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Lipids associated with tissue transglutaminase
Biochimica et Biophvsica Acta 923 (I987) 42-45 Elsevier
42
BBA 22476
Lipids associated with tissue transglutaminase Jolan Harsfalvi, Gabriella Arato and Laszlo Fesus 2nd Department of Medicine, 3rd Department of Medicine and Department of Bioptlvsics, Universi O, School of Medicine, H-4012 Debrecen (Hungao') (Received 28 July 1986)
Key words: Transglutaminase; Neutral lipid; Phospholipid: Covalent modification: Lauric acid; Membrane association
A substantial amount of lipids (cholesterol and its esters, mono-, di- and triacyiglycerols, free fatty acids and the phospholipids phosphatidylethanolamine and phosphatidylinositol) was found associated with tissue transglutaminase purified to apparent homogeneity from guinea pig liver. Removal of lipids results in an increased tendency of the enzyme for self-association and a decreased stability. Lauric acid was detected following hydroxylamine treatment of the enzyme, suggesting the occurrence of a fatty acid-type, covalent, posttranslational modification of transglutaminase. The results provide support for the idea that part of tissue transglutaminase may be localized in the cell membrane. Introduction Transglutaminases are Ca2+-dependent enzymes catalyzing an acyl transfer reaction between peptide-bound glutamine and the primary amino group of various amines including the e-amino group of properly positioned lysine in substrate proteins [1]. Five isoenzyme forms of transglutaminase have been described so far, namely blood coagulation factor XIII in plasma, the epidermal, prostatic and hair follicle transglutaminases, and the ubiquitous tissue ('liver') type in cells [1-3]. Varying portions of the cellular transglutaminase have been found associated with the membrane fraction following tissue or cell homogenization [3-8], suggesting that under certain conditions this enzyme is localized in or associated with the cell membrane in living cells and tissues. There are no biochemical data, however, to explain the mechanism of the membrane localization Correspondence: Dr. L. Fesus, Department of Biophysics, University Medical School of Debrecen, H-4012 Debrecen, Hungary.
of transglutaminases. We have recently found that a specific interaction occurs between purified liver transglutaminase and small unilamellar phos± pholipid vesicles at the lipid phase transition [9]. Now we present data showing that a substantial amount of lipid material, including covalently bound fatty acid, is associated with purified, guinea pig liver transglutaminase. Materials and Methods
Transglutaminase. Guinea pig liver transglutaminase was purified according to Connellan et al. [10]. Its activity was assayed by measuring ammonia released during the transfer reaction between benzyloxycarbonyl-L-glutamylglycine and methylamine [9]. Electrophoretic behavior was studied using alkaline urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [11,12]. Lipid extraction and analysis. Lipids were extracted from transglutaminase protein solution by chloroform/methanol (2:1, v/v; [13]). Mild extraction was carried out at 4°C by light petroleum/n-butanol (7 : 3, v/v; [14]). Organic solvents
0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
43
were evaporated, the dry lipid was dissolved in chloroform and stored under nitrogen at - 3 0 ° C until the analysis. Aliquots were used for quantitation of total lipids and phospholipids [15,16], then a portion was spotted on a high performance thin layer plate (HPTLC plates silicagel 60 MERCK) to determine lipid pattern. The plate was developed in light petroleum/diethyl ether/acetic acid (82 : 18 : 1, v/v). Phospholipids which remained in the origin were scraped from the previous plate, resolved, applied to another plate and separated in chloroform/methanol/isopropanol/0.25 % aqueous potassium chloride/acetic acid (60 : 18 : 50 : 12: 36, v/v). Lipids were detected by conventional staining procedures [17] and identified comparing them