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A procedure for the preparation of [32P]phosphatidic acid
ANALYTICAL BIOCHEMISTRY155, 119-122 (1986)
A Procedure R. KENNEDY Department
of Biochemistry,
for the Preparation KELLER, University
of [32P]Phosphatidic
Acid’
W. LEE ADAIR, AND NANCY CAFMEYER of South Florida
College
of Medicine,
Tampa,
Florida
33612
Received December 2, 1985 The phosphorylation procedure of F. Cramer, W. Rittersdorf, and W. Bohm [( 1961) Chem. 654, 1801 using bis(triethylammonium) phosphate and trichloroacetonitrile was shown to be effective in the synthesis of [32P]phosphatidic acid. From diacylglyceride and 0.5 mCi HP*POd, 25-50 pCi of labeled material (sp act = 1 mCi/pmol) can be prepared in 2 h. The product was shown to be radiochemically pure by both TLC and HPLC. L- and DL-[32P]dip~mitoyl phosphatidic acid prepared using this procedure were shown to be hydrolyzed by rat liver microsomes at apprOXlnK& the same K&S. 0 1986 Academic Press, Inc. KEY WORDS: lipids; phospholipids; phosphatases; organic synthesis: radioactive. Ber.
We recently completed an investigation of the substrate specificity of an activity in rat liver microsomes which carries out the dephosphorylation of dolichyl phosphate. As part of this study, we wished to examine whether the phosphatase which acted on dolichyl phosphate was the same as that responsible for the hydrolysis of PA*. We therefore sought a rapid procedure to monitor the hydrolysis of PA. We favored a radiochemical assay for 32Pi release over the classical colorimetric procedure (1) since the former offers greater speed, specificity, and sensitivity. In addition, we found that the presence of detergents such as Triton X-100 interfered with the calorimetric assay. Two procedures are presently available for the preparation of [32P]PA. In the chemical method of Hokin et al. (Z), 32P is added to PC& to form 32POC13 in an exchange reaction. This procedure is not easily adaptable to a microscale and thus the product generated from ordinary working levels ( 1- 10 mCi) of 3zPi would have a relatively low spe’ This work was supported by NIH Grants GM25364 and CA28781. This work was performed when R.K.K. and W.L.A. were Research Career Development Awardees of the National Institutes of Health. * Abbreviation used: PA, phosphatidic acid.
cific activity (which, because of the exchange reaction, would have to be determined empirically). In addition, the volatility of 32POC13 presents a potential safety hazard. In the enzymatic preparation of [32P]PA, the product is generated from [y-32P]ATP, d&y&de, and a crude cell fraction [bovine brain cytosol(3) and Escherichia coli cytosol (4) have been used] which serves as a source of diglyceride kinase. In addition to the expense of the preparation or purchase of [-Y-~*P]ATP, this procedure has the added disadvantage of requiring preparation of an enzyme source and then the purification and determination of the specific activity of the [32P]PA. In the present report we show that [32P]PA can easily be prepared from diacylglycerol and “Pi by a modification of the procedure of Cramer et al. (5) in which bis(triethylammonium) phosphate serves as the phosphorylating agent and trichloroacetonitrile serves as the condensing agent. The procedure is rapid and generates a radiochemically pure product of known specific activity in good yield. By preparing both L- and DL-[32P]PA, we were able to demonstrate for the first time that rat liver microsomes show no stereospecificity toward hydrolysis of the two isomers of PA. 119
0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
120
KELLER, MATERIALS
AND
Chemicals. Triethylamine,
ADAIR, AND CAFh4EYER
METHODS
