Biochimica et Biophysica Acta, 379 (1975) 598-605
(~? Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
BBA 36950 BIOLOGICAL AND I M M U N O L O G I C A L ACTIVITY OF P R O T H R O M B I N IN R O U G H AND S M O O T H MICROSOMES ISOLATED FROM RAT LIVER
T. L. JANSON and L. HELGELAND Department of Biochem&try, University of Oslo, Blindern (Norway)
(Received May 10th, 1974)
SUMMARY 1. The presence of biological and immunological activity of prothrombin in sonicates from rough and smooth microsomes has been investigated. 2. Biological activity of prothrombin was detected in both the rough and smooth microsomal fraction. The specific and the total activity of prothrombin in the sonicates from smooth microsomes were 3-4-fold higher than in the corresponding fraction from rough microsomes. 3. Prothrombin could be identified in both microsomal fractions by double immunodiffusion. 4. The presence of a macromolecular inhibitor of blood coagulation in the microsomes is reported.
INTRODUCTION The biosynthesis of glycoproteins in rat liver has for some years been extensively studied [1 ] and at present there is much evidence in favour of a stepwise addition of carbohydrates to the polypeptide chain as this moves through the endoplasmic reticutum [1-4]. In accordance with this a different degree of finalization of a glycoprotein would be expected in the rough and smooth microsomes. The purpose of the present work was to investigate at which submicrosomal site biological activity of a glycoprotein could be detected. Prothrombin, a vitamin K-dependent plasma glycoprotein containing about 10 ~o carbohydrate and which has been shown to be localized in the microsomes [5, 6], was chosen since the biological activity can be easily measured by a specific and sensitive coagulation test. As the concentration of prothrombin in the sonicates of the microsomes was very low, the extracts had to be concentrated to obtain reliable analyses of prothrombin. During experiments carried out to reduce the volume of the sonicates, an inhibitor of blood coagulation appeared. The results obtained in the present investigation indicate that both the rough and the smooth microsomes contained detectable amounts of protein molecules exhibiting the biological and immunological activity of prothrombin. By far the highest biological activity was, however, found in the smooth microsomes.
599 MATERIALS AND METHODS
Animals. Male albino rats, 150-200 g, of the Wistar strain (Mollegaard, Havrup, Denmark) were used. The animals were starved for 20 h before being killed by decapitation. Chemicals. CsC1 suprapure from Merck A.G. was used for the isolation of rough and smooth microsomes. Agarose was obtained from Behringwerke A.G. Aquacide lI A grade was purchased from Calbiochem. Freund's Complete Adjuvant was produced by Difco Laboratories. Russel's Viper Venom (Stypven) was obtained from Burroughs Wellcome. Preparation and characterization of the microsomal fractions. The preparation of rough and smooth microsomes was carried out as described by Helgeland et al. [4] using the method of Bergstrand and Dallner [7]. The microsome fractions were washed once with 0.25 M sucrose and the pellets of smooth and rough microsomes (derived from 30 g of liver) were suspended in 1.5 ml of a solution of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HCl buffer pH 7.4 [6]. The suspensions were sonicated for five periods of 30 s in a Branson sonifier at a setting of 1.5 while being cooled in a salt/ice mixture. Intervals of 1 min were included to keep the temperature below 8-10 °C during the treatment. The resulting suspensions were centrifuged for 1 h at 105 000 × g. The rough and smooth microsomes were characterized as described previously [4]. Total microsomes from perfused and nonperfused livers were prepared as described by Bergstrand and Dallner [7]. The livers were perfused through the portal vein with 0.25 M sucrose. Concentration of the sonicates. The sonicates were concentrated either by using a 50 ~ solution of aquacide in a mixture of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HCl buffer pH 7.4 or by dialysis of the sonicates against distilled water followed by lyophilization and dissolving in a small volume of a solution of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HC1 buffer pH 7.4. Analytical methods. Prothrombin was measured using the one-stage method of Hjort et al. [8]. The test was made specific for