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Enzymes associated with nuclear ribosomes
392
SHORT COMMUNICATIONS
useful technique for separation of polynucleotides, especially for easily handling various kinds and relatively large amounts of sample. This work was aided in part b y a grant from the Rockefeller Foundation.
Department o[ Biochemistry, Medical School, Kanazawa University, Kanazawa, Ishikawa (Japan)
K E N I C H I MATSUBARA Y A S U Y U K I TAKAGI
i 1~. L. SINSHEIMER, J. Mol. Biol., i (1959) 432 j . !V[ARMUR AND D. LANE, Proc. Natl. Acad. Sci. U.S., 46 (196o) 453. z 1). DOTY, J. MARMUR, J. EIGNER AND C. SCHILDKRAUT, Proc. Natl. Acad. Sci. U.S., 46 (196o) 461. 4 F. J. BOLLUM, J. Biol. Chem., 235 (1959) 2399. 5 A. KORNBERG, Science, 131 (196o) 15o 3. 8 ]3. D. HALL AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 47 (I961) 137. S. S. COHEN, J. Biol. Chem., 146 (1942) 471. 8 E. CHARGAFF AND S. ZAMENHOF, J. Biol. Chem., 173 (1948) 327 . 9 E. CHARGAFF AND H. F. SAIDEL, J. Biol. Chem., 177 (1949) 417 . 10 N. HI AND I. WATANABE, Bull. Chem. Soc. Japan, 24 (1951) 21o. 11 A. V~TACKER, D. HARTMANN AND H. STRUCK, Z. Natur]orsch., i 2 b (1957) 181. 12 M. SEKIGUCHI, A. TAKETO AND Y. TAKAGI, Biochim. Biophys. Acta, 45 (196°) 199. 13 G. D. GUTHRIE AND R. L. SINSHEIMER, J. Mol. Biol., 2 (196o) 297-
Received August 28th, 1961 Biochim. Biophys. Acta, 55 (1962) 3 8 9 - 3 9 2
Enzymes ossocioted with nucleor ribosomes It has now been established that R N P particles are intimately involved in the biosynthesis of proteins. Apart from the demonstrations suggesting that serum albumin 1 and hemoglobin ~ are synthesized in the microsomes, several enzymes have also been reported to be associated with the ribosomes. Thus, chymotrypsinogen 3, amylase 4, and RNAase 5 activities were found in guinea-pig pancreas ribosomes, and RNAase 6 and fl-galactosidase e, 7 were shown to be present in Escherichia coli ribosomes. These and other findings suggest that ribosomal particles are the sites where active protein synthesis takes place. Evidence has recently been accumulated indicating that in the cell nucleus, protein synthesis seems to be following about the same pattern as in the cytoplasm s-12. The nuclear R N P requires a supply of energy and a soluble enzyme fraction for its incorporation of radioactive amino acids. The characteristics of the purified nuclear ribosomes of calf thymus are also to a large extent similar to those of the cytoplasmic R N P particles12,13. Additional evidence, revealing that biologically active proteins are closely associated with nuclear ribosomes, has now been obtained by the finding of a number of enzymes in the nuclear particles, and is reported in the present communication. Calf-thymus nuclei were isolated according to the procedure of ALLFREY et al. 14, and the crude R N P was obtained by ultracentrifugation of the nuclear saline extract is. Abbreviation: RNP, ribonucleoprotein.