to standards. Cholesterol and its esters, tri-, di- and monoacylglycerols, and fatty acids were obtained from Sigma; phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from Applied Science. All materials were reagent grade, and the organic solvents were destilled before use. Protein-bound fatty acid. Transglutaminase, which had been extracted by light petroleum/nbutanol, was treated with hydroxylamine (1 mol/1, pH 7.0, for 6 h) then the protein-free aqueous solution obtained by centrifree Micropartition (Amicon) separation was extracted with eleven parts of chloroform/methanol (2:1, v/v) and analyzed by gas chromatography. Samples treated with diazomethane/diethyl ether solution to transform fatty acid hydrroxamates to fatty acid methyl esters, were injected into the gas chromatograph (Hewlett Packard, 5840 A) equipped with flame ionization detector (3000C), and analyzed at 105-300°C (heating rate 5 Cdeg/min) on a column packed with 10% OV-1 on Gaschrom Q 80-100 mesh. Results and Discussion
A substantial amount of lipids could be extracted from the purified transglutaminase solution by chloroform/methanol; varying from batch to batch it was in the range of 13-120 /~g/mg transglutaminase protein. Qualitative analysis of the extracted material showed mono-, di- and triacylglycerols, cholesterol and its esters, free fatty acids and phospholipids (Fig. la). The latter constituted about 2-5% of the total lipid by weight
a
b
! I
I
1
2
3
4
5
6 TGose
1
2
3
t
TGose
Fig. i. Thin layer chromatography of lipids extracted from transglutaminase (Tgase). Panel a: neutral lipids; cholesterol ester (1), triacylglycerol (2), free fatty acid (3), cholesterol (4), diacylglycerol (5), monoacylglycerol (6) are standards. Panel b: phospholipids, obtained from origin of the previous plate; phosphatidylethanolamine (1), phosphatidylserine (2), phosphatidylinositol (3) are standards. 200 ~g extracted lipid was applied onto the first plate.
and was mainly phosphatidylethanolamine and phosphatidylinositol (Fig. lb). The procedures for the purification of the guinea pig liver transglutaminase involves (following homogenization of liver in sucrose and ultracentrifugation) three ion exchange chromatography steps and one gel filtration step, a precipitation with ammonium sulfate and another with protamine sulfate [10]. The fact that the purified protein still carries lipids suggests a strong association between them and the possibility that some of the enzyme molecules may be originally localized in a lipid environment and then released during homogenization. The extraction and analytical procedures were repeated using different batches of transglutaminase preparations including one which was a kind gift of Dr. S.I. Chung (Enzyme Chemistry Section, NIDR, NIH, Bethesda, MD); the results were essentially the same, differing only in the quantity of total lipids associated with the enzyme. As shown in Fig. 2, the electrophoretic behavior of transglutaminase did not change following the extraction of lipids and their storage at + 4 ° C in the presence of EGTA and dithiothreitol. An increased tendency for self-association was observed during incubation of the enzyme at 37 ° C following lipid extraction (Fig. 2a). Specific activity of the enzyme decreased about 50% following
44
b
a
l
p__~
,,-~p
1
1
l
o~
o! ca 0a
o ~
ac
d
ABCD
a
c
mi
d
A B
~
-,-80
CD
Fig. 2. Polyacrylamide gel electrophoresis of transglutaminase. Panel a: alkaline urea gel (10% acrylamide): a, c, d columns before, and A, B, C, D columns after delipidation and incubation at 37 °C in the absence of Ca 2 ~ for 0 (a,A), 20 (B), 60 (c,C) rain or in the presence of 10 m M Ca 2+ for 60 rain (d, D). Panel b: sodium dodecyl sulfate gel (4% acrylamide): a. c , d, A, B, C, D samples were prepared parallel to those for alkaline urea polyacrylamide gel electrophoresis, p indicates high molecular weight polymers not entering separating gels. 80 shows the position of 80 k D a protein (molecular mass of transglutaminase) as determined by molecular weight standards.