prothrombin by adding Factor X (Stuart) to the test solution [9]. One unit of prothrombin was arbitrarily chosen as the amount of prothrombin present in 1 ml of standard human plasma diluted 1:1000. Protein was determined by the method of Lowry et al. [10] with crystalline bovine serum albumin as standard. Preparation of crude rat prothrombin and antiserum against crude rat prothrombin. Prothrombin was isolated from rat plasma essentially as described by Li and Olson [11]. Blood was drawn by heart puncture. The product obtained revealed four bands on polyacrylamide gel-disc electrophoresis at pH 9.1. Antisera to crude prothrombin was raised in rabbits by injecting 1.2 mg of crude prothrombin in 1 ml of 0.9 ~ NaC1 and 1 ml of Freund's Complete Adjuvant intramuscularly. Booster doses with half the amount of antigen were given 3, 5 and 6 weeks later. Blood was collected one week after the final injection. Purification of rat prothrombin. Prothrombin was further purified by disc electrophoresis of crude prothrombin in 5 ~ gel pH 9.1 and 4 °C as described by Davis [12]. 75 #g of protein was applied on each gel (0.5 cm x 7.0 cm). The pro-
600 thrombin bands were carefully sliced out and the prothrombin eluted by crushing the gel in 0.5 ml of a solution of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HC1 buffer pH 7.4 and left overnight by 4 °C. The gel was removed by centrifugation. The purified prothrombin appeared homogeneous by polyacrylamide disc electrophoresis pH 9.1 and by immunoelectrophoresis pH 8.6 as described by Scheidegger [13]. Double immunodiffusion. The double immunodiffusion was performed according to Ouchterlony [14]. 1% agarose gel in barbiturate buffer pH 8.6 was used. The gels were of 2 mm thickness. The diameters of the wells were 2 mm and 6-7 mm apart. 5-7 #1 of antigen solution were applied in each well. The precipitates were stained with Amido black. RESULTS
On the inhibitory activity in sonicates from rough and smooth microsomes on the bioassay of prothrombin To obtain reliable analyses of prothrombin, the volumes of the sonicates from the microsomes had to be reduced. It is seen from Fig. 1, however, that the recovery of prothrombin activity decreased with the degree of concentration of the sonicates. Upon dilution to the original volume, a complete recovery of the prothrombin activity was obtained. This result indicates that the reduced prothrombin activity is due to an inhibitory effect on the coagulation assay as a result of the concentration procedure. The inhibition did not seem to be a mere effect of the increased concen-
Rough m i c r o s o m e s
Smooth
mic rosomes
v
lOO
£ E
8c
o
S 6o Q.
)
Q 0
4c
a: 2c
0
I 1
I 2
I 3
I 4
Concentration
I 5
I 1
of p r o t h r o r n b i n
I 2
I 3 (arbitrary
I 4
| 5
I-
units)
Fig. 1. Recovery of prothrombin activity by concentrating sonicates of rough and smooth microsomes. The sonicates of rough and smooth microsomes were concentrated by: treatment by aquacide ( 0 ) and dialysis and lyophilization ( 0 ) . The prothrombin activity in the original sonicates was chosen as 100%.
601 TABLE I BIOLOGICAL ACTIVITY OF PROTHROMBIN IN SONICATES OF MICROSOMES AND CYTOSOL ISOLATED FROM PERFUSED AND NON-PERFUSED RAT LIVERS Total activity is expressed as units of protbrombin/g of liver. N.D., no detectable activity. The values from two experiments are given.
Non-perfused livers Perfused livers
Total activity in sonicates
Total activity in cytosol
0.81 0.87
6.3
0.78 0.81
7.7 N.D.
t r a t i o n o f protein, as it was shown t h a t s e r u m a l b u m i n o f a c o r r e s p o n d i n g concent r a t i o n d i d n o t i n h i b i t the c o a g u l a t i o n process. The i n h i b i t i o n m i g h t m o s t p r o b a b l y be e x p l a i n e d b y the presence o f an i n h i b i t o r o f b l o o d c o a g u l a t i o n in the microsomes. W h e t h e r the sonicates were c o n c e n t r a t e d by a q u a c i d e o r b y dialysis a g a i n s t distilled w a t e r a n d subsequent l y o p h i l i z a t i o n , the same i n h i b i t i o n was o b t a i n e d . It a p p e a r s f r o m Fig. 1 t h a t the i n h i b i t i o n was m o r e p r o n o u n c e d in the sonicates f r o m r o u g h m i c r o s o m e s . It has recently been shown in this l a b o r a t o r y t h a t the i n h i b i t o r y effect is due to a heat-stable, m a c r o m o l e c u l a r i n h i b i t o r o f the t h r o m b i n - f i b r i n o g e n reaction [241.