Biochim. Biophys. Acta, 55 (I962) 392-395
393
SHORT COMMUNICATIONS
The yield was usually about 25 %, based on the total protein content of the saline extract. The nuclear ribosomes were prepared as previously described 1~. To recycle the nuclear ribosomes, the particles were suspended in o.I m M MgCI~, ultracentrifuged, and collected as a pellet. This procedure was repeated twice, and the recycled pellets were dispersed in distilled water for enzyme assay. Some of the nuclear ribosomes were lost in the ultracentrifugal supernatant after the recycling steps. Sedimentation patterns of the supernatant showed mostly I6-S particles and a very small amount of 33-S material. The bulk of the sedimented nuclear ribosomes contained 63-S and 46-S, and some 33-S particles. Malate dehydrogenasO 6 was determined by measuring the reduction of DPN, using sodium malate as the substrate. Lactate, glutamate is and fl-hydroxybutyrylCoA 17 dehydrogenases were determined b y following the oxidation of D P N H , using pyruvate, ~-ketoglutarate and acetoacetyl-CoA, respectively, as the substrates. Glucose-6-phosphatO a and isocitratO 9 dehydrogenases were determined b y the reduction of TPN, using glucose 6-phosphate and isocitrate, respectively, as the substrates. Mg2+-activated ATPase s° was assayed b y following the liberation of inorganic phosphate in the presence of ATP-generating system; and fl-glucuronidase 21 was determined b y measuring the splitting of phenolphthalein from phenolphtalein glucuronide. RNAase activity was tested according to ELSON6. BOLTON et al. 7 and ELSONs have reported the latent RNAase activity of E . coli ribosomes. The RNAase is latent in that its activity can be demonstrated only after the R N P is dissociated b y urea. It was earlier observed z2 that the crude nuclear R N P showed the same self-degradation when subjected to ELSON'S urea treatment on the nuclear preparation. With the purified nuclear ribosomal particles, the latent RNAase activity can again be shown in the similar manner. Fig. I shows such RNAase activity
0.12 -t
E
o 0.10 <1 E
0.08
.9 0
& 0.06 ,11
3 0.04 i
"O < 0.02
t
2
/
i
4 6 Tim e (h)
8
Fig. I. Release of nuclear r i b o s o m a l R N A a s e b y urea a t 37 °. The i n c u b a t i o n m i x t u r e contained 526/~g n u c l e a r r i b o s o m e s a n d 178 # g nuclear r i b o s o m a l R N A in 4 M u r e a - o . 2 M NaCl-o.o 5 M Tris--HC1 (pH 8.o). The curve w a s o b t a i n e d b y s u b s t r a c t i n g the acid-soluble values of b o t h the n u c l e a r rlabosomes ( w i t h o u t urea) an(t the nuclear r i b o s o m a l R N A controls.
Biochim. Biophys. Acta, 55 (1962) 392-395
394
SHORT COMMUNICATIONS
of a purified nuclear ribosomal preparation, in the presence of added nuclear ribosomal RNA. The release of RNAase activity b y urea, it m a y be noted, was not very rapid at 37 °. It took several hours for the latent RNAase activity to reach its maximum. Whether this indicates a somewhat different configuration of the nuclear ribosomal particles is not known at present. Table I shows the other enzymic activities present in the nuclear RNP's. Of the enzymes assayed, all can be found in the crude nuclear RNP. ATPase obviously TABLE I ENZYMIC ACTIVITIES OF ~OCLEAR R N P ' s (m/~moles/mg protein/h) Enzyme
Lactate dehydrogenase Glutamate dehydrogenase Malate dehydrogenase Glucose-6-phosphate d e h y d r o g e n a s e Isocitrate dehydrogenase f l - H y d r o x y b u t y r y l - C o A dehydrogenase ATPase fl-Glucuronidase ( × lO 3)
Crude R N P
773 ° 276 4342 88 771 248 720 27.8
Nuclear ribosomes
212 18 12o 6. 3 1.6 27 o 6.9