1,2dipalmitoylsn-glycerol, and 1,2-dipalmitoyl-rat-glycerol were from Sigma Chemical Company. Trichloroacetonitrile, acetonitrile, and ethylene dichloride were from Aldrich. The acetonitrile and ethylene dichloride were stored over 4pm molecular sieves. H332P04 (carrier free, in 0.02 N HCl) was from ICN. Phosphoric acid (crystalline) was obtained from Tridom. Preparation of phosphorylating reagent. Bis(triethylammonium) [32P]phosphate was prepared as follows. H3P04 (0.55 pmol) and H332P04 (0.55 mCi) were taken to dryness under a stream of argon or nitrogen at 50°C in a 10 X 75-mm disposable glass test tube. The residue was treated with toluene (0.2 ml) and taken to dryness as above. This treatment was then repeated to remove the last traces of water and HCl. After addition of 2 pmol triethylamine in 55 ~1 acetonitrile, the tube was capped and vortexed well. P.eparation of 32P-dipalmitoyl phosphatidic acid. A solution of dipalmitin (sn or rat) containing 1.5 pmol (0.85 mg) in chloroform/ methanol (2/ 1) was taken to dryness in a 13 X loo-mm screw-capped tube under a stream of argon. The residue was dissolved in 50 ~1 ethylene dichloride containing 3 pmol trichloroacetonitrile. Bis(triethylammonium) [32P]phosphate (0.5 pmol, 0.5 mCi) in 50 ~1 acetonitrile was then added in a single portion. The tube was capped (using a Teflon-lined cap) and heated at 50°C for 1 h. After cooling, the reaction mixture was diluted with 2 ml chloroform, 1 ml methanol and 0.75 ml water. Following centrifugation, the upper phase was removed and the lower phase washed with an equal volume of 50% methanol. The lower phase was then applied to a 1 X 2-cm column of DEAE-cellulose (acetate form) equilibrated with chloroform/methanol (2/l). After being rinsed with 10 ml chloroform/methanol (2/ I), the product was eluted with 10 ml 0.1 M ammonium ac’tate in chloroform/methanol (2/ 1). One-quartc vol of water was added and the resulting lower -base, containing the la-
beled product, was collected after centrifugation and stored at -20°C. The final yield, based on added radioactivity, was 5- 10%. Thin layer chromatography. TLC was carried out on precoated plastic-backed plates of silica gel 60 (EM Reagents). The solvent was chloroform/methanol/ammonia/ water (65/35/2/2). Following TLC, the plate was dried well with a heat gun to remove ammonia, developed in an iodine vapor tank, and photographed using the instant copy technique of Engstrom et al. (6).
FIG. I. Upper: TLC analysis of [“PIPA prepared as described under Materials and Methods. Standards included 5 pg each of dolichyl phosphate (Dol-P), phosphatidylcholine (PC), and phosphatidic acid. Lower: Radiochromatogram scan of lane containing [“PIPA. 0, origin; SF, solvent front.
PREPARATION A 210
,
OF [32P]PHOSPHATIDIC 32
Relative
nm
3.995
I_
P cpm
--
5.755 8 $==y==STOP
121
ACID
J w
FIG. 2. HPLC analysis of [‘*PIPA. Labeled material (20,000 cpm) was mixed with 5 pg of standard dioleoyl phosphatidic acid and injected into the HPLC. Upper panel: On-line radioactivity monitoring was carried out with a scintillant flow rate of 3 ml/min. Lower panel: On-line uv monitoring.
High-pressure
liquid
chromatography.
HPLC was performed on a Laboratory Data Control instrument equipped with on-line uv monitoring (2 10 nm). On-line radiochemical monitoring was carried out using a Flow-One monitor (Radiomatic Instruments, Tampa, Fla.). Integration and analysis of the uv output was achieved with a Shimadzu C-R3A Chromatopac. Analysis of PA was carried out on 5-pm silica columns as described for the chromatography of dolichyl phosphate (7). The mobile phase of hexane/isopropanol/ 1.4 M H3P04 (90/ 1O/OS) was pumped at a flow rate of 1 ml/min. Microsomes. Microsomes were prepared from the livers of fasted rats according to Snider et al. (8). The microsomal pellet was dissolved in 0.25 M sucrose to give a final concentration of 1 mg protein/ml. Assay for hydrolysis of [j2P]PA. [32P]PA (200,000 cpm) in chloroform/methanol was mixed with 40 ~1 of 1% Triton X- 100 in chloroform and the solution taken to dryness under argon. The residue was dissolved in 300 ~1 of 0.01 M Tris-Cl, pH 7.0. Water and microsomes (usually 20 fig protein) were then added to give a final volume of 0.4 ml. Aliquots were removed at various times and dispensed into 2 ml chloroform/methanol (2/l). The samples were then treated with 0.4 ml of 0.15 M KCl/5 mM Na,HPO, and centrifuged at 1000 rpm for 5 min. One-half-milliliter aliquots of the upper phase were mixed with 4 ml of Scintiverse E (Fisher Scientific) for de-
termination of released 32Pi by liquid scintillation analysis. RESULTS AND DISCUSSION Analysis of chemically prepared [“‘PIPA.