Identification of prothrombin in sonicates of rough and smooth microsomes by measuring the biological activity T a b l e I shows t h a t the a m o u n t o f p r o t h r o m b i n in the sonicate f r o m microsomes is the same w h e t h e r the livers have been perfused or n o t a l t h o u g h it was found t h a t the c y t o s o l isolated f r o m n o n - p e r f u s e d livers c o n t a i n e d a b o u t 7 units/g o f liver whereas the cytosol f r o m perfused livers did n o t c o n t a i n a n y detectable a m o u n t o f prothrombin. I n o r d e r to o b t a i n reliable analyses o f the p r o t h r o m b i n c o n t e n t o f the m i c r o s o m a l extracts, the i n h i b i t o r h a d to be r e m o v e d . This was achieved by a d s o r b i n g the p r o t h r o m b i n on b a r i u m citrate [24]. T h e precipitate was subsequently dissolved in a solution o f 0.03 M s o d i u m citrate, 0.23 M s o d i u m c h l o r i d e a n d 0.05 M E D T A ,
TABLE II BIOLOGICAL ACTIVITY OF PROTHROMBIN IN SONICATES FROM ROUGH AND SMOOTH MICROSOMES Rough and smooth microsomes from 30 g of liver were sonicated as described in Methods. The prothrombin of 1.0 ml of the sonicate fractions was adsorbed on barium citrate. The precipitate was dissolved in sodium citrate, sodium chloride and EDTA and the solution subjected to gel filtration on a Sephadex G-50 column [24]. Specific activity is expressed as units of prothrombin/mg of protein in the eluate from the Sephadex column. Total activity is expressed as units of prothrombin/g of liver. The values are the means of three experiments zk S.D. Fraction
Specific activity
Total activity
Rough microsomes Smooth microsomes
5.9 4- 0.96 22.8 4- 2.0
0.11 ± 0.009 0.29 4- 0.036
602 and the solution gel filtrated on a Sephadex G-50 Fine column equilibrated with the test buffer. By this procedure a sufficient degree of concentration was also obtained [24]. As shown in Table II, sonicates from both rough and smooth microsomes contained biological activity of prothrombin, the specific and the total activity of the latter fraction being 3-4 fold higher. Similar ratios were found when testing the activity of untreated sonicates. Only a rough estimate of the prothrombin activities in the untreated sonicates could be obtained, however, as the clotting times ( > 90 s) exceeded the limit of reliable measurements, but it suggests that there is no difference in the adsorption of prothrombin from rough and smooth sonicates to barium citrate.
Immunological activity of prothrombin in sonicates of rough and smooth microsomes detected by double immunodiffusion In order to identify prothrombin in the microsomal fractions by immunochemical methods, antiserum to crude prothrombin was used. Fig. 2a shows that the antiserum contained antibodies to all the four components of crude prothrombin, including prothrombin, whereas the purified prothrombin gave one precipitation line with the antiserum. This line formed complete identity with one of the arcs from rat plasma and one from the crude prothrombin. This is in accordance with the homogeneity of purified prothrombin observed by disc electrophoresis and immuneelectrophoresis (see Methods). The purified prothrombin was accordingly accepted as satisfactorily pure to be used in the double-immunodiffusion assay for testing the immunological activity of prothrombin from the microsomal fractions. Fig. 2b shows that the sonicates from both the rough and smooth microsomes formed one precipitation
(b)
(a)
(o ©~ CrPr
Ab
0
0
0
0
0
© Pr
PI
R
Pr
PI
S
Pr
Fig. 2. Double immunodiffusion of (a) different preparations of prothrombin against antiserum to crude prothrombin, (bl) sonicates of rough and smooth microsomes, concentrated about 45 times, against antiserum to crude prothrombin. The photograph was taken after staining (b2) prothrombin from sonicates of rough and smooth microsomes concentrated about 45 times after purification by adsorption to and elution from barium citrate. The photograph was taken before staining. Ab, antiserum 5/d; Pl, rat plasma 25 units (2a), 5 units (2b); CrPr, crude prothrombin 28 units; Pr, purified prothrombin 4.3 units; R, rough sonicate 0.5 units; S, smooth sonicate 1.4 units.
603 line against the antiserum. This line exhibited complete identity with the precipitation line from the purified prothrombin and one of the lines from rat plasma. The result indicates that prothrombin molecules exhibiting immunological activity are present in sonicates of both rough and smooth microsomes. DISCUSSION The rough and smooth microsomes prepared in this investigation were characterized as described in recent work from this laboratory [4]. As reported previously [4], the contamination of the microsomal fractions by mitochondria and lysosomes was negligable as judged by the succinic dehydrogenase and acid phosphatase activity respectively. Accordingly the prothrombin activity detected in the rough and smooth microsomes could not be due to the presence of lysosomal cathepsin C which has been proposed [15] to convert residual prothrombin in the reagents to thrombin, giving an apparent prothrombin activity. By homogenization of the liver the channels of the endoplasmic reticulum are