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Some chemical and physical properties of microsomes
Experimental
459
Cell Research, 8, 459-465 (1955)
SOME CHEMICAL AND PHYSICAL OF MICROSOMES’ L. G. AROOD and LORRAIIiE Deportment
PROPERTIES
ROMANCHEK
of Psychiatry and Depnrtment ofBiochemistry, Unitzrsity College of Medicine, Chicago, Ill., U.S.A.
of Illinois,
Received August 12, 1054
TIIP: ground suhstanre of most cells is beIievcd to consist of an elaborate framework of fihrils comprised of submicroscopic particles arranged in headed chains (13, 4). This reticulate structure is estremely plastic, usually assuming the: orientation and configuration intrinsic in the pattern and physical forcrs within the cell (13, 4). In addition to providing a structural framework for the: cell, the c’ytoplasmic fihrils may comprisr units of functional activity such as motility, contractility, and conduction. 1.lusrle contraction is due to the folding of the l)olypcptide chains of m?ofibrils (19), \\hilc the ncurofihrils have long been suspected of hcing involved in nerve conduction (11). hlonn6 is of the opinion that rncrgy-yielding processes kvithin the sublnicroscopic particles arc essential for maintaining the dynamic state of thv fihrils. l’hc prcsencc of aclenosincttriphosl)hatase (A’l’Pasc) in the microsomcs of li\or (15), nerve (l), and niusclc (8) has been cstahlished; and hIonn6 c.oncluded that .4’1’I’asc I\as present in the cytoplasmic fibrils in general since they arc contractile and eahihit hirefringencc of flow (12). ‘I’hc present communication deals \vith an attempt to compare and charactcrizc thr isolated microsomcs from dilfcrtbnt tissues and to rcconstitutc them into fihriliar structures rcsemhling the c\-toplasmic fibrils in uivo. \\‘hen microsomcs from rat liver, kidney, musrlc, brain, and nerve wcrc prcparcd in 0.25 hl suvrosr by diflc~rcntial ccntrifugation, they \vere found to he sphcrcs 20 1.50 111~ in diamctcr (1) and to cshihit hirefringence of Ilo\\-. ‘I’hc scdimrnted microsomcs formed a sol \vhich hehavrd like a thisotropic gel \yhen rxtruded into lvatcr, in mut+h the same manner as attoniy0sin. METHODS Isolation of the microsome fraction of rat tissues was done after the method of Hogcboom cl al. (7). I Iomogcnatcs of the various tissues in 0.25 hl sucrnse were first sedimcntetl al 15,000 x g to rernovc mitochontlria, nuclei, ant1 stroma an11 then at 100,000 i( g to isolate the microsorncs. Centrifugation was (lone in the Spinco Xtoclel I ‘I‘his work ~&IS supported University of Illinois.
by a contract
twtwec~n the Office
of Saval
I~rsrwch
Experimertltrl
and the
Cell Research 8
460
L. G. Abood and L. Romanchek
L ultracentrifuge at -5 degrees. The hydrophilic microsomal sol was drawn into a 0.5 ml hypodermic syringe whose tip was drawn out to a 0.1 mm bore, and when the sol was carefully extruded into 1O-8 M magnesium chloride (pH 6.0-7.0), fine gelatinous threads resembling actomyosin were formed. The histochemical technique for ATPase was that of Gomori (6). Rat sciatic nerves were placed in Maximow depression slides and then dissected into single fibers or bundles of usually less then 5 fibers. A medium containing 0.02 M ATP, 0.001 M magnesium chloride, 0.02 M calcium chloride, and histidine buffer, pH 7.5 was then added to the slide and the system incubated for 20 minutes at 37 degrees. Preliminary fixation of the nerves in ice-cold acetone or 70 per cent alcohol did not in any way improve the histochemical technique. ATPase activity was determined on the microsome fraction according to the method of DuBois and Potter (3), except that histidine buffer replaced Verona1 and 0.001 M magnesium chloride was substituted for calcium chloride. Adenylic kinase was determined by the method of Kornberg (9), and adenylic deaminase was measured by following the disappearance of adenylic acid at 260 rnp in the Beckman DU Spectrophotometer. Separation of the “acid-insoluble” fractions was done following the method of Schneider (18). Sodium and potassium were removed from the microsomes by boiling in water for 10 minutes and then determined on the filtrate, using the Beckman flame photometer. Silica glassware, which was low in sodium and potassium, was used whenever analysis for the alkali metals was made. RESULTS
When
AND
DISCUSSION
particles exhibiting double refraction of flow are forced through a tube they align themselves parallel to the direction of flow (5). With
narrow
TABLE Acid
insoluble
components
I
and electrolytes
Wmg % dry dry wgt. I wgt. I
in microsomes
Micromoles/micromoles
of various
of total
tissues.