Recycyled nuclear ribosomes
43 25 14 o o
o
5.2
is not associated with the free nuclear ribosomes, since no activity could be detected in the purified particles. This also shows the advantage of using nuclear ribosomes for incorporation studies. The purification process to obtain freed nuclear ribosomal particles causes some reduction of enzymic activities ranging from complete or extensive removal, as in the case of ATPase and isocitrate dehydrogenase, to about 25 O//oremoval for/3-glucuronidase. Most of the enzymic activities must then be retained in the deoxycholate-soluble fraction, which, while still having a significant amount of RNP, contained also other proteins. Part of these activities probably also could be carried along from the nuclear soluble fraction. Recycling the nuclear ribosomes resulted in the disappearance of glucose-6phosphate and isocitrate dehydrogenase activfties. It also significantly reduced the lactate and malate dehydrogenase activities. About 75 ~ of/5-glucuronidase activity, however, still remained after re-ultracentrifugation. Glutamate dehydrogenase activity, on the other hand, was concentrated to about 1.4-fold. Since the recycling procedure partially dissociated the particles and caused the loss of 1/4 of the nuclear ribosomes as particles of low sedimentation coefficients, this change in nuclearribosomal enzymic activity m a y suggest that enzymes were released into the supernatant, or that different enzymes were linked with particles of different sizes. The enzymic activities of the nuclear R N P ' s varied somewhat from one preparation to another, perhaps because of physiological differences in the animals. Nevertheless, these variations are not considered excessive, and the activities of the various enzymes assayed remained in nearly constant proportions with each other. The fact that purified nuclear ribosomes, even after recycling, still retained significant enzymic activities, shows the intimate association between those enzymes so far assayed and the nuclear ribosomal particles. All the enzymic activities found in the nuclear ribosomes of calf thymus, exBiochim. Biophys. Acta, 55 (1962) 392 395
SHORT COMMUNICATIONS
395
cept glucose-6-phosphate and fl-hydroxybutyryl-CoA dehydrogenases, have been reported to be present in the nuclear fraction of various tissues ~3. The demonstration that the nuclear ribosomes have glucose-6-phosphate and fl-hydroxybutyryl-CoA dehydrogenase activities shows that the cell nucleus of calf thymus contains these two enzymes. The presence of glutamate dehydrogenase activity in the nuclear ribosomes needs some clarification. It has been thought that the nuclear glutamate dehydrogenase activity is due to mitochondrial contamination. We have shown here that the activity was concentrated after recycling. This would suggest that it is indeed present in the calf-thymus nuclei. Failure to detect glutamate dehydrogenase activity in isolated nuclei probably is due to its low activity, particularly when assayed in the presence of large amounts of other proteins such as histones. This investigation was supported by a grant from the U.S. Public Health Service (RG-7923).
Henry Shaw School o/Botany and Adophus Busch I l l Laboratory o/ Molecular Biology, Washington University, Department o/Medicine, St. Louis University School o/ Medicine, St. Louis, Mo. (U.S.A.)
TUNG-YUE WANG KUANG MEI WANG