supposed to be disrupted and forming vesicles by a pinching-off process [16, 17]. Morgan and Peters [18] have shown for rat liver transferrin and albumin that negligable amounts of nonluminal proteins are incorporated in the content of the vesicles by this process. In agreement with this we found (Table I) that the amount of prothrombin present in the sonicates from microsomes isolated from perfused (no prothrombin in the cytosol) and nonperfused livers (7 units of prothrombin in the cytosol per g of liver) is the same. Taken together, these results indicate that the prothrombin identified in the microsomal fractions represents luminal prothrombin. By the procedure applied for isolating rough and smooth microsomes [7], the degree of cross contamination of the fractions is reduced to a satisfactory low level. Bergstrand and Dallner [7] found by electron microscope studies only a small contamination, about 5 9/0, of the rough microsomes by the smooth microsomes. Consistent with this we found only traces of protein bound sialic acid in sonicates from rough microsomes, whereas sonicates from smooth microsomes contained significantly large amounts [4]. Thus, the prothrombin activity of the rough microsomal fraction was too high to be explained by a contamination of the rough microsomes by the smooth microsomes (Table II). The presence of prothrombin activity in rough microsomes shows that the prothrombin precursor molecules at this subcellular site have acquired a structure reacting in the coagulation process. The specific activity is very low, however, compared to the corresponding activity of the smooth fraction. The difference between the specific activity of the prothrombin in the rough and smooth sonicates could be explained in several ways. One possibility is that biological active prothrombin precursor molecules in the rough fraction are attached to the membranes. This is in accordance with the hypothesis recently suggested by Redman and Cherian [3] for the secretory pathway of plasma glycoproteins in the endoplasmic reticulum whereby the nascent glycoproteins are attached to membranes of the rough endoplasmic reticulum, being transferred to the lumen of the smooth endoplasmic reticulum and Golgi apparatus after being completed. However, our recent studies of protein-bound carbohydrates in sonicates from rough and smooth microsomes do not favour this hypothesis since substantial amounts of glycoproteins in the lumen of both fractions
604 were found [4]. A possible interpretation of the low prothrombin activity of the rough sonicate could be that uncompleted prothrombin molecules are present, being less biologically active than the prothrombin in smooth microsomes. Since we have shown (Johansen, ~ and Helgeland, L., unpublished results) that all the neuraminic acid and substantial amounts of mannose, galactose and glucosamine can be removed from pure bovine prothrombin without affecting the clotting activity (also found for less extensively degraded thrombin [19] and prothrombin [20]), it seems unlikely that incomplete glycosylation of the prothrombin precursor molecules could explain the low prothrombin activity of the rough microsomes. Vitamin K seems to be active in a metabolic step in the biosynthesis of prothrombin in which the calcium binding sites of the molecules are formed [21]. Because of the significantly higher biological activity of prothrombin in the smooth microsomes it is tempting to suggest the site of action of vitamin K to be mainly the smooth fraction of the endoplasmic reticulum. The results presented in this paper strongly indicate that the subcellular site of finalization of prothrombin is the smooth endoplasmic reticulum (and Golgi apparatus). The identification of prothrombin in sonicates from rough and smooth microsomes by double immunodiffusion shows that prothrombin molecules of immunological identity to plasma prothrombin are present in both fractions. This is in agreement with observations made by Simkin and Jamieson [22] for acidic glycoproteins of guinea pig liver, which were identified in rough and smooth microsomes by immunochemical techniques. Kim et al. [23], however, found that mucin molecules of immunological identity to purified mucin from rat small intestinal mucosa could only be detected in smooth microsomes. As mucin contains 80 % carbohydrate, rough and smooth microsomes contain most probably mucin precursors which are very different. Furthermore, it is reasonable to assume that antibody against the carbohydrate component of the mucin molecule is formed. This might explain that immunological identity to completed mucin only could be observed in the smooth microsomes. ACKNOWLEDGEMENTS The authors wish to thank Professor S. G. Laland for his interest in this work. Financial support from The Norwegian Research Council for Science and the Humanities is gratefully acknowledged. REFERENCES 1 Spiro, R. G. (1970) Annu. Rev. Biochem. 39, 599-638 2 Molnar, J., Tetas, M. and Chao, H. (1969) Biochem. Biophys. Res. Commun. 37, 684-690 3 Redman, C. M. and Cherian, M. G. (1972) J. Cell. Biol. 52, 231-245 4 Helgeland, L., Christensen, T. B. and Janson, T. L. (1972) Biochim. Biophys. Acta 286, 62-71 5 Goswami, P. and Munro, H. N. (1962) Biochim. Biophys. Acta 55, 410-412 6 Helgeland, L. and Laland, S. (1962) Biochim. Biophys. Acta 62, 200-202 7 Bergstrand, A. and Dallner, G. (1969) Anal. Biochem. 29, 351-356 8 Hjort, P., Rapaport, S. J. and Owren, P. A. (1955) J. Lab. Clin. Med. 46, 89-97 9 Hasselback, R. and Hjort, P. (1960) J. Appl. Physiol. 15, 945-948 10 Lowry, O. H., Rosebrough, N. J., Farr, A. I. and Randall, R. J. (1951) J. Biol. Chem, 193,265-275 11 Li, L.-F. and Olson, R. E. (1967) J. Biol. Chem. 242, 5611-5616 12 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427
