702
BIOCHEMICAL SOCIETY TRANSACTIONS
solution, which prompted an immediate discharge from the cutaneous granular glands. The skin was then cut into small pieces and cultured in a nutrient/salts medium in a manner similar to that described by Baily (1971). One of the amino acids in the medium was replaced by the 3H-labelled amino acid. The skin cultures were incubated for up to 7 days at 28°C. After 3 and 7 days, samples of skin were first treated with an adrenaline solution, then dried in vacuo and extracted with methanol. The adrenaline solution, the methanol extract and the remaining medium were examined for the presence of jH-labelled caerulein. Thesampleswere mixed with an authentic sample of caerulein and examined by paper electrophoresis or paper chromatography. Samples from the adrenaline solution and the methanol extracts after 3 or 7 days incubation gave no radioactive peaks corresponding to the caerulein marker. However, samples of the medium in which the skins had incubated for 3 days gave a small peak of radioactivity which co-migrated with caerulein. When this solution was kept at 37°C over a period of several hours it was found that the amount of radioactivity which co-migrated with caerulein increased considerably. This suggests that caerulein matures during this period from a precursor molecule. We thank the Medical .Research Council for generous financial support. Anastasi, A., Erspamer, V. & Endean, R. (1967) Experientia 23, 699-700 Anastasi, A,, Erspamer, V. & Endean, R. (1968) Arch. Biochem. Biophys. 125, 57-68 Baily, P. J. (1971) Br. J. Dermatol. 85, 264-271 Dockray, G. J. & Hopkins, C. R. (1975) J. Cell Biol. 64, 724-733 Gregory,H., Hardy, P. M., Jones, D. S., Kenner, G. W. & Sheppard,R. C. (1964)Nature(London) 204,931-933 Mutt, V. & Jorpes, E. (1971) Biochem. J. 125, 5 7 ~ - 5 8 ~ Paul, J. (1972) Cell and Tissue Culture, 4th edn., p. 109, Churchill-Livingstone, Edinburgh and London
Membrane Lipid Metabolism in Acholeplasma laidlawii A during Normal and Temperature-Shift-Down Conditions ANDERS CHRISTIANSSON and &E WIESLANDER Department of ibficrobicdogy, University of Lund, Sohegatan 21, S-223 62 Lund, Sweden Experiments with Acholeplasma laidlawii have contributed considerably to our understanding of membrane structure and function (Razin, 1975). The membrane-lipid metabolism of Acholeplasma cells is, however, not as well known as that for fatty acid auxotrophs of Escherichia coli used in similar membrane studies. Previous studies by us (Wieslander & Rilfors, 1977) have shown that in A. Iaidlawii A strain EF22 the membrane lipid content (and especially the glucolipid content) varies as a consequence of saturated or unsaturated fatty acid supplementation to a lipid-depleted growth medium. In the present investigation the temperature-shift-down technique was used to establish the impact of a factor that affects membrane viscosity (temperature) on the lipid metabolism and lipid relationships in A . laidlawii. Acholeplarma 1aidlawiiA strain EF22 was grown statically in a lipid-depleted tryptose/ bovine serum albumin medium (Razin & Rottem, 1975). Each litre of medium was supplemented witheither a mixture of palmiticacid(75jimol)plus oleicacid (75jimol), or oleic acid alone (15OjimoIJ. These were also given together with 8mg of cholesterol per litre. Membrane lipids were labelled by adding I4C- and/or jH-labelled fatty acids to the medium. Cultures were grown 12h at 37"C, divided into twoportions, one of which was rapidly cooled to 17°C and the other was maintained at 37°C. Samples for lipid and protein analysis were removed periodically and centrifuged. The cells were extracted with chloroform/methanol (2: 1, v/v) and the lipid extract was then purified from contaminants by Sephadex column chromatography (Wells & Dittmer, 1963). 1977
703
568th MEETING, ABERDEEN 2.5
2.0
7.0
!-
I
1
16
20
I
1
(b)
I .5
v)
't -
I*o
9 .-a 0.5 %
0
-1
12
16
20
24
I2
16
20
24
I2
24
2.0
I.o
12
16
20
24
Period of growth (h) Fig. 1. Metabohm of major membrane polar lipids of Acholeplasma laidlawii ajier temperature shijbdown The amount of each lipid at 12h was assigned the relative value of 1.0, and lipid variations during subsequent growth was related to this value. Supplementations were: (a) 75p~-palmitic acid plus 75p~-oleicacid; (6) as in (a) plus 8mgofcholesterol/litre; (c) 150p~-oleicacid; (d) as in (c) plus 8mg of cholesterol/litre. 0, Monoglucosyl Growth at 37°C; diglyceride; V, diglucosyl diglyceride; 0 , phosphatidylglycetol.-, --_-,growth after temperature shift-down to 17°C at 12h. T.1.c. was carried out on silica-gel H plates buffered with 1% (w/v) Na2B407,10H20 in the solvent system chloroform/methanol/water (65 :25: 4, by vol.). Neutral lipids were separated as described by Kates (1972~).Lipids were detected by specific reagents (Kates, 19726) or by radioautography. Fatty acid composition and lipid content were determined by liquid-scintillation counting of radioactivity. The temperature range of membrane lipid phase transition was determined from changes in ASoowith temperature (MacDonald et al., 1976). Endogenous lipolytic activity was assayed by incubating osmotically lysed membranes (Razin, 1975) in buffer (Rottem et al., 1973). Lipid synthesis was also followed by giving a pulse of ~n-['~C]glycerol3-phosphate immediately before shift.