p. N. CAMPBELL, O. GREENGARD AND B. A. I{ERNOT, Biochem. J., 74 (196o) lO 7. R. SCHWEET, H. LAMFROM AND E. ALLEN, Proc. Natl. Aead. Sci. U.S., 44 (1958) lO29. p. SIEKEVITZ AND Cr. E. PALLADE, f . Biophys. Biochem. Cytol., 4 (1958) 557. A. K. LAIRED AND A. D. BARTON, Biochim. Biophys. Acta, 25 (1957) 56. A. J. MORRIS AND S. R. DICKMAN, f . Biol. Chem., 235 (196o) 14o 4. D. ELSON, Biochim. Biophys. Aeta, 36 (1959) 372. E. T. BOLTON, R. J. BRITTEN, D. B. COWIE B. J. MCCARTHY, K. McQUILLEN AND R. B. ROBERTS, A n n u a l Report o/ the Biophysics Section. Yearb. Carneg. Inst., 58 (1959) 259. s j . H. FRENSTER, V. G-. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 46 (196o) 259. J. H. FRENSTER, V. O. ALLFREY AND A. E. MIRSKY, Biochim. Biophys. Acta, 47 (1961) 13o 10 j. W. HOPKINS, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1461. tx T. Y. ~,VANG, Biochim. Biophys. Acta, 49 (1961) lO8. 12 T. Y. WANG, Biochim. Biophys. Acta, 51 (1961) 18o. 1~ T. Y. WANG, Biochim. Biophys. Acta, 53 (1961) 158. 14 V. G. ALLFREY, A. E. MIRSKY AND S. OSAWA, J. Gen. Physiol., 4 ° (1957) 451. is T. Y. "WANG, Biochim. Biophys. Acta, 49 (1961) 239. 13 O. H. LOWRY, in S. P. COLOWlCK AND N. O. KAPLAN., Methods in Enzymology, Vol. 4, Academic Press Inc., New York, 1957, p. 366. 1~ F. LYNEN AND S. OCHOA, Biochim. Biophys. Acta, 12 (1953) 299. 13 M. V. BUELL, O. H. LOWRY, N. R. ROBERTS, M. L. "~V. CHANG AND N. KAPLAN, f . Biol. Chem., 232 (1958 ) 979. l t t ~-~. t~. ROBERTS, 1~. R. CODHO, O. H. LOWRY AND E. J. CRAWFORD, J. Neurochem., 3 (1958)1o9. 20 M. E. PULLMAN, H. S. PENEFSKY, A. DATTA AND E. RACHER, f . Biol. Chem., 235 (196o) 3322. 21 p. TALALAY, \V. H. FISHMAN AND C. HUGGINS, J. Biol. Chem., 166 (1946) 757. 23 T. Y. \VANG, Biochim. Biophys. Acta, 45 (1961) 8. 23 D. B. ROODYN, in G. H. BOURNE AND J. F. DANIELLI, International Reviews on Cytology, Vol. 8, Academic Press Inc., N e w York, 196o, p. 279. 1 2 3 4 5 6
Received October i8th, 1961 Biochim. Biophys. Acta, 55 (1962) 392-395
SHORT COMMUNICATIONS
useful technique for separation of polynucleotides, especially for easily handling various kinds and relatively large amounts of sample. This work was aided in part b y a grant from the Rockefeller Foundation.
Department o[ Biochemistry, Medical School, Kanazawa University, Kanazawa, Ishikawa (Japan)
K E N I C H I MATSUBARA Y A S U Y U K I TAKAGI
i 1~. L. SINSHEIMER, J. Mol. Biol., i (1959) 432 j . !V[ARMUR AND D. LANE, Proc. Natl. Acad. Sci. U.S., 46 (196o) 453. z 1). DOTY, J. MARMUR, J. EIGNER AND C. SCHILDKRAUT, Proc. Natl. Acad. Sci. U.S., 46 (196o) 461. 4 F. J. BOLLUM, J. Biol. Chem., 235 (1959) 2399. 5 A. KORNBERG, Science, 131 (196o) 15o 3. 8 ]3. D. HALL AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 47 (I961) 137. S. S. COHEN, J. Biol. Chem., 146 (1942) 471. 8 E. CHARGAFF AND S. ZAMENHOF, J. Biol. Chem., 173 (1948) 327 . 9 E. CHARGAFF AND H. F. SAIDEL, J. Biol. Chem., 177 (1949) 417 . 10 N. HI AND I. WATANABE, Bull. Chem. Soc. Japan, 24 (1951) 21o. 11 A. V~TACKER, D. HARTMANN AND H. STRUCK, Z. Natur]orsch., i 2 b (1957) 181. 12 M. SEKIGUCHI, A. TAKETO AND Y. TAKAGI, Biochim. Biophys. Acta, 45 (196°) 199. 13 G. D. GUTHRIE AND R. L. SINSHEIMER, J. Mol. Biol., 2 (196o) 297-
Received August 28th, 1961 Biochim. Biophys. Acta, 55 (1962) 3 8 9 - 3 9 2
Enzymes ossocioted with nucleor ribosomes It has now been established that R N P particles are intimately involved in the biosynthesis of proteins. Apart from the demonstrations suggesting that serum albumin 1 and hemoglobin ~ are synthesized in the microsomes, several enzymes have also been reported to be associated with the ribosomes. Thus, chymotrypsinogen 3, amylase 4, and RNAase 5 activities were found in guinea-pig pancreas ribosomes, and RNAase 6 and fl-galactosidase e, 7 were shown to be present in Escherichia coli ribosomes. These and