605 13 Scheidegger, J. J. (1955) Int. Arch. Allergy Appl. Immunol. 7, 103-110 14 Ouchterlony, O. (1962) (Kallos, P. and Waksman, B. H. eds.) Progress in Allergy Vol. 6, pp. 30154, Karger, New York 15 Johnston, M. F. M., Kipfer, R. K. and Olson, R. E. (1972) J. Biol. Chem. 247, 3987-3993 16 Palade, G. E. and Siekevitz, P. (1956) J. Biophys. Biochem. Cytol. 2, 171-200 (plates 28-34) 17 Dallner, G. and Ernster, L. (1968) J. Histochem. Cytochem. 16, 611-632 18 Morgan, E. H. and Peters, Jr, T. (1971) J. Biol. Chem. 246, 3508-3511 19 Skaug, K. and Christensen, T. B. (1971) Biochim. Biophys. Acta 230, 627-629 20 Nelsestuen, G. L. and Suttie, J. W. (1971) Biochem. Biophys. Res. Comm. 45, 198 21 Suttie, J. W., Nelsestuen, G. L. and Shah, D. V. (1973) Thrombos. Diathes. Haemorrh. Suppl. 54, 37-49 22 Simkin, J. L. and Jamieson, J. C. (1967) Biochem. J. 103, 153-164 23 Kim, Y. S., Perdomo, J. and Nordberg, J. (1971) J. Biol. Chem. 246, 5466 5476 24 Helgeland, L. (1975) Biochim. Biophys. Acta, in the press
(~? Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
BBA 36950 BIOLOGICAL AND I M M U N O L O G I C A L ACTIVITY OF P R O T H R O M B I N IN R O U G H AND S M O O T H MICROSOMES ISOLATED FROM RAT LIVER
T. L. JANSON and L. HELGELAND Department of Biochem&try, University of Oslo, Blindern (Norway)
(Received May 10th, 1974)
SUMMARY 1. The presence of biological and immunological activity of prothrombin in sonicates from rough and smooth microsomes has been investigated. 2. Biological activity of prothrombin was detected in both the rough and smooth microsomal fraction. The specific and the total activity of prothrombin in the sonicates from smooth microsomes were 3-4-fold higher than in the corresponding fraction from rough microsomes. 3. Prothrombin could be identified in both microsomal fractions by double immunodiffusion. 4. The presence of a macromolecular inhibitor of blood coagulation in the microsomes is reported.
INTRODUCTION The biosynthesis of glycoproteins in rat liver has for some years been extensively studied [1 ] and at present there is much evidence in favour of a stepwise addition of carbohydrates to the polypeptide chain as this moves through the endoplasmic reticutum [1-4]. In accordance with this a different degree of finalization of a glycoprotein would be expected in the rough and smooth microsomes. The purpose of the present work was to investigate at which submicrosomal site biological activity of a glycoprotein could be detected. Prothrombin, a vitamin K-dependent plasma glycoprotein containing about 10 ~o carbohydrate and which has been shown to be localized in the microsomes [5, 6], was chosen since the biological activity can be easily measured by a specific and sensitive coagulation test. As the concentration of prothrombin in the sonicates of the microsomes was very low, the extracts had to be concentrated to obtain reliable analyses of prothrombin. During experiments carried out to reduce the volume of the sonicates, an inhibitor of blood coagulation appeared. The results obtained in the present investigation indicate that both the rough and the smooth microsomes contained detectable amounts of protein molecules exhibiting the biological and immunological activity of prothrombin. By far the highest biological activity was, however, found in the smooth microsomes.
599 MATERIALS AND METHODS
Animals. Male albino rats, 150-200 g, of the Wistar strain (Mollegaard, Havrup, Denmark) were used. The animals were starved for 20 h before being killed by decapitation. Chemicals. CsC1 suprapure from Merck A.G. was used for the isolation of rough and smooth microsomes. Agarose was obtained from Behringwerke A.G. Aquacide lI A grade was purchased from Calbiochem. Freund's Complete Adjuvant was produced by Difco Laboratories. Russel's Viper Venom (Stypven) was obtained from Burroughs Wellcome. Preparation and characterization of the microsomal fractions. The preparation of rough and smooth microsomes was carried out as described by Helgeland et al. [4] using the method of Bergstrand and Dallner [7]. The microsome fractions were washed once with 0.25 M sucrose and the pellets of smooth and rough microsomes (derived from 30 g of liver) were suspended in 1.5 ml of a solution of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HCl buffer pH 7.4 [6]. The suspensions were sonicated for five periods of 30 s in a Branson sonifier at a setting of 1.5 while being cooled in a salt/ice mixture. Intervals of 1 min were included to keep the temperature below 8-10 °C during the treatment. The resulting suspensions were centrifuged for 1 h at 105 000 × g. The rough and smooth