the removal of lipids (data not shown). Enzymatic self cross-linkage [18] of the intact transglutaminase as well as the enzyme treated with organic solvent occurred when incubations were carried out at 37 ° C in the presence of Ca 2 +. Following the treatment of lipid-depleted transglutaminase protein with hydroxylamine (a m e t h o d generally used to uncouple ester-linked fatty acids from proteins [19]), a fatty acid could be detected in the c h l o r o f o r m / m e t h a n o l extract of the protein-free solution. Using appropriate standards the fatty acid was identified as lauric acid (Fig. 3). This was 0.5-1.4 mol per mol transglutaminase (using 80000 D a as the molecular mass of the enzyme in the calculation). It was not found without the addition of hydroxylamine. Recently, fatty acid modification of several viral and cellular proteins (including oncogen coded m e m b r a n e proteins) have been observed [20-24] and these data have been convincingly used to explain the membrane localization of these proteins. Protein-bound fatty acids may serve as signals for m e m b r a n e targeting and contribute to either an anchorage to the inner surface of the cell m e m b r a n e or to a
5
1"0
75 "20 rain • Retention time
Fig. 3. Gas chromatography profile of the chloroform/ methanol extract prepared from the protein-free aqueous solution of hydroxylamine-treated transglutaminase (300 ~g protein). Before hydroxylamine treatment the enzyme was ex-
tracted with light petroleum/n-butanol (7:3, v/v) at 4°C). $ with number represents the retention time of saturated fatty acid standards with given carbon atoms as determined in a separate run. tight binding of integral m e m b r a n e proteins to specific lipid structures in the bilayer of the membrane. In the case of transglutaminases, several investigators have demonstrated varying degrees of association of this enzyme with the m e m b r a n e fraction prepared from cells and tissues [3-8,25, 26]. Although the tissue (liver) type of transglutaminase seems to be mostly cytoplasmic in cultured cells [27], quite a high proportion of it was found in the m e m b r a n e fraction prepared from tissues such as liver [4,25] and lung [8]. The localization of epidermal transglutaminase is almost exclusively the cell m e m b r a n e [3,5,26]. Since we used a tissue to purify the enzyme it is not possible to decide what cell type was the source of the enzyme-lipid complex. However, our transglutaminase preparation is antigenically homogeneous: it does not cross-react with antibodies raised against epidermal, hair follicle and blood coagulation factor X I I I transglutaminases (data not shown). Therefore, the most likely explanation
45 of our f i n d i n g is that tissue t r a n s g l u t a m i n a s e in liver is partially m e m b r a n e - a s s o c i a t e d [4,25], because of fatty acylation a n d the o b s e r v a t i o n that a p o r t i o n of the m e m b r a n e - l o c a l i z e d t r a n s g l u t a m inase is released d u r i n g the h o m o g e n i z a t i o n procedure carrying some of the lipids with which it was associated in the bilayer. We have previously shown that an interaction takes place b e t w e e n purified liver t r a n s g l u t a m inase a n d small u n i l a m e l l a r p h o s p h o l i p i d vesicles at the lipid phase transition [9]. T h e activity of t r a n s g l u t a m i n a s e inserted into the lipid layer is greatly reduced [9]. Interestingly, a n increased t r a n s g l u t a m i n a s e activity can be measured in lysates of several kinds of cells shortly following their s t i m u l a t i o n ; e.g., in lymphocytes treated with mitogens [28], in macrophages d u r i n g the process of i m m u n e - c o m p l e x - i n d u c e d phagocytosis [29], in mast cells u p o n i m m u n o l o g i c a n d n o n - i m m u n o logic stimuli to release h i s t a m i n e [30], in isoprot e r e n o l - s t i m u l a t e d glioma cells [31], in sympathetic ganglion after a x o t o m y [32], a n d in liver following h e p a t e c t o m y [33]. Since the i n h i b i t i o n of p r o t e i n synthesis did n o t prevent the increased activities, it is possible that enzyme molecules are released from the lipid e n v i r o n m e n t related to cell activation. As a consequence, more catalytically active t r a n s g l u t a m i n a s e molecules are available, the m e a s u r a b l e activity is higher a n d a n increased a m o u n t of t r a n s g l u t a m i n a s e p r o d u c t (like p r o t e i n b o u n d ~/-glutamylhistamine in mast cells [30]) can be detected. I n such a n interpretation, m e m b r a n e - b o u n d t r a n s g l u t a m i n a s e m a y represent a properly positioned inactive ('cryptic') e n z y m e pool which is readily activated by its release from the m e m b r a n e a n d the increasing Ca 2+ c o n c e n t r a tion [34] d u r i n g the biochemical process of transm e m b r a n e signaling.
Acknowledgement We gratefully acknowledge the help of J. H a r a n g i (Biochemical D e p a r t m e n t of L. K o s s u t h University) in p e r f o r m i n g the gas chromatographic analysis.
