast cells were obtained from the peritoneal fluid of 5o0 normal, male SpragueDawley rats by the method of PADAWAR AND GORDON~, using a 0. 9 % NaCI solution containing o.15 % disodium ethylenediaminetetraacetate for flushing the peritoneal cavity. Approximately I. IOs m a s t cells were isolated from the peritoneal fluid of each rat. Differential counts indicated that these cells comprised 9 ° to 98 % of the total cell population of each preparation.
VOL. 31 (I959)
PRELIMINARY NOTES
279
The cells were washed with acetone, dissolved in a 2 % NaOH and dialyzed against distilled water for 72 h. The dialyzed material was digested at 37 ° with 5 mg crystalline trypsin in o.I M phosphate buffer, pH 7.8, for 5 days in a manner similar to that described previouslys. M t e r proteolysis, the extract was dialyzed against running tap water for 24h. (NH,),SO, was added to 0.6 satn. and the small precipitate that formed was removed by filtration. The filtrate was dialyzed thoroughly against distilled water and concentrated to a volume of 25 ml. Approximately one-half of the material was passed through a small column of Dowex-5 o (H+). Nitrogen, hexosamine and uronic acid (by the carbazole method) were determined on the effluent; ester sutfate, uronic acid and the specific rotation were measured prior to treatment with the ion-exchange resin. The analyses are shown in Table I. The amino sugar was identified as glucosamine by the method of STOFFYN AND JEANLOZTM. Paper chromatography, using a 40 % aqueous solution of tert.-butanol as the solvent system TM, showed the presence of a single substance with a mobility TABLE I ANALYSES OF HEPARIN OF RAT MAST CELLS Values expressed as m o l a r ratios w i t h h e x o s a m i n e t a k e n as I .oo Pr~aration Mast cell Na h e p a r i n * (I45 U / m g )
Hexosamin@*
N***
Uronic a~id~
S04~§
[¢1]D
I.OO i.oo
i. 78 1.42
1.38 1.39
3.13
-~- 54.0
* A c o m m e r c i a l p r e p a r a t i o n obtained f r o m General Biochemicals, Inc. ** A modification of t h e ELSO~-MORGAN m e t h o d ~ following hydrolysis in 4 N HC1 for 2 4 h t 100 °. *** B y the micro-Kjeldahl method. § B y t h e carbazole reaction l°. §| B y a colorimetric reaction with b a r i u m chloranilate n following h y d r o l y s i s in N HC1 for 2 h a t IO0 °.
identical to that of a commercial preparation of heparin. The rate of hydrolysis x4,16 was similar to that found for heparin and much slower than that of either the chondroitinsulfuric or hyaluronic acids. When the effect of the mucopolysacchafide on coagulation of blood was compared with that of a commercial preparation of beef heparin, it was found to possess only IO % of the activity of heparin. That rat heparin has a lower anticoagulant activity than beef or dog heparin has been noted by others TM. Although the anticoagulant activity of the substance is lower than that found in a preparation of beef heparin, the compound isolated from the mast cells behaved like heparin in all other respects. There was no indication for the presence of any other mucopolysaccharide. This is in marked disagreement with the view of ASBOEHANSENe regarding the mast cell as a source of hyaluronic acid. KORN4 has reported the presence of a chondroitinsulfuric acid fraction as well as heparin in mast-cell tumors of mice. MAGNUSSON AND LARSSON3 alSO obtained evidence of a sulfated mucopolysaccharide which contained galactosamine as the amino sugar in a preparation from a dog mastocytoma. These findings do not necessarily disagree with the present observation since the mast cells in tumors are held together by connective
280
PRELIMINARY
NOTES
V0L. 31 (1959)
tissue which undoubtedly contains chondroitinsulfuric acid. On the other hand, the mast cells harvested from the peritoneal fluid of rats as described in the present paper, were free of all other connective tissue components. The authors are indebted to Mrs. KATHRYN F. DEWEY for valuable technical assistance. This work was supported by grants from the National Heart Institute, U.S. Public Health Service (No. H-3II) and the Chicago Heart Association.
LaRabida-University o] Chicago Institute and the Departments o] Biochemistry and Pediatrics, University o] Chicago, Chicago, Ill. (U.S.A.)
SARA SCHILLER ALBERT DORFMAN
j . E. JORPES, Heparin, 2 n d ed., O x f o r d U n i v e r s i t y Press, L o n d o n , 1946. 2 j . E. JoRPES, E. ODEBLAD AND H. BOSTROM, Acta Haematol., 9 (1953) 273. 3 S. MAGNUSSON AND B. LARSSON, Acta Chem. Seand., 9 (1955) 534. * E. D. KORN, J. Am. Chem. Sos., 8o (1958) 152o. s L. SPOLTER AND W. MARK, Federation Pros., 17 (1958) 314. 6 G. ASBOE-HANSEN, Ann. Rheumatic Diseases, 9 (195 o) 1497 j . PADAWAR AND A. S. GORDON, Proc. Soc. Exptl. Biol. Med., 88 (1955) 29. 8 S. SCHILLER, M. B. MATHEWS, H. JEFFERSON, J. LUDOWlEG AND A. DORFMAN, J. Biol. Chem., 211 (1954) 717 . 9 L. A. ELSON AND W. T. J. MORGAN, Biochem. J., 27 (1933) 1824. i0 Z. DlSCHE, J. Biol. Chem., 167 (1947) 189. 11 R. J. BERTOLACINI AND J. E. BARNEY, Anal. Chem., 29 (I957) 281. 12 p. j . STOFFYN AND R. W. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. xa j . E. SCOTT, Thesis, M a n c h e s t e r U n i v e r s i t y (1956). la j . E. JORPES AND S. GARDELL, J. Biol. Chem., 176 (1948) 267. is j . A. CIFONELLI AND A. DORFMAN, J. Biol. Chem., 231 (1958) i i . is F. C. MONKHOUSE, R. G. MAcKNESON AND G. BAMBERS, Proc. Soc. Exptl. Biol. Med., 95 (I957) 489 • 1
Received August 29th, 1958
Biosynthesis of mucopolysaccharides in bovine cornea Mucopolysaccharides are present in high concentration in the cornea, where together with the collagen fibers they are intimately involved in maintaining the transparency and structure of this specialized tissue. MEYER and coworkers z, ~ have separated crude corneal polysacchafide material into three acid mucopolysacchafide fractions, which together comprise about 2 % of the dry weight of this tissue: (I) keratosulfate, a polymer containing N-acetylglucosamine, galactose, and sulfate in equimolar proportions, (2) a chondroitin sulfate, and (3) a chondroitin. In the present communication, evidence is presented for the biosynthesis of these compounds in isolated adult corneas from 14C-labeled glucose. The general procedure employed was as foUows. Individual adult-bovine corneas were incubated with shaking in air at 38 ° in 3 ml of Krebs-Ringer carbonate buffer plus L-glutamine and uniformly labeled D-glucose. After thorough washing with distilled water, each cornea was hydrolyzed in I ml 4 N HC1 at 96-1oo ° in sealed tubes for 16 h. The hexosamines were isolated in pure form by adsorption and elution