microsomes were characterized as described previously [4]. Total microsomes from perfused and nonperfused livers were prepared as described by Bergstrand and Dallner [7]. The livers were perfused through the portal vein with 0.25 M sucrose. Concentration of the sonicates. The sonicates were concentrated either by using a 50 ~ solution of aquacide in a mixture of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HCl buffer pH 7.4 or by dialysis of the sonicates against distilled water followed by lyophilization and dissolving in a small volume of a solution of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HC1 buffer pH 7.4. Analytical methods. Prothrombin was measured using the one-stage method of Hjort et al. [8]. The test was made specific for prothrombin by adding Factor X (Stuart) to the test solution [9]. One unit of prothrombin was arbitrarily chosen as the amount of prothrombin present in 1 ml of standard human plasma diluted 1:1000. Protein was determined by the method of Lowry et al. [10] with crystalline bovine serum albumin as standard. Preparation of crude rat prothrombin and antiserum against crude rat prothrombin. Prothrombin was isolated from rat plasma essentially as described by Li and Olson [11]. Blood was drawn by heart puncture. The product obtained revealed four bands on polyacrylamide gel-disc electrophoresis at pH 9.1. Antisera to crude prothrombin was raised in rabbits by injecting 1.2 mg of crude prothrombin in 1 ml of 0.9 ~ NaC1 and 1 ml of Freund's Complete Adjuvant intramuscularly. Booster doses with half the amount of antigen were given 3, 5 and 6 weeks later. Blood was collected one week after the final injection. Purification of rat prothrombin. Prothrombin was further purified by disc electrophoresis of crude prothrombin in 5 ~ gel pH 9.1 and 4 °C as described by Davis [12]. 75 #g of protein was applied on each gel (0.5 cm x 7.0 cm). The pro-
600 thrombin bands were carefully sliced out and the prothrombin eluted by crushing the gel in 0.5 ml of a solution of 0.12 M sodium chloride, 6.06 mM sodium citrate and 5.14 mM veronal/HC1 buffer pH 7.4 and left overnight by 4 °C. The gel was removed by centrifugation. The purified prothrombin appeared homogeneous by polyacrylamide disc electrophoresis pH 9.1 and by immunoelectrophoresis pH 8.6 as described by Scheidegger [13]. Double immunodiffusion. The double immunodiffusion was performed according to Ouchterlony [14]. 1% agarose gel in barbiturate buffer pH 8.6 was used. The gels were of 2 mm thickness. The diameters of the wells were 2 mm and 6-7 mm apart. 5-7 #1 of antigen solution were applied in each well. The precipitates were stained with Amido black. RESULTS
On the inhibitory activity in sonicates from rough and smooth microsomes on the bioassay of prothrombin To obtain reliable analyses of prothrombin, the volumes of the sonicates from the microsomes had to be reduced. It is seen from Fig. 1, however, that the recovery of prothrombin activity decreased with the degree of concentration of the sonicates. Upon dilution to the original volume, a complete recovery of the prothrombin activity was obtained. This result indicates that the reduced prothrombin activity is due to an inhibitory effect on the coagulation assay as a result of the concentration procedure. The inhibition did not seem to be a mere effect of the increased concen-
Rough m i c r o s o m e s
Smooth
mic rosomes
v
lOO
£ E
8c
o
S 6o Q.
)
Q 0
4c
a: 2c
0
I 1
I 2
I 3
I 4
Concentration
I 5
I 1
of p r o t h r o r n b i n
I 2
I 3 (arbitrary
I 4
| 5
I-
units)
Fig. 1. Recovery of prothrombin activity by concentrating sonicates of rough and smooth microsomes. The sonicates of rough and smooth microsomes were concentrated by: treatment by aquacide ( 0 ) and dialysis and lyophilization ( 0 ) . The prothrombin activity in the original sonicates was chosen as 100%.
601 TABLE I BIOLOGICAL ACTIVITY OF PROTHROMBIN IN SONICATES OF MICROSOMES AND CYTOSOL ISOLATED FROM PERFUSED AND NON-PERFUSED RAT LIVERS Total activity is expressed as units of protbrombin/g of liver. N.D., no detectable activity. The values from two experiments are given.
Non-perfused livers Perfused livers
Total activity in sonicates
Total activity in cytosol
0.81 0.87
6.3
0.78 0.81
7.7 N.D.
t r a t i o n o f protein, as it was shown t h a t s e r u m a l b u m i n o f a c o r r e s p o n d i n g concent r a t i o n d i d n o t i n h i b i t the c o a g u l a t i o n process. The i n h i b i t i o n m i g h t m o s t p r o b a b l y be e x p l a i n e d b y the presence o f an i n h i b i t o r o f b l o o d c o a g u l a t i o n in the microsomes. W h e t h e r the sonicates were c o n c e n t r a t e d by a q u a c i d e o r b y dialysis a g a i n s t distilled w a t e r a n d subsequent l y o p h i l i z a t i o n , the same i n h i b i t i o n was o b t a i n e d . It a p p e a r s f r o m Fig. 1 t h a t the i n h i b i t i o n was m o r e p r o n o u n c e d in the sonicates f r o m r o u g h m i c r o s o m e s . It has recently been shown in this l a b o r a t o r y t h a t the i n h i b i t o r y effect is due to a heat-stable, m a c r o m o l e c u l a r i n h i b i t o r o f the t h r o m b i n - f i b r i n o g e n reaction [241.