Vol. 5
704
BIOCHEMlCAL SOCIETY TRANSACTIONS
Table 1. Effect of temperature shijt-down on incorporation of unsaturated (oleic acid) compared with saturated (galmitic acid)fatty acids into the diyerent major membrane lipid species The values shown are the fraction (mo1/100mol) of oleic acid in each lipid. Growth medium was supplemented with 75p~-palmitic acid plus 75p~-oleic acid. Abbreviations : PG, phosphatidylglycerol; DGDG, diglucosyl diglyceride; MGDG, monoglucosyl diglyceride; ND,not determined. Incubation tern-. Period of perature (“C) growth@) 37 17 DGDG 37 17 MGDG 37 17 Lipid PG
.#
,. 12 37 37 41 41 34 34
13
42
14 55 39 45 43
44
41
44 40
43
37
15
41
16 57 40 48 43 45 45
19 59 43 46 43
44 52
24 61 69 47 ND 42 50
After temperature shift-down, only palmitic acid plus oleic acid supplements gave a lag in the growth curve (AS4,,). With all other supplements, growth continued uninterrupted at a decreased rate that was approximately the same for all shifted cultures. Lipid analysis revealed complex and sometimes drastic changes in lipid and fatty acid composition during the growth cycle at 37°C and after temperature shift-down. Our strain contains the following lipids :mono- and di-glucosyldiglyceride,phosphatidylglycerol, glycerophosphorylmono- and glycerophosphoryldi-glucosyl diglyceride, a more fully acylated glycosyl diglyceride (glycolipid X ) (Wieslander & Rilfors, 1972). Induced changes in the relative amounts of notably monoglucosyl diglyceride and diglucosyl diglyceride were evident during growth at 37°C (Fig. 1). After temperature shift-down to 17°C the changes in lipid content in all cultures (ratio monoglucosyl diglyceride/diglucosyl diglyceride) were similar to the pattern shown by cultures containing a saturated fatty acid (palmitic acid) at 37°C. Saturated fatty acids or a temperature decrease are known to lower membrane fluidity and permeability (Ruin, 1975). Although structurally similar, monoglucosyl diglyceride and diglucosyl diglyceride have different physical appearance (phase structure) (Wieslander, 1976). Table 1reveals an adaptable mechanism for rapidly increasing the extent of membranelipid unsaturation at lower temperatures. The dissimilarities of these changes between thelipid speciesindicates a subtle regulatory mechanism for this adaptation, since several lipids are precursors to each other. Prolonged incubation of membranes did not reveal any significant endogenous lipolyticenzyme activities, which could influence these observed fatty acid changes. Pulse experiments with a lipid backbone precursor, sn-glycerol 3-phosphate, indicated an active synthesis de nouo of all lipids after temperature shift-down. It is therefore likely that the changes in fatty acid composition are to a large extent associated with synthesis of entirely new lipid molecules. If this is so, our organisms, in common with E. coli, seems to lack a recycling mechanism for sparing of lipid-metabolism components. Obviously, in addition to the expected changes in unsaturation, this organism also possesses an enzymic mechanism that regulates membrane polar-lipid-head synthesis as a response to temperature and other viscosity parameters. Kates, M. (1972~)Techniques of Lipidology, pp. 435-441, North-Holland, Amsterdam Kates, M. (19726) Techniques of Lipidology, pp. 502-51 1, North-Holland, Amsterdam MacDonald,R. C., MacDonald, R. C., Simon, S. A. & Baer, E. (1 976) Biochemistry 15,885-891 Ran’n, S . (1975) Prog. Surf. Membr. Sci. 9,257-311 1977
568th MEETING, ABERDEEN
705
RaZia, S. 8c Rottem, S. (1975) in Biochemicul Methods in Membrune Studies (A. H . Maddy, ed.),Chapman and Hall, London Rottem, S.,Hasin, M. & Razin, S. (1973) Biochim. Biophys. Actu 323,520-531 Wells, M. A. & Dittmer, J. C. (1963) Biochemistry 2, 1259-1263 Wieslander, A. (1976) Proc. SOC.Gen. Microbiol, 3, 164 Wieslander, A. & Rilfors, L. (1977) Biochim. Biophys. Acru in the press
Uridine Diphosphate GlucuronyltransferaseActivity in Nuclei and Nuclear Envelopes of Rat Liver and its Apparent Induction by Phenobarbital G. J. WISHART and D. J. FRY Department of Biochemistry and Department of Anatomy, University of Dundee, Dundee DD1 4HN, Scotland, U.K.