other findings suggest that ribosomal particles are the sites where active protein synthesis takes place. Evidence has recently been accumulated indicating that in the cell nucleus, protein synthesis seems to be following about the same pattern as in the cytoplasm s-12. The nuclear R N P requires a supply of energy and a soluble enzyme fraction for its incorporation of radioactive amino acids. The characteristics of the purified nuclear ribosomes of calf thymus are also to a large extent similar to those of the cytoplasmic R N P particles12,13. Additional evidence, revealing that biologically active proteins are closely associated with nuclear ribosomes, has now been obtained by the finding of a number of enzymes in the nuclear particles, and is reported in the present communication. Calf-thymus nuclei were isolated according to the procedure of ALLFREY et al. 14, and the crude R N P was obtained by ultracentrifugation of the nuclear saline extract is. Abbreviation: RNP, ribonucleoprotein.
Biochim. Biophys. Acta, 55 (I962) 392-395
393
SHORT COMMUNICATIONS
The yield was usually about 25 %, based on the total protein content of the saline extract. The nuclear ribosomes were prepared as previously described 1~. To recycle the nuclear ribosomes, the particles were suspended in o.I m M MgCI~, ultracentrifuged, and collected as a pellet. This procedure was repeated twice, and the recycled pellets were dispersed in distilled water for enzyme assay. Some of the nuclear ribosomes were lost in the ultracentrifugal supernatant after the recycling steps. Sedimentation patterns of the supernatant showed mostly I6-S particles and a very small amount of 33-S material. The bulk of the sedimented nuclear ribosomes contained 63-S and 46-S, and some 33-S particles. Malate dehydrogenasO 6 was determined by measuring the reduction of DPN, using sodium malate as the substrate. Lactate, glutamate is and fl-hydroxybutyrylCoA 17 dehydrogenases were determined b y following the oxidation of D P N H , using pyruvate, ~-ketoglutarate and acetoacetyl-CoA, respectively, as the substrates. Glucose-6-phosphatO a and isocitratO 9 dehydrogenases were determined b y the reduction of TPN, using glucose 6-phosphate and isocitrate, respectively, as the substrates. Mg2+-activated ATPase s° was assayed b y following the liberation of inorganic phosphate in the presence of ATP-generating system; and fl-glucuronidase 21 was determined b y measuring the splitting of phenolphthalein from phenolphtalein glucuronide. RNAase activity was tested according to ELSON6. BOLTON et al. 7 and ELSONs have reported the latent RNAase activity of E . coli ribosomes. The RNAase is latent in that its activity can be demonstrated only after the R N P is dissociated b y urea. It was earlier observed z2 that the crude nuclear R N P showed the same self-degradation when subjected to ELSON'S urea treatment on the nuclear preparation. With the purified nuclear ribosomal particles, the latent RNAase activity can again be shown in the similar manner. Fig. I shows such RNAase activity
0.12 -t
E
o 0.10 <1 E
0.08
.9 0
& 0.06 ,11
3 0.04 i
"O < 0.02
t
2
/
i
4 6 Tim e (h)
8
Fig. I. Release of nuclear r i b o s o m a l R N A a s e b y urea a t 37 °. The i n c u b a t i o n m i x t u r e contained 526/~g n u c l e a r r i b o s o m e s a n d 178 # g nuclear r i b o s o m a l R N A in 4 M u r e a - o . 2 M NaCl-o.o 5 M Tris--HC1 (pH 8.o). The curve w a s o b t a i n e d b y s u b s t r a c t i n g the acid-soluble values of b o t h the n u c l e a r rlabosomes ( w i t h o u t urea) an(t the nuclear r i b o s o m a l R N A controls.
