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Light-activated adenyl cyclase from Trichoderma viride
FEMS Mierobiuh~gy Letters 93 ( 19021 275-278 ,¢;, 1092 Federatitm nf European Microbiological Societies 11378-1097/92/505,0tl Published by Elsevier
275
FEMSLE 04910
Light-activated adenyl cyclase from Trichoderma viride Nade~,da Kolarova, Jana Hapiovfi, Miroslav Gregik btstmtte of (.Twnti.slD', Slocak Acadt, my of Sciettct.x, Bratishwa, Czechostolakia Received 28 January 1992 Revision received 19 March 19'92 Accepted 21} March 1992
Key words: Adenyl cyclase; cAMP-phosphodiesterase; Photo-induced conidiation; Trichoderma t'iride
1. SUMMARY The effect of light on adenyl cyclase (E.C. 4.6.1.1) and 3':5'-cyclic-AMP-phosphodiesterase (E.C. 3.1.4.17) activity of Trichoderma ciride was investigated. Adenyl cyclase proved to be a membrane-associated enzyme, requiring Mn 2÷ and was activated by light. In contrast, 3' :5'-cyclicAMP-phosphodiesterase showed no light-stimulated activity. The activity of 3' :5'-cyclic-AMPphosphodiesterase was present mainly in the cytosol and was stimulated by Mg z÷.
tion across cell membranes, involves generation and accumulation of cAMP in the cell [2]. It :s well documented that the intracellular concentra~tion of cAMP is determined by a balance between the activitics of adenyl cyclase (AC) and 3' :5'-cyclic-AMP-phosphodiesterase (PDE) [3]. Therefore, the aim of the present investigation was to obtain more information about the effects of light on activities of adenyl cyclase and 3' :5'c'¢clic-AMP-phosphodiesterase, both of which are responsible for the level of cAMP in Trichoderma t'iride.
2, INTRODUCTION
3. MATERIALS AND METHODS
The fungus Trichoderma ciride grows as a vegetative mycelium in the dark, provided the nutrient supply is sufficient. A short pulse of white or blue light causes the sporulation of T. ciride [1]. However, the signal pathways of light-induced sporulation are only partially understood. One of the pathways, which is transducing the informa-
3.1. Fungtrs Trichoderma ciride Person ex S.F. Gray, strain no. F-534 from Czechoslovak Collection of Microorganisms (T.G. Masaryk University, Brno) was used. The fungus was maintained on agar slants containing salts, glucose and yeast autolysate [4].
Correspondence to." N. Kolarova, Inslitule tlf Chemistry, Slo. yak Academy of Sciences, Dfibravsk;i cesta 9, 842 38 Bratislava. Czechc.~i-ovakia.
3.2. Cultivation The fungus was grown in shake flasks wrapped in aluminium foil on a rotary shaker at 28°C in
27f~ the medium as described by Mandcls and Andreotti [5].
3.3. Photoinduction The dark-grown mycelium of T. f'iride was illuminated under a fluore~ent lamp by a white light of 1.2 klx intensity. The control experiments remained in the dark and all operations requiring darkness were performed under a red safelight, 3.4. Preparation of membrane and cytosolic fraction The membrane and the cytosolie fraction from myeelia of Trichoderma I'iride were prepared by the procedure described by Gre~ik et al. [6]. 3.5. Adenylate cyclase actit'ity AC activity was determined by measuring cAMP formation from ATP. Assay medium contained 109 mM Hepes buffer, 4.5 mM MgCI 2, l mM ATP, 1).5 mM MnCI,. Incubation was initiated by the addition of membrane or cytosolie fraction (20-40 #g protein) to the reaction mixture in a final volume of 0,3 ml. The reaction was terminated by heating for 3 min at 100°C. For determination of cAMP concentration the radioimmunoassay method by means of a ~zsI kit (Institute for Research, Production and Application of Radioisotopes, Prague) was applied. ATP was removed by adsorption on alumina [7]. 3.6. cAMP-plmsphodiesterase activity PDE activity was assayed in a final volume of 100 ,u.I containing about I0/xg protein in 95 ~zl extraction buffer: 20 mM Tris" HCI (pH 7.5), 3 mM MgCi 2, 1 mM PMSF, 1 mM EGTA. The reaction was initiated by adding the substrate: 3 ~1 100 mM 3',5'-.cAMP and 2 ~1 1 mM [3H]cAMP (9.62.10 °~ Bq mmol-*) and after 30 rain incubation at 25°C it was stopped by placing the test tube into a bath of boiling water for 2 rain. Reaction products were identified by chromatography on PEI cellulose plates [8]. The spots on developed chr~matograms were marked under UV light at 254 nm and cut out. The radioactivity associated with each spot was measured in a LKB 1214 Rackbeta liquid scintillation system. The value of radioactivity is expressed in DPM.