The preparation of [32P]PA described under Materials and Methods yields 25-50 PCi of purified material from 1.5 pmol dipalmitin and 0.5 mCi 32Pi.The product, which can be prepared and purified in 2 h, has a specific activity of 1 mCi/pmol, 10,000 times greater than that reported for the original 32POC13 procedure (2). In addition, unlike the POCl3 procedure, the specific activity of the product
Y 0.5
1.0 Time
1.5
2.0
2.5
(h)
FIG. 3. Time course of hydrolysis of D,L-[“PIPA (solid circles) and L-[‘*PIPA (open circles). Reactions were carried out using 100 pg microsomal protein. For details, see Materials and Methods.
122
KELLER.
ADAIR, AND CAFMEYER
can be predetermined by adjusting the specific activity of the starting H332P04. The radiochemical purity of the product is demonstrated by TLC analysis in Fig. 1 and HPLC analysis in Fig. 2. Use of [32P]PA to assayfor PA hydrolysis.
We used rat liver microsomes as a source of hydrolase activity to test the biological activity of the chemically prepared [32P]PA. When assayed as described under Materials and Methods with various concentrations of [32P]PA, release of 32P into the upper phase was found to be saturable and exhibit a K,,, of 28 PM (data not shown). This value is similar to those reported by others for microsomal phosphatidate phosphatase (9,lO). The 32P released was in the form of inorganic phosphate, since assay of release using ammonium molybdate followed by isobutanol treatment, which specifically extracts Pi (1 I), yielded the same results as the chloroform/methanol extraction (data not shown). It is to be noted, however, that others ( 12,13) have shown that Pi release does not necessarily reflect phosphatidate phosphatase activity but can also be due to the combined action of lipase and glycerol-3phosphate phosphohydrolase. The availability of both 1,2-dipalmitoyl-snglycerol and 1,2-dipalmitoyl-rat-glycerol allowed us to prepare racemic (i.e. DL) [32P]PA as well as L-[~~P]PA and therefore test, for the first time, the susceptibility to hydrolysis of the D form. Fig. 3 shows that under the conditions of assay, both L- and DL-[~~P]PA are hydrolyzed to completion and at similar rates, indicating that there is no preference for the naturally occurring L isomer. Since phospholipases are specific for the L isomer (14), the
data suggest that the primary enzyme responsible for the release of 32Pi under our assay conditions is phosphatidate phosphatase. The availability of a facile procedure for the preparation of [32P]PA should aid in future in vitro studies of phosphatidic acid metabolism. ACKNOWLEDGMENT We acknowledge the assistance of Susan D. Brennan in the preparation of labeled phosphatidic acid.
REFERENCES 1. Hajra, A. K., and Agranoff, B. W. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 14, pp. 185-188, Academic Press, New York. 2. Hokin, L. E., Hokin, M. R., and Mathison, D. (1963) Biochim. Biophys. Acta 67,485-491. 3. Hosaka, K., Yamashita, S., and Numa, S. (1975) J. Biochem. 77,50 l-509. 4. Pieringer, R. A., and Kunnes, R. S. (1965) J. Biol. Chem. 240,2833. 5. Cramer, F., Rittersdorf, W., and Bohm, W. (1961) Chem. Ber. 654, 180-188. 6. Engstrom, N., Hellgren, L., and Vincent, J. (1980) J. Chromatogr. 189,284. 7. Keller, R. K., Fuller, M. S., Rottler, G. D., and Connelly. L. W. (1985) Anal. Biochem. 147, 166-173. 8. Snider, M. D., Sultzman, L. A., and Robbins, P. W. (1980)
Cell 21,385-392.
Caras, I., and Shapiro, B. (1975) Biochim. Biophys. Acta 409, 201-211. 10. Hosaka, K., Yamashita, S., and Numa, S. (1975) J. Biochem. 77,50 I-509. II. Berenblum, I., and Chain, E. (1938) Biochem. J. 32, 9.
295-298.
Sturton, R. G., and Brindley, D. N. ( 1978) Biochem. J. 171,263-266. 13. Smith, M. E., Sedgwick, B., Brindley, D. N., and Hubscher, G. (1967) Eur. J. Biochem. 3, 70-77. 14. VanDeenen, L. L. M., and DeHaas, G. H. (1963) Biochim. Biophys. Acta 70, 538. 12.