The presence of UDP-glucuronyltransferase (EC 2.4.1.17) activity in chick-embryo liver nuclei and nuclear envelope and its inducibility by phenobarbital has been previously reported (Fry & Wishart, 1976). This enzyme has also been found in adult rat liver, with 64-76 % in the microsomal and 14-19 % in the unpuri6ed nuclear fractions (Amar-Costesec et ul., 1974). In view of the heterogeneity of such unpurified nuclear fractions and the high microsomal activity, much, if not all, of such apparent nuclear activity could be due to microsomal contamination. In the present study a reliable estimate of nuclear and nuclearenvelopeactivity was sought. Purified nuclear preparations have been prepared, with the degree of contamination of these quantified by electronmicroscope morphometry (Weibel & Bolender, 1972), and the nuclear envelopes were isolated from these fractions as described previously (Fry & Wishart, 1976). The results on the nuclear and nuclearenvelope preparations were compared with those on standard microsomal preparations and on microsomal preparations subjected to the nuclear-envelope-isolation procedures (‘treated microsomes’). As the microsomal enzyme is latent in brokencell preparations, such comparison has little meaning without somestandardization of the degree to which the latent enzyme has been activated (L.eakey &Donald, 1976). All fractionswere therefore assayed for glucuronyltransferase activity, by the procedure described by Winsnes (1969) with o-aminophenolas substrate, at a range of digitonin concentrations, so that estimates of maximal activity could be made. Female Wistar rats (3 months old) were used for all experiments and the effect of phenobarbital was determined by comparing results on phenobarbital-treated and control rats sampled on the same day, and with their liver fractions prepared in parallel. Maximal phenobarbital induction was achieved by introducing 1-2g/l into the drinking water for over 3 weeks before the experiment, or by injecting 100mg/kgintraperitoneally daily for 3 days and assaying on the fifth day. In control rat livers the purified nuclear fractions contained less than 10% nonnuclear membraneand showed few non-membranouscontaminants.The purified nuclear fraction contained 19.6% of the total homogenate DNA, but its maximal glucuronyltransferase activity was only 0.5% of the total. The true nuclear glucuronyltransferase activity therefore appears to be approx. 2.5% of the homogenate activity, a value substantially less than indicated by crude cell fractionation. The nuclear envelopesretained over 80% of the total nuclear activity, indicatingthat the envelope is the primary, if not the sole, location of the nuclear enzyme. Maximal nuclear, nuclear-envelope and microsoma1 glucuronyltransferaseactivities per mg of phospholipid were comparable (230nmol of o-aminophenylglucuronide/hper mg of phospholipid).Similarresults were obtained when 5-hydroxytryptamine or bilirubin was used as substrate instead of o-aminophenol. With o-aminophenol as substrate, the maximal activity obtained after digitonin treatment of ‘treated microsome’ preparations was nearly twice that obtained with the standard microsomal preparations, and it seems that the envelopisolation procedure used (low ionic concentration, pH8.5, deoxyribonuclease)has an activating VOl. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
solution, which prompted an immediate discharge from the cutaneous granular glands. The skin was then cut into small pieces and cultured in a nutrient/salts medium in a manner similar to that described by Baily (1971). One of the amino acids in the medium was replaced by the 3H-labelled amino acid. The skin cultures were incubated for up to 7 days at 28°C. After 3 and 7 days, samples of skin were first treated with an adrenaline solution, then dried in vacuo and extracted with methanol. The adrenaline solution, the methanol extract and the remaining medium were examined for the presence of jH-labelled caerulein. Thesampleswere mixed with an authentic sample of caerulein and examined by paper electrophoresis or paper chromatography. Samples from the adrenaline solution and the methanol extracts after 3 or 7 days incubation gave no radioactive peaks corresponding to the caerulein marker. However, samples of the medium in which the skins had incubated for 3 days gave a small peak of radioactivity which co-migrated with caerulein. When this solution was kept at 37°C over a period of several hours it was found that the amount of radioactivity which co-migrated with caerulein increased considerably. This suggests that caerulein matures during this period from a precursor molecule. We thank the Medical .Research Council for generous financial support. Anastasi, A., Erspamer, V. & Endean, R. (1967) Experientia 23, 699-700 Anastasi, A,, Erspamer, V. & Endean, R. (1968) Arch. Biochem. Biophys. 