Identification of prothrombin in sonicates of rough and smooth microsomes by measuring the biological activity T a b l e I shows t h a t the a m o u n t o f p r o t h r o m b i n in the sonicate f r o m microsomes is the same w h e t h e r the livers have been perfused or n o t a l t h o u g h it was found t h a t the c y t o s o l isolated f r o m n o n - p e r f u s e d livers c o n t a i n e d a b o u t 7 units/g o f liver whereas the cytosol f r o m perfused livers did n o t c o n t a i n a n y detectable a m o u n t o f prothrombin. I n o r d e r to o b t a i n reliable analyses o f the p r o t h r o m b i n c o n t e n t o f the m i c r o s o m a l extracts, the i n h i b i t o r h a d to be r e m o v e d . This was achieved by a d s o r b i n g the p r o t h r o m b i n on b a r i u m citrate [24]. T h e precipitate was subsequently dissolved in a solution o f 0.03 M s o d i u m citrate, 0.23 M s o d i u m c h l o r i d e a n d 0.05 M E D T A ,
TABLE II BIOLOGICAL ACTIVITY OF PROTHROMBIN IN SONICATES FROM ROUGH AND SMOOTH MICROSOMES Rough and smooth microsomes from 30 g of liver were sonicated as described in Methods. The prothrombin of 1.0 ml of the sonicate fractions was adsorbed on barium citrate. The precipitate was dissolved in sodium citrate, sodium chloride and EDTA and the solution subjected to gel filtration on a Sephadex G-50 column [24]. Specific activity is expressed as units of prothrombin/mg of protein in the eluate from the Sephadex column. Total activity is expressed as units of prothrombin/g of liver. The values are the means of three experiments zk S.D. Fraction
Specific activity
Total activity
Rough microsomes Smooth microsomes
5.9 4- 0.96 22.8 4- 2.0
0.11 ± 0.009 0.29 4- 0.036
602 and the solution gel filtrated on a Sephadex G-50 Fine column equilibrated with the test buffer. By this procedure a sufficient degree of concentration was also obtained [24]. As shown in Table II, sonicates from both rough and smooth microsomes contained biological activity of prothrombin, the specific and the total activity of the latter fraction being 3-4 fold higher. Similar ratios were found when testing the activity of untreated sonicates. Only a rough estimate of the prothrombin activities in the untreated sonicates could be obtained, however, as the clotting times ( > 90 s) exceeded the limit of reliable measurements, but it suggests that there is no difference in the adsorption of prothrombin from rough and smooth sonicates to barium citrate.
Immunological activity of prothrombin in sonicates of rough and smooth microsomes detected by double immunodiffusion In order to identify prothrombin in the microsomal fractions by immunochemical methods, antiserum to crude prothrombin was used. Fig. 2a shows that the antiserum contained antibodies to all the four components of crude prothrombin, including prothrombin, whereas the purified prothrombin gave one precipitation line with the antiserum. This line formed complete identity with one of the arcs from rat plasma and one from the crude prothrombin. This is in accordance with the homogeneity of purified prothrombin observed by disc electrophoresis and immuneelectrophoresis (see Methods). The purified prothrombin was accordingly accepted as satisfactorily pure to be used in the double-immunodiffusion assay for testing the immunological activity of prothrombin from the microsomal fractions. Fig. 2b shows that the sonicates from both the rough and smooth microsomes formed one precipitation
(b)
(a)
(o ©~ CrPr
Ab
0
0
0
0
0
© Pr
PI
R
Pr
PI
S
Pr
Fig. 2. Double immunodiffusion of (a) different preparations of prothrombin against antiserum to crude prothrombin, (bl) sonicates of rough and smooth microsomes, concentrated about 45 times, against antiserum to crude prothrombin. The photograph was taken after staining (b2) prothrombin from sonicates of rough and smooth microsomes concentrated about 45 times after purification by adsorption to and elution from barium citrate. The photograph was taken before staining. Ab, antiserum 5/d; Pl, rat plasma 25 units (2a), 5 units (2b); CrPr, crude prothrombin 28 units; Pr, purified prothrombin 4.3 units; R, rough sonicate 0.5 units; S, smooth sonicate 1.4 units.