Biochim. Biophys. Acta, 55 (1962) 392-395
394
SHORT COMMUNICATIONS
of a purified nuclear ribosomal preparation, in the presence of added nuclear ribosomal RNA. The release of RNAase activity b y urea, it m a y be noted, was not very rapid at 37 °. It took several hours for the latent RNAase activity to reach its maximum. Whether this indicates a somewhat different configuration of the nuclear ribosomal particles is not known at present. Table I shows the other enzymic activities present in the nuclear RNP's. Of the enzymes assayed, all can be found in the crude nuclear RNP. ATPase obviously TABLE I ENZYMIC ACTIVITIES OF ~OCLEAR R N P ' s (m/~moles/mg protein/h) Enzyme
Lactate dehydrogenase Glutamate dehydrogenase Malate dehydrogenase Glucose-6-phosphate d e h y d r o g e n a s e Isocitrate dehydrogenase f l - H y d r o x y b u t y r y l - C o A dehydrogenase ATPase fl-Glucuronidase ( × lO 3)
Crude R N P
773 ° 276 4342 88 771 248 720 27.8
Nuclear ribosomes
212 18 12o 6. 3 1.6 27 o 6.9
Recycyled nuclear ribosomes
43 25 14 o o
o
5.2
is not associated with the free nuclear ribosomes, since no activity could be detected in the purified particles. This also shows the advantage of using nuclear ribosomes for incorporation studies. The purification process to obtain freed nuclear ribosomal particles causes some reduction of enzymic activities ranging from complete or extensive removal, as in the case of ATPase and isocitrate dehydrogenase, to about 25 O//oremoval for/3-glucuronidase. Most of the enzymic activities must then be retained in the deoxycholate-soluble fraction, which, while still having a significant amount of RNP, contained also other proteins. Part of these activities probably also could be carried along from the nuclear soluble fraction. Recycling the nuclear ribosomes resulted in the disappearance of glucose-6phosphate and isocitrate dehydrogenase activfties. It also significantly reduced the lactate and malate dehydrogenase activities. About 75 ~ of/5-glucuronidase activity, however, still remained after re-ultracentrifugation. Glutamate dehydrogenase activity, on the other hand, was concentrated to about 1.4-fold. Since the recycling procedure partially dissociated the particles and caused the loss of 1/4 of the nuclear ribosomes as particles of low sedimentation coefficients, this change in nuclearribosomal enzymic activity m a y suggest that enzymes were released into the supernatant, or that different enzymes were linked with particles of different sizes. The enzymic activities of the nuclear R N P ' s varied somewhat from one preparation to another, perhaps because of physiological differences in the animals. Nevertheless, these variations are not considered excessive, and the activities of the various enzymes assayed remained in nearly constant proportions with each other. The fact that purified nuclear ribosomes, even after recycling, still retained significant enzymic activities, shows the intimate association between those enzymes so far assayed and the nuclear ribosomal particles. All the enzymic activities found in the nuclear ribosomes of calf thymus, exBiochim. Biophys. Acta, 55 (1962) 392 395
SHORT COMMUNICATIONS
395
cept glucose-6-phosphate and fl-hydroxybutyryl-CoA dehydrogenases, have been reported to be present in the nuclear fraction of various tissues ~3. The demonstration that the nuclear ribosomes have glucose-6-phosphate and fl-hydroxybutyryl-CoA dehydrogenase activities shows that the cell nucleus of calf thymus contains these two enzymes. The presence of glutamate dehydrogenase activity in the nuclear ribosomes needs some clarification. It has been thought that the nuclear glutamate dehydrogenase activity is due to mitochondrial contamination. We have shown here that the activity was concentrated after recycling. This would suggest that it is indeed present in the calf-thymus nuclei. Failure to detect glutamate dehydrogenase activity in isolated nuclei probably is due to its low activity, particularly when assayed in the presence of large amounts of other proteins such as histones. This investigation was supported by a grant from the U.S. Public Health Service (RG-7923).