125, 57-68 Baily, P. J. (1971) Br. J. Dermatol. 85, 264-271 Dockray, G. J. & Hopkins, C. R. (1975) J. Cell Biol. 64, 724-733 Gregory,H., Hardy, P. M., Jones, D. S., Kenner, G. W. & Sheppard,R. C. (1964)Nature(London) 204,931-933 Mutt, V. & Jorpes, E. (1971) Biochem. J. 125, 5 7 ~ - 5 8 ~ Paul, J. (1972) Cell and Tissue Culture, 4th edn., p. 109, Churchill-Livingstone, Edinburgh and London
Membrane Lipid Metabolism in Acholeplasma laidlawii A during Normal and Temperature-Shift-Down Conditions ANDERS CHRISTIANSSON and &E WIESLANDER Department of ibficrobicdogy, University of Lund, Sohegatan 21, S-223 62 Lund, Sweden Experiments with Acholeplasma laidlawii have contributed considerably to our understanding of membrane structure and function (Razin, 1975). The membrane-lipid metabolism of Acholeplasma cells is, however, not as well known as that for fatty acid auxotrophs of Escherichia coli used in similar membrane studies. Previous studies by us (Wieslander & Rilfors, 1977) have shown that in A. Iaidlawii A strain EF22 the membrane lipid content (and especially the glucolipid content) varies as a consequence of saturated or unsaturated fatty acid supplementation to a lipid-depleted growth medium. In the present investigation the temperature-shift-down technique was used to establish the impact of a factor that affects membrane viscosity (temperature) on the lipid metabolism and lipid relationships in A . laidlawii. Acholeplarma 1aidlawiiA strain EF22 was grown statically in a lipid-depleted tryptose/ bovine serum albumin medium (Razin & Rottem, 1975). Each litre of medium was supplemented witheither a mixture of palmiticacid(75jimol)plus oleicacid (75jimol), or oleic acid alone (15OjimoIJ. These were also given together with 8mg of cholesterol per litre. Membrane lipids were labelled by adding I4C- and/or jH-labelled fatty acids to the medium. Cultures were grown 12h at 37"C, divided into twoportions, one of which was rapidly cooled to 17°C and the other was maintained at 37°C. Samples for lipid and protein analysis were removed periodically and centrifuged. The cells were extracted with chloroform/methanol (2: 1, v/v) and the lipid extract was then purified from contaminants by Sephadex column chromatography (Wells & Dittmer, 1963). 1977
703
568th MEETING, ABERDEEN 2.5
2.0
7.0
!-
I
1
16
20
I
1
(b)
I .5
v)
't -
I*o
9 .-a 0.5 %
0
-1
12
16
20
24
I2
16
20
24
I2
24
2.0
I.o
12
16
20
24
Period of growth (h) Fig. 1. Metabohm of major membrane polar lipids of Acholeplasma laidlawii ajier temperature shijbdown The amount of each lipid at 12h was assigned the relative value of 1.0, and lipid variations during subsequent growth was related to this value. Supplementations were: (a) 75p~-palmitic acid plus 75p~-oleicacid; (6) as in (a) plus 8mgofcholesterol/litre; (c) 150p~-oleicacid; (d) as in (c) plus 8mg of cholesterol/litre. 0, Monoglucosyl Growth at 37°C; diglyceride; V, diglucosyl diglyceride; 0 , phosphatidylglycetol.-, --_-,growth after temperature shift-down to 17°C at 12h. T.1.c. was carried out on silica-gel H plates buffered with 1% (w/v) Na2B407,10H20 in the solvent system chloroform/methanol/water (65 :25: 4, by vol.). Neutral lipids were separated as described by Kates (1972~).Lipids were detected by specific reagents (Kates, 19726) or by radioautography. Fatty acid composition and lipid content were determined by liquid-scintillation counting of radioactivity. The temperature range of membrane lipid phase transition was determined from changes in ASoowith temperature (MacDonald et al., 1976). Endogenous lipolytic activity was assayed by incubating osmotically lysed membranes (Razin, 1975) in buffer (Rottem et al., 1973). Lipid synthesis was also followed by giving a pulse of ~n-['~C]glycerol3-phosphate immediately before shift.