A Procedure R. KENNEDY Department
of Biochemistry,
for the Preparation KELLER, University
of [32P]Phosphatidic
Acid’
W. LEE ADAIR, AND NANCY CAFMEYER of South Florida
College
of Medicine,
Tampa,
Florida
33612
Received December 2, 1985 The phosphorylation procedure of F. Cramer, W. Rittersdorf, and W. Bohm [( 1961) Chem. 654, 1801 using bis(triethylammonium) phosphate and trichloroacetonitrile was shown to be effective in the synthesis of [32P]phosphatidic acid. From diacylglyceride and 0.5 mCi HP*POd, 25-50 pCi of labeled material (sp act = 1 mCi/pmol) can be prepared in 2 h. The product was shown to be radiochemically pure by both TLC and HPLC. L- and DL-[32P]dip~mitoyl phosphatidic acid prepared using this procedure were shown to be hydrolyzed by rat liver microsomes at apprOXlnK& the same K&S. 0 1986 Academic Press, Inc. KEY WORDS: lipids; phospholipids; phosphatases; organic synthesis: radioactive. Ber.
We recently completed an investigation of the substrate specificity of an activity in rat liver microsomes which carries out the dephosphorylation of dolichyl phosphate. As part of this study, we wished to examine whether the phosphatase which acted on dolichyl phosphate was the same as that responsible for the hydrolysis of PA*. We therefore sought a rapid procedure to monitor the hydrolysis of PA. We favored a radiochemical assay for 32Pi release over the classical colorimetric procedure (1) since the former offers greater speed, specificity, and sensitivity. In addition, we found that the presence of detergents such as Triton X-100 interfered with the calorimetric assay. Two procedures are presently available for the preparation of [32P]PA. In the chemical method of Hokin et al. (Z), 32P is added to PC& to form 32POC13 in an exchange reaction. This procedure is not easily adaptable to a microscale and thus the product generated from ordinary working levels ( 1- 10 mCi) of 3zPi would have a relatively low spe’ This work was supported by NIH Grants GM25364 and CA28781. This work was performed when R.K.K. and W.L.A. were Research Career Development Awardees of the National Institutes of Health. * Abbreviation used: PA, phosphatidic acid.
cific activity (which, because of the exchange reaction, would have to be determined empirically). In addition, the volatility of 32POC13 presents a potential safety hazard. In the enzymatic preparation of [32P]PA, the product is generated from [y-32P]ATP, d&y&de, and a crude cell fraction [bovine brain cytosol(3) and Escherichia coli cytosol (4) have been used] which serves as a source of diglyceride kinase. In addition to the expense of the preparation or purchase of [-Y-~*P]ATP, this procedure has the added disadvantage of requiring preparation of an enzyme source and then the purification and determination of the specific activity of the [32P]PA. In the present report we show that [32P]PA can easily be prepared from diacylglycerol and “Pi by a modification of the procedure of Cramer et al. (5) in which bis(triethylammonium) phosphate serves as the phosphorylating agent and trichloroacetonitrile serves as the condensing agent. The procedure is rapid and generates a radiochemically pure product of known specific activity in good yield. By preparing both L- and DL-[32P]PA, we were able to demonstrate for the first time that rat liver microsomes show no stereospecificity toward hydrolysis of the two isomers of PA. 119
0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
120
KELLER, MATERIALS
AND
Chemicals. Triethylamine,
ADAIR, AND CAFh4EYER
METHODS