Protein was determined by the method of Lowry [9], using bovine serum albumin as a standard.
4. RESULTS AND DISCUSSION A previous study [4] has shown that illumination of dark-grown mycelia of T. t'iride results in a significant change in their intracellular cAMP level. In an attempt to explain the light-induced increase of cAMP content in cells, the influence of illumination of T. t'iride on both the activity of adenyl cyclase (AC) and that of the 3' :5'-eyclicAMP-phosphodlesterase (PDE) has been investigated. Methods which in previous work [6] were applied successfully to the isolation of membrane and cytosollc fractions from T. t'iride were used also in investigation of localization of AC and PDE in this fungus. It could be expected that, as is usually found, AC will be localized predominantly in the cell surface membranes of microorganisms [10], On the other hand, Reigh et al. [11] have reported that in Neurospora crassa AC is only partially associated with the sedimenting membrane fraction. Besides the membrane-bound AC they have also purified a soluble AC stimulated by ealmodulin. In contrast to Reigh el al. [11] in the previous experiment with 1". ,'iride, 90% of AC activity was found in the membrane fraction. Because of low stability of isolated AC [12], a further purification of this enzyme was omitted. The data in Table 1 show the in vitro effects of Mn 2÷ and GTP on the activity of membranebound AC of 1". eiride. The latter activity was higher if Mn "+ ions were present in the assay medium. Nevertheless, in the presence of Mg-'* ions, GTP did not increase the activity of AC in the way shown in iV. crassa [13]. While the AC activity was found to be located mainly in the membrane fraction, PDE activity was demonstrated to be predominantly present in the cytosolic fraction (Fig. 1). Part of the product of PDE activity (5'-AMP) was further acted upon by non-specific phosphatase to adenosine as shown in Fig. 1. The majority of PDE exhibited
277 Table I Effect tff Mn z+ and GTP on adenylalecyclase activity in the membrane fraction of 1-. riride. Added eompot, nd
Adenylate eycla~ aclivity (pmol/mg o f protein x min)
No addition +~ GTP 0.5 mM MnCI, 0.5raM MnCI, + GTP
7.4 _+t.01 I 1,(12+_ 1.17 44.9 ± 5.01 41.fl5 _+4.68
~' 2a u
Assays were performed after 15 rain at 25+C, under iaboratot3' light. Values represent means_+SD of activities in at least three different experiments. :' The basic mixture contains 100 mM llepes buffer. 4.5 mM MgCI: and I mM ATP.
15
"-
8
'~2
16
20
T IME I m.t:n]
an absolute but non-specific requit~'ment for ions of free bivalent metals. Nevertheless, PDE from T. riride was found to be maximally activated
Fig. 2. Activation of T. riride membrane adenylate cycl:~.m hy light. Experimental conditions were as de~ribed in MxrEm,~l.s ANt> METEtODS. illumination of AC was performed under fluorescent light (+:3). All operation of dark control was performed under a red safelight t4,).
~z'~ D P M
7G
60
50
3~
+ ReOCt ion
memOrQne
eytosoi,¢
ITII~ tLJr e.
f roc'~ion
f rOOt ,On
¢~It +feee extract
: t t~me 0 i'/11n
J--J- c A M P (~l- 0denos]de
m-
5 AMF'
Fig. I. Distribution of PDE assayed in the presence of 3 mM MgCI,. The dat.'i represent the means of three differen! experiments. The experimental conditions are described in MATERIAl..':, ANn M~xnot~s. 1011% of DPM is total rudioactivity o{ a s:~mple applied on the pbte.