Vol. 5
704
BIOCHEMlCAL SOCIETY TRANSACTIONS
Table 1. Effect of temperature shijt-down on incorporation of unsaturated (oleic acid) compared with saturated (galmitic acid)fatty acids into the diyerent major membrane lipid species The values shown are the fraction (mo1/100mol) of oleic acid in each lipid. Growth medium was supplemented with 75p~-palmitic acid plus 75p~-oleic acid. Abbreviations : PG, phosphatidylglycerol; DGDG, diglucosyl diglyceride; MGDG, monoglucosyl diglyceride; ND,not determined. Incubation tern-. Period of perature (“C) growth@) 37 17 DGDG 37 17 MGDG 37 17 Lipid PG
.#
,. 12 37 37 41 41 34 34
13
42
14 55 39 45 43
44
41
44 40
43
37
15
41
16 57 40 48 43 45 45
19 59 43 46 43
44 52
24 61 69 47 ND 42 50
After temperature shift-down, only palmitic acid plus oleic acid supplements gave a lag in the growth curve (AS4,,). With all other supplements, growth continued uninterrupted at a decreased rate that was approximately the same for all shifted cultures. Lipid analysis revealed complex and sometimes drastic changes in lipid and fatty acid composition during the growth cycle at 37°C and after temperature shift-down. Our strain contains the following lipids :mono- and di-glucosyldiglyceride,phosphatidylglycerol, glycerophosphorylmono- and glycerophosphoryldi-glucosyl diglyceride, a more fully acylated glycosyl diglyceride (glycolipid X ) (Wieslander & Rilfors, 1972). Induced changes in the relative amounts of notably monoglucosyl diglyceride and diglucosyl diglyceride were evident during growth at 37°C (Fig. 1). After temperature shift-down to 17°C the changes in lipid content in all cultures (ratio monoglucosyl diglyceride/diglucosyl diglyceride) were similar to the pattern shown by cultures containing a saturated fatty acid (palmitic acid) at 37°C. Saturated fatty acids or a temperature decrease are known to lower membrane fluidity and permeability (Ruin, 1975). Although structurally similar, monoglucosyl diglyceride and diglucosyl diglyceride have different physical appearance (phase structure) (Wieslander, 1976). Table 1reveals an adaptable mechanism for rapidly increasing the extent of membranelipid unsaturation at lower temperatures. The dissimilarities of these changes between thelipid speciesindicates a subtle regulatory mechanism for this adaptation, since several lipids are precursors to each other. Prolonged incubation of membranes did not reveal any significant endogenous lipolyticenzyme activities, which could influence these observed fatty acid changes. Pulse experiments with a lipid backbone precursor, sn-glycerol 3-phosphate, indicated an active synthesis de nouo of all lipids after temperature shift-down. It is therefore likely that the changes in fatty acid composition are to a large extent associated with synthesis of entirely new lipid molecules. If this is so, our organisms, in common with E. coli, seems to lack a recycling mechanism for sparing of lipid-metabolism components. Obviously, in addition to the expected changes in unsaturation, this organism also possesses an enzymic mechanism that regulates membrane polar-lipid-head synthesis as a response to temperature and other viscosity parameters. Kates, M. (1972~)Techniques of Lipidology, pp. 435-441, North-Holland, Amsterdam Kates, M. (19726) Techniques of Lipidology, pp. 502-51 1, North-Holland, Amsterdam MacDonald,R. C., MacDonald, R. C., Simon, S. A. & Baer, E. (1 976) Biochemistry 15,885-891 Ran’n, S . (1975) Prog. Surf. Membr. Sci. 9,257-311 1977
568th MEETING, ABERDEEN
705
RaZia, S. 8c Rottem, S. (1975) in Biochemicul Methods in Membrune Studies (A. H . Maddy, ed.),Chapman and Hall, London Rottem, S.,Hasin, M. & Razin, S. (1973) Biochim. Biophys. Actu 323,520-531 Wells, M. A. & Dittmer, J. C. (1963) Biochemistry 2, 1259-1263 Wieslander, A. (1976) Proc. SOC.Gen. Microbiol, 3, 164 Wieslander, A. & Rilfors, L. (1977) Biochim. Biophys. Acru in the press