1,2dipalmitoylsn-glycerol, and 1,2-dipalmitoyl-rat-glycerol were from Sigma Chemical Company. Trichloroacetonitrile, acetonitrile, and ethylene dichloride were from Aldrich. The acetonitrile and ethylene dichloride were stored over 4pm molecular sieves. H332P04 (carrier free, in 0.02 N HCl) was from ICN. Phosphoric acid (crystalline) was obtained from Tridom. Preparation of phosphorylating reagent. Bis(triethylammonium) [32P]phosphate was prepared as follows. H3P04 (0.55 pmol) and H332P04 (0.55 mCi) were taken to dryness under a stream of argon or nitrogen at 50°C in a 10 X 75-mm disposable glass test tube. The residue was treated with toluene (0.2 ml) and taken to dryness as above. This treatment was then repeated to remove the last traces of water and HCl. After addition of 2 pmol triethylamine in 55 ~1 acetonitrile, the tube was capped and vortexed well. P.eparation of 32P-dipalmitoyl phosphatidic acid. A solution of dipalmitin (sn or rat) containing 1.5 pmol (0.85 mg) in chloroform/ methanol (2/ 1) was taken to dryness in a 13 X loo-mm screw-capped tube under a stream of argon. The residue was dissolved in 50 ~1 ethylene dichloride containing 3 pmol trichloroacetonitrile. Bis(triethylammonium) [32P]phosphate (0.5 pmol, 0.5 mCi) in 50 ~1 acetonitrile was then added in a single portion. The tube was capped (using a Teflon-lined cap) and heated at 50°C for 1 h. After cooling, the reaction mixture was diluted with 2 ml chloroform, 1 ml methanol and 0.75 ml water. Following centrifugation, the upper phase was removed and the lower phase washed with an equal volume of 50% methanol. The lower phase was then applied to a 1 X 2-cm column of DEAE-cellulose (acetate form) equilibrated with chloroform/methanol (2/l). After being rinsed with 10 ml chloroform/methanol (2/ I), the product was eluted with 10 ml 0.1 M ammonium ac’tate in chloroform/methanol (2/ 1). One-quartc vol of water was added and the resulting lower -base, containing the la-
beled product, was collected after centrifugation and stored at -20°C. The final yield, based on added radioactivity, was 5- 10%. Thin layer chromatography. TLC was carried out on precoated plastic-backed plates of silica gel 60 (EM Reagents). The solvent was chloroform/methanol/ammonia/ water (65/35/2/2). Following TLC, the plate was dried well with a heat gun to remove ammonia, developed in an iodine vapor tank, and photographed using the instant copy technique of Engstrom et al. (6).
FIG. I. Upper: TLC analysis of [“PIPA prepared as described under Materials and Methods. Standards included 5 pg each of dolichyl phosphate (Dol-P), phosphatidylcholine (PC), and phosphatidic acid. Lower: Radiochromatogram scan of lane containing [“PIPA. 0, origin; SF, solvent front.
PREPARATION A 210
,
OF [32P]PHOSPHATIDIC 32
Relative
nm
3.995
I_
P cpm
--
5.755 8 $==y==STOP
121
ACID
J w
FIG. 2. HPLC analysis of [‘*PIPA. Labeled material (20,000 cpm) was mixed with 5 pg of standard dioleoyl phosphatidic acid and injected into the HPLC. Upper panel: On-line radioactivity monitoring was carried out with a scintillant flow rate of 3 ml/min. Lower panel: On-line uv monitoring.
High-pressure
liquid
chromatography.
HPLC was performed on a Laboratory Data Control instrument equipped with on-line uv monitoring (2 10 nm). On-line radiochemical monitoring was carried out using a Flow-One monitor (Radiomatic Instruments, Tampa, Fla.). Integration and analysis of the uv output was achieved with a Shimadzu C-R3A Chromatopac. Analysis of PA was carried out on 5-pm silica columns as described for the chromatography of dolichyl phosphate (7). The mobile phase of hexane/isopropanol/ 1.4 M H3P04 (90/ 1O/OS) was pumped at a flow rate of 1 ml/min. Microsomes. Microsomes were prepared from the livers of fasted rats according to Snider et al. (8). The microsomal pellet was dissolved in 0.25 M sucrose to give a final concentration of 1 mg protein/ml. Assay for hydrolysis of [j2P]PA. [32P]PA (200,000 cpm) in chloroform/methanol was mixed with 40 ~1 of 1% Triton X- 100 in chloroform and the solution taken to dryness under argon. The residue was dissolved in 300 ~1 of 0.01 M Tris-Cl, pH 7.0. Water and microsomes (usually 20 fig protein) were then added to give a final volume of 0.4 ml. Aliquots were removed at various times and dispensed into 2 ml chloroform/methanol (2/l). The samples were then treated with 0.4 ml of 0.15 M KCl/5 mM Na,HPO, and centrifuged at 1000 rpm for 5 min. One-half-milliliter aliquots of the upper phase were mixed with 4 ml of Scintiverse E (Fisher Scientific) for de-
termination of released 32Pi by liquid scintillation analysis. RESULTS AND DISCUSSION Analysis of chemically prepared [“‘PIPA.