with Mg -'+ as divalent cationic ligand, whereas with 0.5 mM MnCI, ions in the assay system, PDE was much less activated (unpublished results). In order to investigate a possible effect of light on AC and/or PDE both the membrane and the cytosolic fractions were isolated in a dark room and thc preparations were divided into two parts. A part was illuminated by white light of 1.2 klx intensity, to estimate AC and PDE activities immediately after illumination of cytosol/or membrane fraction. In the remainder of the preparation AC and PDE activities were assayed in the darkness. Figure 2 shows the light-sensitive linear increase of cAMP production by membranebound AC of 7". ciride. Illumination of the membrane fraction caused a two-fold increase of cAMP level as compared to the dark control. lllumination of cytosolic fraction had no effect on PDE activity (data not shown). In cell-free extracts the increase in cAMP level after exposure to light proceeded in two steps with two peaks (Fig. 3), similar to that which has been reported in experiments performed in viva [6]. The addition of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX) to the reaction mixture had no effect on the time
278 5O
c
/ ,- t,0
cAMP-dependent phosphorylation in control of cell proliferation and differentiation has been shown in yeast [14].
o,.
~o
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ACKNOWLEDGEM ENTS
20
i
We would like to thank Dr. V. Farka~ for his continuous support and encouraging discussions.
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1D
•
•
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S
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Fig, 3. Effect of tight and IBMX on the time course of cAMP level in the cell-free extract of T. riride. AC aclivity was as~ycd in :u reaction mixture containing in final volume of 0.3 ml I(X) mM Hepcs (pH 0.5), II.5 mM MnCI,, 3 mM MgCI2, i mM PMSF, I mM EGTA and approximatdy 40 ~g ol" protein. Reaction was initiated by addition of I mM ATP. cAMP ~as determined by radioimmunoassay. A concentration of 1 mM IBMX was used. (i) dark control: (©) illuminated cell-free extract without IBMX; ( × ) illuminated cell-free extract in the presence of IBMX.
course of cAMP concentration after illumination (Fig. 3). The present results indicate the possibility that the decrease of the cAMP level in the second minute after illumination may not be caused by PDE, but probably by binding of cAMP to cytosolic cAMP-dependent protein kinases. These results arc supported by our preliminary observation [6] of light-stimulated cAMP-dependent phosphorylation of at least two proteins in the cell-free extract from T. riride. These two phosphorylated proteins most probably result from two different cytosolic activities of two different cytosolic cAMP-dependent protein kinases. A similar important regulatory role of
REFERENCES [I] Belina, V. and Spi~iakov~i. J. (1976) Folia Microbiol. 21. 362-37O. [2] Nishizuka, Y. (198fi)Science 233, 305-312. [3] Helmreich, E.JM. and Pleuffcr, T. (1985)Trcnd~, Pharmac~)l. Sci. 6, 438-443. [4] Gre.~k, M., Kolanwa, N. and Farka~, V. (1988) Exp. Mycol. 12, 295-301. [5] Mandels, M. and Andreotti. R.E. (1978) Proce.~s Bioehem. 13, fi-13. [6] Greg[k. M., Kolarm'a, N. and FarkaL V. (1980) FEBS Lelt. 24~;, 185-1~7. [7] Volker, T.T., Viratetle, O.M., Delaage, M.A. and Libboues~, J. (1985) Anal. Biechem. 144, 347-355. [8] Pitk~nen. A., Halonen, T.O.. Kilpcl~inen, H.O. and Riekkinen, P.J. (1984)Anal. Biochem. 137, 397-401. [9} Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randal, R.J. (1951)J. Biol. Chem, 193, 265-268. [lO] Robison. G.A., Butcher, R.W. and Sutherland, E.W. (1971) Cyclic AMP. pp. 531. Academic Press, New York, NY. [1 I] Reigh, J.A., T~llez-1fi6n, M.T., Flawifi, M.M. and Torres. I|.N. (1984) Biochem. J. 221,541-543. [12] Janssens, P.M.W., Van E.~sen. H.W., Guijl. J.J.M., De Waal. A. and Van Driel, R. (197) MoL Cell. Biochem. 76. 55-65. [13] Rosenherg, G.B. and Pall, M L (1983) Arch, Biuchem. Biophvs. 22t, 243-253, [14] Malsumoto, K, Uno, 1. and Ishikawa, T. (1983) Cell 32, 417 -423.