References 1 Folk, J.E. (1980) Annu. Rev. Biochem. 49, 517-531 2 Chung, S.I. (1975) in Isoenzymes, Vol. 1 (Market, C.L., ed.), pp. 259-272, Academic Press, New York
3 Thacher, S.M. and Rice, R.H. (1985) Cell 40, 685-695 4 Slife, C.W., Dorsett, M.D., Bouquett, G.T., Register, A., Taylor, E. and Conroy, S. (1985) Arch. Biochem Biophys. 241,329-336 5 Lichti, U., Ben, T. and Yuspa, S.H. (1985) J. Biol. Chem. 260, 1422-1426 6 Schmidt, R., Reichert, U., Michael, S., Shroot, B. and Bouclier, M. (1985) FEBS Lett. 186, 201-204 7 Bures, M.D., Goldsmith, L.A. and Stone, K.R. (1980) Invest. Urol. 17, 298-304 8 Chang, S.K. and Chung, S.I. (1986) J. Biol. Chem. 261, 8112-8121 9 Fesus, L., Horvath, A. and Harsfalvi, J. (1983) FEBS Lett. 155, 1-5 10 Connellan, J.M., Chung, S.I., Whetzel, N.K., Bradley, L.M. and Folk, J.E. (1970) J. Biol. Chem. 246, 1093-1096 11 Head, J.F. and Perry, S.V. (1974) Biochem. J. 137, 145-153 12 Laemmli, U.K. (1970) Nature 277, 680-685 13 Bowyer, D.E. and King, J.P. (1977) J. Chromatogr. 143, 473-490 14 Emmelot, T.P. and Bos, C.J. (1968) Biochim. Biophys. Acta 150, 341-353 15 Bragdon, J.H. (1951) J. Biol. Chem. 190, 513 517 16 Itaya, K. and Ui, M. (1966) Clin. Chim. Acta 14, 361-366 17 Kates, M. (1972) in Techniques of lipidology (Work, T.S. and Burden, R., eds.), pp. 335-441, North-Holland, Amsterdam 18 Birckbichler, P.J., Orr, G.R., Carter, H.A. and Patterson, M.F., Jr. (1977) Biochim. Biophys. Res. Commun. 78, 1-5 19 Schlesinger, M.J. and Magee, A.I. (1982) Biophys. J. 37, 126-127 20 Schlesinger, M.J. (1981) Annu. Rev. Biochem. 50, 193-206 21 Dahl, C.E., Dahl, J.S. and Bloch, K. (1983) J. Biol. Chem. 258, 11814-11818 22 Henderson, L.E., Krutzsch, H.C. and Oroszlan, S. (1983) Proc. Natl. Acad. Sci. USA 80, 339-343 23 Berger, M. and Schmidt, M.F.G. (1984) J. Biol. Chem. 259, 5364-5367 24 Kaufman, J.F., Krangel, M.S. and Strominger, J.L. (1984) J. Biol. Chem. 259, 7230-7238 25 Barnes, R.N., Bungay, P.J., Elliott, B.M., Walton, P.L. and Griffin, M. (1985) Carcinogenesis 6, 459-463 26 Simon, M. and Green, H. (1985) Cell 40, 677-683 27 Davies, P.J.A., Cornwell, M.M., Johnson, J.D., Reggioni, A., Myer, M. and Murtaugh, M.P. (1984) Diab. Care 7, Suppl. 1, 35-41 28 Novogrodsky, A., Quittener, S., Rubin, A.L. and Stenzel, K.H. (1978) Proc. Natl. Acad. Sci. USA 75, 1157-1161 29 Fesus, L., Sandor, M., Horvath, A., Bagyinka, C., Erdei, A. and Gergely, A. (1981) Mol. Immunol. 18, 633-638 30 Fesus, L., Szucs, E.F., Barrett, K., Metcalfe, D.E. and Folk, J.E. (1985) J. Biol. Chem. 260, 13771-13779 3i Komer, G. and Bachrach, U. (1985) J. Cell. Physiol. 124, 379-385 32 Gilad, G.M., Varon, L.E. and Gila& V.H. (1985) J. Neurochem. 44, 1385-1390 33 Haddox, M.K. and Russel, D.H. (1981) Proc. Natl. Acad. Sci. USA 78, 1712-1716 34 Fesus, L., Harsfalvi, J., Horvath, A. and Sandor, M. (1984) Mol. Immunol. 21, 1161-1165
42
BBA 22476
Lipids associated with tissue transglutaminase Jolan Harsfalvi, Gabriella Arato and Laszlo Fesus 2nd Department of Medicine, 3rd Department of Medicine and Department of Bioptlvsics, Universi O, School of Medicine, H-4012 Debrecen (Hungao') (Received 28 July 1986)
Key words: Transglutaminase; Neutral lipid; Phospholipid: Covalent modification: Lauric acid; Membrane association