The preparation of [32P]PA described under Materials and Methods yields 25-50 PCi of purified material from 1.5 pmol dipalmitin and 0.5 mCi 32Pi.The product, which can be prepared and purified in 2 h, has a specific activity of 1 mCi/pmol, 10,000 times greater than that reported for the original 32POC13 procedure (2). In addition, unlike the POCl3 procedure, the specific activity of the product
Y 0.5
1.0 Time
1.5
2.0
2.5
(h)
FIG. 3. Time course of hydrolysis of D,L-[“PIPA (solid circles) and L-[‘*PIPA (open circles). Reactions were carried out using 100 pg microsomal protein. For details, see Materials and Methods.
122
KELLER.
ADAIR, AND CAFMEYER
can be predetermined by adjusting the specific activity of the starting H332P04. The radiochemical purity of the product is demonstrated by TLC analysis in Fig. 1 and HPLC analysis in Fig. 2. Use of [32P]PA to assayfor PA hydrolysis.
We used rat liver microsomes as a source of hydrolase activity to test the biological activity of the chemically prepared [32P]PA. When assayed as described under Materials and Methods with various concentrations of [32P]PA, release of 32P into the upper phase was found to be saturable and exhibit a K,,, of 28 PM (data not shown). This value is similar to those reported by others for microsomal phosphatidate phosphatase (9,lO). The 32P released was in the form of inorganic phosphate, since assay of release using ammonium molybdate followed by isobutanol treatment, which specifically extracts Pi (1 I), yielded the same results as the chloroform/methanol extraction (data not shown). It is to be noted, however, that others ( 12,13) have shown that Pi release does not necessarily reflect phosphatidate phosphatase activity but can also be due to the combined action of lipase and glycerol-3phosphate phosphohydrolase. The availability of both 1,2-dipalmitoyl-snglycerol and 1,2-dipalmitoyl-rat-glycerol allowed us to prepare racemic (i.e. DL) [32P]PA as well as L-[~~P]PA and therefore test, for the first time, the susceptibility to hydrolysis of the D form. Fig. 3 shows that under the conditions of assay, both L- and DL-[~~P]PA are hydrolyzed to completion and at similar rates, indicating that there is no preference for the naturally occurring L isomer. Since phospholipases are specific for the L isomer (14), the
data suggest that the primary enzyme responsible for the release of 32Pi under our assay conditions is phosphatidate phosphatase. The availability of a facile procedure for the preparation of [32P]PA should aid in future in vitro studies of phosphatidic acid metabolism. ACKNOWLEDGMENT We acknowledge the assistance of Susan D. Brennan in the preparation of labeled phosphatidic acid.
REFERENCES 1. Hajra, A. K., and Agranoff, B. W. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 14, pp. 185-188, Academic Press, New York. 2. Hokin, L. E., Hokin, M. R., and Mathison, D. (1963) Biochim. Biophys. Acta 67,485-491. 3. Hosaka, K., Yamashita, S., and Numa, S. (1975) J. Biochem. 77,50 l-509. 4. Pieringer, R. A., and Kunnes, R. S. (1965) J. Biol. Chem. 240,2833. 5. Cramer, F., Rittersdorf, W., and Bohm, W. (1961) Chem. Ber. 654, 180-188. 6. Engstrom, N., Hellgren, L., and Vincent, J. (1980) J. Chromatogr. 189,284. 7. Keller, R. K., Fuller, M. S., Rottler, G. D., and Connelly. L. W. (1985) Anal. Biochem. 147, 166-173. 8. Snider, M. D., Sultzman, L. A., and Robbins, P. W. (1980)
Cell 21,385-392.
Caras, I., and Shapiro, B. (1975) Biochim. Biophys. Acta 409, 201-211. 10. Hosaka, K., Yamashita, S., and Numa, S. (1975) J. Biochem. 77,50 I-509. II. Berenblum, I., and Chain, E. (1938) Biochem. J. 32, 9.
295-298.
Sturton, R. G., and Brindley, D. N. ( 1978) Biochem. J. 171,263-266. 13. Smith, M. E., Sedgwick, B., Brindley, D. N., and Hubscher, G. (1967) Eur. J. Biochem. 3, 70-77. 14. VanDeenen, L. L. M., and DeHaas, G. H. (1963) Biochim. Biophys. Acta 70, 538. 12.