275
FEMSLE 04910
Light-activated adenyl cyclase from Trichoderma viride Nade~,da Kolarova, Jana Hapiovfi, Miroslav Gregik btstmtte of (.Twnti.slD', Slocak Acadt, my of Sciettct.x, Bratishwa, Czechostolakia Received 28 January 1992 Revision received 19 March 19'92 Accepted 21} March 1992
Key words: Adenyl cyclase; cAMP-phosphodiesterase; Photo-induced conidiation; Trichoderma t'iride
1. SUMMARY The effect of light on adenyl cyclase (E.C. 4.6.1.1) and 3':5'-cyclic-AMP-phosphodiesterase (E.C. 3.1.4.17) activity of Trichoderma ciride was investigated. Adenyl cyclase proved to be a membrane-associated enzyme, requiring Mn 2÷ and was activated by light. In contrast, 3' :5'-cyclicAMP-phosphodiesterase showed no light-stimulated activity. The activity of 3' :5'-cyclic-AMPphosphodiesterase was present mainly in the cytosol and was stimulated by Mg z÷.
tion across cell membranes, involves generation and accumulation of cAMP in the cell [2]. It :s well documented that the intracellular concentra~tion of cAMP is determined by a balance between the activitics of adenyl cyclase (AC) and 3' :5'-cyclic-AMP-phosphodiesterase (PDE) [3]. Therefore, the aim of the present investigation was to obtain more information about the effects of light on activities of adenyl cyclase and 3' :5'c'¢clic-AMP-phosphodiesterase, both of which are responsible for the level of cAMP in Trichoderma t'iride.
2, INTRODUCTION
3. MATERIALS AND METHODS
The fungus Trichoderma ciride grows as a vegetative mycelium in the dark, provided the nutrient supply is sufficient. A short pulse of white or blue light causes the sporulation of T. ciride [1]. However, the signal pathways of light-induced sporulation are only partially understood. One of the pathways, which is transducing the informa-
3.1. Fungtrs Trichoderma ciride Person ex S.F. Gray, strain no. F-534 from Czechoslovak Collection of Microorganisms (T.G. Masaryk University, Brno) was used. The fungus was maintained on agar slants containing salts, glucose and yeast autolysate [4].
Correspondence to." N. Kolarova, Inslitule tlf Chemistry, Slo. yak Academy of Sciences, Dfibravsk;i cesta 9, 842 38 Bratislava. Czechc.~i-ovakia.
3.2. Cultivation The fungus was grown in shake flasks wrapped in aluminium foil on a rotary shaker at 28°C in
27f~ the medium as described by Mandcls and Andreotti [5].
3.3. Photoinduction The dark-grown mycelium of T. f'iride was illuminated under a fluore~ent lamp by a white light of 1.2 klx intensity. The control experiments remained in the dark and all operations requiring darkness were performed under a red safelight, 3.4. Preparation of membrane and cytosolic fraction The membrane and the cytosolie fraction from myeelia of Trichoderma I'iride were prepared by the procedure described by Gre~ik et al. [6]. 3.5. Adenylate cyclase actit'ity AC activity was determined by measuring cAMP formation from ATP. Assay medium contained 109 mM Hepes buffer, 4.5 mM MgCI 2, l mM ATP, 1).5 mM MnCI,. Incubation was initiated by the addition of membrane or cytosolie fraction (20-40 #g protein) to the reaction mixture in a final volume of 0,3 ml. The reaction was terminated by heating for 3 min at 100°C. For determination of cAMP concentration the radioimmunoassay method by means of a ~zsI kit (Institute for Research, Production and Application of Radioisotopes, Prague) was applied. ATP was removed by adsorption on alumina [7]. 3.6. cAMP-plmsphodiesterase activity PDE activity was assayed in a final volume of 100 ,u.I containing about I0/xg protein in 95 ~zl extraction buffer: 20 mM Tris" HCI (pH 7.5), 3 mM MgCi 2, 1 mM PMSF, 1 mM EGTA. The reaction was initiated by adding the substrate: 3 ~1 100 mM 3',5'-.cAMP and 2 ~1 1 mM [3H]cAMP (9.62.10 °~ Bq mmol-*) and after 30 rain incubation at 25°C it was stopped by placing the test tube into a bath of boiling water for 2 rain. Reaction products were identified by chromatography on PEI cellulose plates [8]. The spots on developed chr~matograms were marked under UV light at 254 nm and cut out. The radioactivity associated with each spot was measured in a LKB 1214 Rackbeta liquid scintillation system. The value of radioactivity is expressed in DPM.