A substantial amount of lipids (cholesterol and its esters, mono-, di- and triacyiglycerols, free fatty acids and the phospholipids phosphatidylethanolamine and phosphatidylinositol) was found associated with tissue transglutaminase purified to apparent homogeneity from guinea pig liver. Removal of lipids results in an increased tendency of the enzyme for self-association and a decreased stability. Lauric acid was detected following hydroxylamine treatment of the enzyme, suggesting the occurrence of a fatty acid-type, covalent, posttranslational modification of transglutaminase. The results provide support for the idea that part of tissue transglutaminase may be localized in the cell membrane. Introduction Transglutaminases are Ca2+-dependent enzymes catalyzing an acyl transfer reaction between peptide-bound glutamine and the primary amino group of various amines including the e-amino group of properly positioned lysine in substrate proteins [1]. Five isoenzyme forms of transglutaminase have been described so far, namely blood coagulation factor XIII in plasma, the epidermal, prostatic and hair follicle transglutaminases, and the ubiquitous tissue ('liver') type in cells [1-3]. Varying portions of the cellular transglutaminase have been found associated with the membrane fraction following tissue or cell homogenization [3-8], suggesting that under certain conditions this enzyme is localized in or associated with the cell membrane in living cells and tissues. There are no biochemical data, however, to explain the mechanism of the membrane localization Correspondence: Dr. L. Fesus, Department of Biophysics, University Medical School of Debrecen, H-4012 Debrecen, Hungary.
of transglutaminases. We have recently found that a specific interaction occurs between purified liver transglutaminase and small unilamellar phos± pholipid vesicles at the lipid phase transition [9]. Now we present data showing that a substantial amount of lipid material, including covalently bound fatty acid, is associated with purified, guinea pig liver transglutaminase. Materials and Methods
Transglutaminase. Guinea pig liver transglutaminase was purified according to Connellan et al. [10]. Its activity was assayed by measuring ammonia released during the transfer reaction between benzyloxycarbonyl-L-glutamylglycine and methylamine [9]. Electrophoretic behavior was studied using alkaline urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [11,12]. Lipid extraction and analysis. Lipids were extracted from transglutaminase protein solution by chloroform/methanol (2:1, v/v; [13]). Mild extraction was carried out at 4°C by light petroleum/n-butanol (7 : 3, v/v; [14]). Organic solvents
0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
43
were evaporated, the dry lipid was dissolved in chloroform and stored under nitrogen at - 3 0 ° C until the analysis. Aliquots were used for quantitation of total lipids and phospholipids [15,16], then a portion was spotted on a high performance thin layer plate (HPTLC plates silicagel 60 MERCK) to determine lipid pattern. The plate was developed in light petroleum/diethyl ether/acetic acid (82 : 18 : 1, v/v). Phospholipids which remained in the origin were scraped from the previous plate, resolved, applied to another plate and separated in chloroform/methanol/isopropanol/0.25 % aqueous potassium chloride/acetic acid (60 : 18 : 50 : 12: 36, v/v). Lipids were detected by conventional staining procedures [17] and identified comparing them to standards. Cholesterol and its esters, tri-, di- and monoacylglycerols, and fatty acids were obtained from Sigma; phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from Applied Science. All materials were reagent grade, and the organic solvents were destilled before use. Protein-bound fatty acid. Transglutaminase, which had been extracted by light petroleum/nbutanol, was treated with hydroxylamine (1 mol/1, pH 7.0, for 6 h) then the protein-free