Henry Shaw School o/Botany and Adophus Busch I l l Laboratory o/ Molecular Biology, Washington University, Department o/Medicine, St. Louis University School o/ Medicine, St. Louis, Mo. (U.S.A.)
TUNG-YUE WANG KUANG MEI WANG
p. N. CAMPBELL, O. GREENGARD AND B. A. I{ERNOT, Biochem. J., 74 (196o) lO 7. R. SCHWEET, H. LAMFROM AND E. ALLEN, Proc. Natl. Aead. Sci. U.S., 44 (1958) lO29. p. SIEKEVITZ AND Cr. E. PALLADE, f . Biophys. Biochem. Cytol., 4 (1958) 557. A. K. LAIRED AND A. D. BARTON, Biochim. Biophys. Acta, 25 (1957) 56. A. J. MORRIS AND S. R. DICKMAN, f . Biol. Chem., 235 (196o) 14o 4. D. ELSON, Biochim. Biophys. Aeta, 36 (1959) 372. E. T. BOLTON, R. J. BRITTEN, D. B. COWIE B. J. MCCARTHY, K. McQUILLEN AND R. B. ROBERTS, A n n u a l Report o/ the Biophysics Section. Yearb. Carneg. Inst., 58 (1959) 259. s j . H. FRENSTER, V. G-. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Sci. U.S., 46 (196o) 259. J. H. FRENSTER, V. O. ALLFREY AND A. E. MIRSKY, Biochim. Biophys. Acta, 47 (1961) 13o 10 j. W. HOPKINS, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1461. tx T. Y. ~,VANG, Biochim. Biophys. Acta, 49 (1961) lO8. 12 T. Y. WANG, Biochim. Biophys. Acta, 51 (1961) 18o. 1~ T. Y. WANG, Biochim. Biophys. Acta, 53 (1961) 158. 14 V. G. ALLFREY, A. E. MIRSKY AND S. OSAWA, J. Gen. Physiol., 4 ° (1957) 451. is T. Y. "WANG, Biochim. Biophys. Acta, 49 (1961) 239. 13 O. H. LOWRY, in S. P. COLOWlCK AND N. O. KAPLAN., Methods in Enzymology, Vol. 4, Academic Press Inc., New York, 1957, p. 366. 1~ F. LYNEN AND S. OCHOA, Biochim. Biophys. Acta, 12 (1953) 299. 13 M. V. BUELL, O. H. LOWRY, N. R. ROBERTS, M. L. "~V. CHANG AND N. KAPLAN, f . Biol. Chem., 232 (1958 ) 979. l t t ~-~. t~. ROBERTS, 1~. R. CODHO, O. H. LOWRY AND E. J. CRAWFORD, J. Neurochem., 3 (1958)1o9. 20 M. E. PULLMAN, H. S. PENEFSKY, A. DATTA AND E. RACHER, f . Biol. Chem., 235 (196o) 3322. 21 p. TALALAY, \V. H. FISHMAN AND C. HUGGINS, J. Biol. Chem., 166 (1946) 757. 23 T. Y. \VANG, Biochim. Biophys. Acta, 45 (1961) 8. 23 D. B. ROODYN, in G. H. BOURNE AND J. F. DANIELLI, International Reviews on Cytology, Vol. 8, Academic Press Inc., N e w York, 196o, p. 279. 1 2 3 4 5 6
Received October i8th, 1961 Biochim. Biophys. Acta, 55 (1962) 392-395