Protein was determined by the method of Lowry [9], using bovine serum albumin as a standard.
4. RESULTS AND DISCUSSION A previous study [4] has shown that illumination of dark-grown mycelia of T. t'iride results in a significant change in their intracellular cAMP level. In an attempt to explain the light-induced increase of cAMP content in cells, the influence of illumination of T. t'iride on both the activity of adenyl cyclase (AC) and that of the 3' :5'-eyclicAMP-phosphodlesterase (PDE) has been investigated. Methods which in previous work [6] were applied successfully to the isolation of membrane and cytosollc fractions from T. t'iride were used also in investigation of localization of AC and PDE in this fungus. It could be expected that, as is usually found, AC will be localized predominantly in the cell surface membranes of microorganisms [10], On the other hand, Reigh et al. [11] have reported that in Neurospora crassa AC is only partially associated with the sedimenting membrane fraction. Besides the membrane-bound AC they have also purified a soluble AC stimulated by ealmodulin. In contrast to Reigh el al. [11] in the previous experiment with 1". ,'iride, 90% of AC activity was found in the membrane fraction. Because of low stability of isolated AC [12], a further purification of this enzyme was omitted. The data in Table 1 show the in vitro effects of Mn 2÷ and GTP on the activity of membranebound AC of 1". eiride. The latter activity was higher if Mn "+ ions were present in the assay medium. Nevertheless, in the presence of Mg-'* ions, GTP did not increase the activity of AC in the way shown in iV. crassa [13]. While the AC activity was found to be located mainly in the membrane fraction, PDE activity was demonstrated to be predominantly present in the cytosolic fraction (Fig. 1). Part of the product of PDE activity (5'-AMP) was further acted upon by non-specific phosphatase to adenosine as shown in Fig. 1. The majority of PDE exhibited
277 Table I Effect tff Mn z+ and GTP on adenylalecyclase activity in the membrane fraction of 1-. riride. Added eompot, nd
Adenylate eycla~ aclivity (pmol/mg o f protein x min)
No addition +~ GTP 0.5 mM MnCI, 0.5raM MnCI, + GTP
7.4 _+t.01 I 1,(12+_ 1.17 44.9 ± 5.01 41.fl5 _+4.68
~' 2a u
Assays were performed after 15 rain at 25+C, under iaboratot3' light. Values represent means_+SD of activities in at least three different experiments. :' The basic mixture contains 100 mM llepes buffer. 4.5 mM MgCI: and I mM ATP.
15
"-
8
'~2
16
20
T IME I m.t:n]
an absolute but non-specific requit~'ment for ions of free bivalent metals. Nevertheless, PDE from T. riride was found to be maximally activated
Fig. 2. Activation of T. riride membrane adenylate cycl:~.m hy light. Experimental conditions were as de~ribed in MxrEm,~l.s ANt> METEtODS. illumination of AC was performed under fluorescent light (+:3). All operation of dark control was performed under a red safelight t4,).
~z'~ D P M
7G
60
50
3~
+ ReOCt ion
memOrQne
eytosoi,¢
ITII~ tLJr e.
f roc'~ion
f rOOt ,On
¢~It +feee extract
: t t~me 0 i'/11n
J--J- c A M P (~l- 0denos]de
m-
5 AMF'
Fig. I. Distribution of PDE assayed in the presence of 3 mM MgCI,. The dat.'i represent the means of three differen! experiments. The experimental conditions are described in MATERIAl..':, ANn M~xnot~s. 1011% of DPM is total rudioactivity o{ a s:~mple applied on the pbte.