aqueous solution obtained by centrifree Micropartition (Amicon) separation was extracted with eleven parts of chloroform/methanol (2:1, v/v) and analyzed by gas chromatography. Samples treated with diazomethane/diethyl ether solution to transform fatty acid hydrroxamates to fatty acid methyl esters, were injected into the gas chromatograph (Hewlett Packard, 5840 A) equipped with flame ionization detector (3000C), and analyzed at 105-300°C (heating rate 5 Cdeg/min) on a column packed with 10% OV-1 on Gaschrom Q 80-100 mesh. Results and Discussion
A substantial amount of lipids could be extracted from the purified transglutaminase solution by chloroform/methanol; varying from batch to batch it was in the range of 13-120 /~g/mg transglutaminase protein. Qualitative analysis of the extracted material showed mono-, di- and triacylglycerols, cholesterol and its esters, free fatty acids and phospholipids (Fig. la). The latter constituted about 2-5% of the total lipid by weight
a
b
! I
I
1
2
3
4
5
6 TGose
1
2
3
t
TGose
Fig. i. Thin layer chromatography of lipids extracted from transglutaminase (Tgase). Panel a: neutral lipids; cholesterol ester (1), triacylglycerol (2), free fatty acid (3), cholesterol (4), diacylglycerol (5), monoacylglycerol (6) are standards. Panel b: phospholipids, obtained from origin of the previous plate; phosphatidylethanolamine (1), phosphatidylserine (2), phosphatidylinositol (3) are standards. 200 ~g extracted lipid was applied onto the first plate.
and was mainly phosphatidylethanolamine and phosphatidylinositol (Fig. lb). The procedures for the purification of the guinea pig liver transglutaminase involves (following homogenization of liver in sucrose and ultracentrifugation) three ion exchange chromatography steps and one gel filtration step, a precipitation with ammonium sulfate and another with protamine sulfate [10]. The fact that the purified protein still carries lipids suggests a strong association between them and the possibility that some of the enzyme molecules may be originally localized in a lipid environment and then released during homogenization. The extraction and analytical procedures were repeated using different batches of transglutaminase preparations including one which was a kind gift of Dr. S.I. Chung (Enzyme Chemistry Section, NIDR, NIH, Bethesda, MD); the results were essentially the same, differing only in the quantity of total lipids associated with the enzyme. As shown in Fig. 2, the electrophoretic behavior of transglutaminase did not change following the extraction of lipids and their storage at + 4 ° C in the presence of EGTA and dithiothreitol. An increased tendency for self-association was observed during incubation of the enzyme at 37 ° C following lipid extraction (Fig. 2a). Specific activity of the enzyme decreased about 50% following
44
b
a
l
p__~
,,-~p
1
1
l
o~
o! ca 0a
o ~
ac
d
ABCD
a
c
mi
d
A B
~
-,-80
CD
Fig. 2. Polyacrylamide gel electrophoresis of transglutaminase. Panel a: alkaline urea gel (10% acrylamide): a, c, d columns before, and A, B, C, D columns after delipidation and incubation at 37 °C in the absence of Ca 2 ~ for 0 (a,A), 20 (B), 60 (c,C) rain or in the presence of 10 m M Ca 2+ for 60 rain (d, D). Panel b: sodium dodecyl sulfate gel (4% acrylamide): a. c , d, A, B, C, D samples were prepared parallel to those for alkaline urea polyacrylamide gel electrophoresis, p indicates high molecular weight polymers not entering separating gels. 80 shows the position of 80 k D a protein (molecular mass of transglutaminase) as determined by molecular weight standards.