603 line against the antiserum. This line exhibited complete identity with the precipitation line from the purified prothrombin and one of the lines from rat plasma. The result indicates that prothrombin molecules exhibiting immunological activity are present in sonicates of both rough and smooth microsomes. DISCUSSION The rough and smooth microsomes prepared in this investigation were characterized as described in recent work from this laboratory [4]. As reported previously [4], the contamination of the microsomal fractions by mitochondria and lysosomes was negligable as judged by the succinic dehydrogenase and acid phosphatase activity respectively. Accordingly the prothrombin activity detected in the rough and smooth microsomes could not be due to the presence of lysosomal cathepsin C which has been proposed [15] to convert residual prothrombin in the reagents to thrombin, giving an apparent prothrombin activity. By homogenization of the liver the channels of the endoplasmic reticulum are supposed to be disrupted and forming vesicles by a pinching-off process [16, 17]. Morgan and Peters [18] have shown for rat liver transferrin and albumin that negligable amounts of nonluminal proteins are incorporated in the content of the vesicles by this process. In agreement with this we found (Table I) that the amount of prothrombin present in the sonicates from microsomes isolated from perfused (no prothrombin in the cytosol) and nonperfused livers (7 units of prothrombin in the cytosol per g of liver) is the same. Taken together, these results indicate that the prothrombin identified in the microsomal fractions represents luminal prothrombin. By the procedure applied for isolating rough and smooth microsomes [7], the degree of cross contamination of the fractions is reduced to a satisfactory low level. Bergstrand and Dallner [7] found by electron microscope studies only a small contamination, about 5 9/0, of the rough microsomes by the smooth microsomes. Consistent with this we found only traces of protein bound sialic acid in sonicates from rough microsomes, whereas sonicates from smooth microsomes contained significantly large amounts [4]. Thus, the prothrombin activity of the rough microsomal fraction was too high to be explained by a contamination of the rough microsomes by the smooth microsomes (Table II). The presence of prothrombin activity in rough microsomes shows that the prothrombin precursor molecules at this subcellular site have acquired a structure reacting in the coagulation process. The specific activity is very low, however, compared to the corresponding activity of the smooth fraction. The difference between the specific activity of the prothrombin in the rough and smooth sonicates could be explained in several ways. One possibility is that biological active prothrombin precursor molecules in the rough fraction are attached to the membranes. This is in accordance with the hypothesis recently suggested by Redman and Cherian [3] for the secretory pathway of plasma glycoproteins in the endoplasmic reticulum whereby the nascent glycoproteins are attached to membranes of the rough endoplasmic reticulum, being transferred to the lumen of the smooth endoplasmic reticulum and Golgi apparatus after being completed. However, our recent studies of protein-bound carbohydrates in sonicates from rough and smooth microsomes do not favour this hypothesis since substantial amounts of glycoproteins in the lumen of both fractions
604 were found [4]. A possible interpretation of the low prothrombin activity of the rough sonicate could be that uncompleted prothrombin molecules are present, being less biologically active than the prothrombin in smooth microsomes. Since we have shown (Johansen, ~ and Helgeland, L., unpublished results) that all the neuraminic acid and substantial amounts of mannose, galactose and glucosamine can be removed from pure bovine prothrombin without affecting the clotting activity (also found for less extensively degraded thrombin [19] and prothrombin [20]), it seems unlikely that incomplete glycosylation of the prothrombin precursor molecules could explain the low prothrombin activity of the rough microsomes. Vitamin K seems to be active in a metabolic step in the biosynthesis of prothrombin in which the calcium binding sites of the molecules are formed [21]. Because of the significantly higher biological activity of prothrombin in the smooth microsomes it is tempting to suggest the site of action of vitamin K to be mainly the smooth fraction of the endoplasmic reticulum. The results presented in this paper strongly indicate that the subcellular site of finalization of prothrombin is the smooth endoplasmic reticulum (and Golgi apparatus). The identification of prothrombin in sonicates from rough and smooth microsomes by double immunodiffusion shows that prothrombin molecules of immunological identity to plasma prothrombin are present in both fractions. This is in agreement with observations made by Simkin and Jamieson [22] for acidic glycoproteins of guinea pig liver, which were identified in rough and smooth microsomes by immunochemical techniques. Kim et al. [23], however, found that mucin molecules of immunological identity to purified mucin from rat small intestinal mucosa could only be detected in smooth microsomes. As mucin contains 80 % carbohydrate, rough and smooth microsomes contain most probably mucin precursors which are very different. Furthermore, it is reasonable to assume that antibody against the carbohydrate component of the mucin molecule is formed. This might explain that immunological identity to completed mucin only could be observed in the smooth microsomes. ACKNOWLEDGEMENTS The authors wish to thank Professor S. G. Laland for his interest in this work. Financial support from The Norwegian Research Council for Science and the Humanities is gratefully acknowledged. REFERENCES 1 Spiro, R. G. (1970) Annu. Rev. Biochem. 39, 599-638 2 Molnar, J., Tetas, M. and Chao, H. (1969) Biochem. Biophys. Res. Commun. 37, 684-690 3 Redman, C. M. and Cherian, M. G. (1972) J. Cell. Biol. 52, 231-245 4 Helgeland, L., Christensen, T. B. and Janson, T. L. (1972) Biochim. Biophys. Acta 286, 62-71 5 Goswami, P. and Munro, H. N. (1962) Biochim. Biophys. Acta 55, 410-412 6 Helgeland, L. and Laland, S. (1962) Biochim. Biophys. Acta 62, 200-202 7 Bergstrand, A. and Dallner, G. (1969) Anal. Biochem. 29, 351-356 8 Hjort, P., Rapaport, S. J. and Owren, P. A. (1955) J. Lab. Clin. Med. 46, 89-97 9 Hasselback, R. and Hjort, P. (1960) J. Appl. Physiol. 15, 945-948 10 Lowry, O. H., Rosebrough, N. J., Farr, A. I. and Randall, R. J. (1951) J. Biol. Chem, 193,265-275 11 Li, L.-F. and Olson, R. E. (1967) J. Biol. Chem. 242, 5611-5616 12 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427
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