with Mg -'+ as divalent cationic ligand, whereas with 0.5 mM MnCI, ions in the assay system, PDE was much less activated (unpublished results). In order to investigate a possible effect of light on AC and/or PDE both the membrane and the cytosolic fractions were isolated in a dark room and thc preparations were divided into two parts. A part was illuminated by white light of 1.2 klx intensity, to estimate AC and PDE activities immediately after illumination of cytosol/or membrane fraction. In the remainder of the preparation AC and PDE activities were assayed in the darkness. Figure 2 shows the light-sensitive linear increase of cAMP production by membranebound AC of 7". ciride. Illumination of the membrane fraction caused a two-fold increase of cAMP level as compared to the dark control. lllumination of cytosolic fraction had no effect on PDE activity (data not shown). In cell-free extracts the increase in cAMP level after exposure to light proceeded in two steps with two peaks (Fig. 3), similar to that which has been reported in experiments performed in viva [6]. The addition of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX) to the reaction mixture had no effect on the time
278 5O
c
/ ,- t,0
cAMP-dependent phosphorylation in control of cell proliferation and differentiation has been shown in yeast [14].
o,.
~o
o 3
ACKNOWLEDGEM ENTS
20
i
We would like to thank Dr. V. Farka~ for his continuous support and encouraging discussions.
O
1D
•
•
,
2
~
•
...m...
S
.n
~
n
8 ~0 T 1ME(minl
Fig, 3. Effect of tight and IBMX on the time course of cAMP level in the cell-free extract of T. riride. AC aclivity was as~ycd in :u reaction mixture containing in final volume of 0.3 ml I(X) mM Hepcs (pH 0.5), II.5 mM MnCI,, 3 mM MgCI2, i mM PMSF, I mM EGTA and approximatdy 40 ~g ol" protein. Reaction was initiated by addition of I mM ATP. cAMP ~as determined by radioimmunoassay. A concentration of 1 mM IBMX was used. (i) dark control: (©) illuminated cell-free extract without IBMX; ( × ) illuminated cell-free extract in the presence of IBMX.
course of cAMP concentration after illumination (Fig. 3). The present results indicate the possibility that the decrease of the cAMP level in the second minute after illumination may not be caused by PDE, but probably by binding of cAMP to cytosolic cAMP-dependent protein kinases. These results arc supported by our preliminary observation [6] of light-stimulated cAMP-dependent phosphorylation of at least two proteins in the cell-free extract from T. riride. These two phosphorylated proteins most probably result from two different cytosolic activities of two different cytosolic cAMP-dependent protein kinases. A similar important regulatory role of
REFERENCES [I] Belina, V. and Spi~iakov~i. J. (1976) Folia Microbiol. 21. 362-37O. [2] Nishizuka, Y. (198fi)Science 233, 305-312. [3] Helmreich, E.JM. and Pleuffcr, T. (1985)Trcnd~, Pharmac~)l. Sci. 6, 438-443. [4] Gre.~k, M., Kolanwa, N. and Farka~, V. (1988) Exp. Mycol. 12, 295-301. [5] Mandels, M. and Andreotti. R.E. (1978) Proce.~s Bioehem. 13, fi-13. [6] Greg[k. M., Kolarm'a, N. and FarkaL V. (1980) FEBS Lelt. 24~;, 185-1~7. [7] Volker, T.T., Viratetle, O.M., Delaage, M.A. and Libboues~, J. (1985) Anal. Biechem. 144, 347-355. [8] Pitk~nen. A., Halonen, T.O.. Kilpcl~inen, H.O. and Riekkinen, P.J. (1984)Anal. Biochem. 137, 397-401. [9} Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randal, R.J. (1951)J. Biol. Chem, 193, 265-268. [lO] Robison. G.A., Butcher, R.W. and Sutherland, E.W. (1971) Cyclic AMP. pp. 531. Academic Press, New York, NY. [1 I] Reigh, J.A., T~llez-1fi6n, M.T., Flawifi, M.M. and Torres. I|.N. (1984) Biochem. J. 221,541-543. [12] Janssens, P.M.W., Van E.~sen. H.W., Guijl. J.J.M., De Waal. A. and Van Driel, R. (197) MoL Cell. Biochem. 76. 55-65. [13] Rosenherg, G.B. and Pall, M L (1983) Arch, Biuchem. Biophvs. 22t, 243-253, [14] Malsumoto, K, Uno, 1. and Ishikawa, T. (1983) Cell 32, 417 -423.