the removal of lipids (data not shown). Enzymatic self cross-linkage [18] of the intact transglutaminase as well as the enzyme treated with organic solvent occurred when incubations were carried out at 37 ° C in the presence of Ca 2 +. Following the treatment of lipid-depleted transglutaminase protein with hydroxylamine (a m e t h o d generally used to uncouple ester-linked fatty acids from proteins [19]), a fatty acid could be detected in the c h l o r o f o r m / m e t h a n o l extract of the protein-free solution. Using appropriate standards the fatty acid was identified as lauric acid (Fig. 3). This was 0.5-1.4 mol per mol transglutaminase (using 80000 D a as the molecular mass of the enzyme in the calculation). It was not found without the addition of hydroxylamine. Recently, fatty acid modification of several viral and cellular proteins (including oncogen coded m e m b r a n e proteins) have been observed [20-24] and these data have been convincingly used to explain the membrane localization of these proteins. Protein-bound fatty acids may serve as signals for m e m b r a n e targeting and contribute to either an anchorage to the inner surface of the cell m e m b r a n e or to a
5
1"0
75 "20 rain • Retention time
Fig. 3. Gas chromatography profile of the chloroform/ methanol extract prepared from the protein-free aqueous solution of hydroxylamine-treated transglutaminase (300 ~g protein). Before hydroxylamine treatment the enzyme was ex-
tracted with light petroleum/n-butanol (7:3, v/v) at 4°C). $ with number represents the retention time of saturated fatty acid standards with given carbon atoms as determined in a separate run. tight binding of integral m e m b r a n e proteins to specific lipid structures in the bilayer of the membrane. In the case of transglutaminases, several investigators have demonstrated varying degrees of association of this enzyme with the m e m b r a n e fraction prepared from cells and tissues [3-8,25, 26]. Although the tissue (liver) type of transglutaminase seems to be mostly cytoplasmic in cultured cells [27], quite a high proportion of it was found in the m e m b r a n e fraction prepared from tissues such as liver [4,25] and lung [8]. The localization of epidermal transglutaminase is almost exclusively the cell m e m b r a n e [3,5,26]. Since we used a tissue to purify the enzyme it is not possible to decide what cell type was the source of the enzyme-lipid complex. However, our transglutaminase preparation is antigenically homogeneous: it does not cross-react with antibodies raised against epidermal, hair follicle and blood coagulation factor X I I I transglutaminases (data not shown). Therefore, the most likely explanation
45 of our f i n d i n g is that tissue t r a n s g l u t a m i n a s e in liver is partially m e m b r a n e - a s s o c i a t e d [4,25], because of fatty acylation a n d the o b s e r v a t i o n that a p o r t i o n of the m e m b r a n e - l o c a l i z e d t r a n s g l u t a m inase is released d u r i n g the h o m o g e n i z a t i o n procedure carrying some of the lipids with which it was associated in the bilayer. We have previously shown that an interaction takes place b e t w e e n purified liver t r a n s g l u t a m inase a n d small u n i l a m e l l a r p h o s p h o l i p i d vesicles at the lipid phase transition [9]. T h e activity of t r a n s g l u t a m i n a s e inserted into the lipid layer is greatly reduced [9]. Interestingly, a n increased t r a n s g l u t a m i n a s e activity can be measured in lysates of several kinds of cells shortly following their s t i m u l a t i o n ; e.g., in lymphocytes treated with mitogens [28], in macrophages d u r i n g the process of i m m u n e - c o m p l e x - i n d u c e d phagocytosis [29], in mast cells u p o n i m m u n o l o g i c a n d n o n - i m m u n o logic stimuli to release h i s t a m i n e [30], in isoprot e r e n o l - s t i m u l a t e d glioma cells [31], in sympathetic ganglion after a x o t o m y [32], a n d in liver following h e p a t e c t o m y [33]. Since the i n h i b i t i o n of p r o t e i n synthesis did n o t prevent the increased activities, it is possible that enzyme molecules are released from the lipid e n v i r o n m e n t related to cell activation. As a consequence, more catalytically active t r a n s g l u t a m i n a s e molecules are available, the m e a s u r a b l e activity is higher a n d a n increased a m o u n t of t r a n s g l u t a m i n a s e p r o d u c t (like p r o t e i n b o u n d ~/-glutamylhistamine in mast cells [30]) can be detected. I n such a n interpretation, m e m b r a n e - b o u n d t r a n s g l u t a m i n a s e m a y represent a properly positioned inactive ('cryptic') e n z y m e pool which is readily activated by its release from the m e m b r a n e a n d the increasing Ca 2+ c o n c e n t r a tion [34] d u r i n g the biochemical process of transm e m b r a n e signaling.
Acknowledgement We gratefully acknowledge the help of J. H a r a n g i (Biochemical D e p a r t m e n t of L. K o s s u t h University) in p e r f o r m i n g the gas chromatographic analysis.
References 1 Folk, J.E. (1980) Annu. Rev. Biochem. 49, 517-531 2 Chung, S.I. (1975) in Isoenzymes, Vol. 1 (Market, C.L., ed.), pp. 259-272, Academic Press, New York
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