Uridine Diphosphate GlucuronyltransferaseActivity in Nuclei and Nuclear Envelopes of Rat Liver and its Apparent Induction by Phenobarbital G. J. WISHART and D. J. FRY Department of Biochemistry and Department of Anatomy, University of Dundee, Dundee DD1 4HN, Scotland, U.K.
The presence of UDP-glucuronyltransferase (EC 2.4.1.17) activity in chick-embryo liver nuclei and nuclear envelope and its inducibility by phenobarbital has been previously reported (Fry & Wishart, 1976). This enzyme has also been found in adult rat liver, with 64-76 % in the microsomal and 14-19 % in the unpuri6ed nuclear fractions (Amar-Costesec et ul., 1974). In view of the heterogeneity of such unpurified nuclear fractions and the high microsomal activity, much, if not all, of such apparent nuclear activity could be due to microsomal contamination. In the present study a reliable estimate of nuclear and nuclearenvelopeactivity was sought. Purified nuclear preparations have been prepared, with the degree of contamination of these quantified by electronmicroscope morphometry (Weibel & Bolender, 1972), and the nuclear envelopes were isolated from these fractions as described previously (Fry & Wishart, 1976). The results on the nuclear and nuclearenvelope preparations were compared with those on standard microsomal preparations and on microsomal preparations subjected to the nuclear-envelope-isolation procedures (‘treated microsomes’). As the microsomal enzyme is latent in brokencell preparations, such comparison has little meaning without somestandardization of the degree to which the latent enzyme has been activated (L.eakey &Donald, 1976). All fractionswere therefore assayed for glucuronyltransferase activity, by the procedure described by Winsnes (1969) with o-aminophenolas substrate, at a range of digitonin concentrations, so that estimates of maximal activity could be made. Female Wistar rats (3 months old) were used for all experiments and the effect of phenobarbital was determined by comparing results on phenobarbital-treated and control rats sampled on the same day, and with their liver fractions prepared in parallel. Maximal phenobarbital induction was achieved by introducing 1-2g/l into the drinking water for over 3 weeks before the experiment, or by injecting 100mg/kgintraperitoneally daily for 3 days and assaying on the fifth day. In control rat livers the purified nuclear fractions contained less than 10% nonnuclear membraneand showed few non-membranouscontaminants.The purified nuclear fraction contained 19.6% of the total homogenate DNA, but its maximal glucuronyltransferase activity was only 0.5% of the total. The true nuclear glucuronyltransferase activity therefore appears to be approx. 2.5% of the homogenate activity, a value substantially less than indicated by crude cell fractionation. The nuclear envelopesretained over 80% of the total nuclear activity, indicatingthat the envelope is the primary, if not the sole, location of the nuclear enzyme. Maximal nuclear, nuclear-envelope and microsoma1 glucuronyltransferaseactivities per mg of phospholipid were comparable (230nmol of o-aminophenylglucuronide/hper mg of phospholipid).Similarresults were obtained when 5-hydroxytryptamine or bilirubin was used as substrate instead of o-aminophenol. With o-aminophenol as substrate, the maximal activity obtained after digitonin treatment of ‘treated microsome’ preparations was nearly twice that obtained with the standard microsomal preparations, and it seems that the envelopisolation procedure used (low ionic concentration, pH8.5, deoxyribonuclease)has an activating VOl. 5