Nitrogen
. . . .
5.7
57
60.0
1.08
16.0
47
14
3.4
Peripheral nerve . . .
5.5
59
50.1
1.00
3.5
40
12
3.4
Muscle
. _ .
6.5
61
40.1
1.20
4.0
44
11
4.0
. . . .
7.0
60
33.3
1.25
4.7
28
9
3.1
. . .
7.5
63
41.5
1.30
5.1
21
7
3.0
Brain
Liver Kidney
Average Experimental
of 4 values
agreeing
Cell Research 8
within
6 per cent.
461
Properties of microsomes
Fig. 1. Unfixed, unstained fiber prepared artificially from nerve fiber microsomes. Magnification 200 x .
the correct salt concentration they would tend to coalesce and form a stable gel if properly oriented. The microsomes of all tissues studied possessed this property of flow birefringence, and as long as the microsomal salt concentration was at least 3 m M/ml, a stable gel formed. The microsomes aligned themselves into numerous parallel fibrils longitudinal to the fiber axis (Figs. 1 and 2). In a thread 0.5 mm in diameter as many as 50 of these tllaments could be observed at magnifications of 200 x . In the absence of magnesium, in 0.25 M sucrose, or in a slightly alkaline solution the threads lacked the lllar structure and would more readily disintegrate. Unlike actomyosin, the threads would not contract upon the addition of ATP. About 60 per cent of the total dry weight of the microsomes from all tissues was comprised of lipid. The phospholipid content of microsomes from nervous tissues was somewhat greater than in the case of the other tissues where the values were quite similar. In general, the nucleic acid Experimenlal
Cell Research 8
462
L. G. Abood and L. Romanchek
Fig. 2. Fibers prepared by extrusion of nerve fiber microsomes into 5 per cent formalin and stained with 0.01 per cent methylene blue. Magnification 200 x .
content of the various tissues was also similar, although slightly lovver in neural tissue. \\‘ith the exception of an extremely high content in brain, the “phosphoprotein” content of the microsomes of various tissues was in close agreement. The potassium and sodium concentration of the microsomes of neural and muscular tissue were similar, being considerably greater than in liver and kidney. A considerable amount of ATPase vvas found to be present in the microsomcs of all tissues (Table II). The activity of brain and kidney was less than half that of muscle and over twice as great as the ATPase activity of liver and peripheral nerve. Adenylic kinase and adenylic deaminase activity of the microsomes seemed to vary from tissue to tissue in the same proportion as ATPase with the exception of kidney. Cytoplasmic fibrils have long been suspected of being comprised of submicroscopic particles, although considerable controversy exists as to the Experimental
Cell Research
8
Properties of microsomes
463
exact nature of the particles involved. MonnE (13) and Runnstrijm (16) are of the opinion that they are equivalent to the microsomes, while FreyWyssling (4) considers it unlikely that morphogenesis and metabolism are performed by the same elements. The cytoplasmic tlbrils, however, are not mere structural elements but appear to be in a state of continual reorganization and reconstitution as determined by the variable morphogenetic and TABLE Enzymes
of adenylate
system
II
in microsomes -
ATP-ase
of various
Adenylate
tissues.
T
kinase
-
Tissue
-
Adenylic deaminase
I-
PM ATP
split/l5’/pM
N
-
I*M ATP formed/lO’/pM
b
r- IFM AA/lO’/pM
Muscle . . . . . .
1.07
0.30
0.0350
Brain
. . . . . .
0.43
0.10
0.0090
. . . . .
0.45
0.053
0.0046
. . . . . .
0.21
0.035
Kidney Liver
Peripheral Average
nerve
.
0.22
-
of 4 values
0.028 -
agreeing
within
N
0.0020
! I
0.0015
8 per cent.
functional requirements of the cell. Among the numerous types of tibrils, known to respond during functional activity are the myofibrils, neurotibrils, epithelial tibrils, and cilia. This activity usually consists of a visible contraction or motility involving rearrangements in polypeptide chains. Polymerization of actomyosin occurs upon the addition of ATP (1 l), and dissociation of the protein complex follows the dephosphorylation of ATP. It is believed that the polypeptide rearrangements associated with most cytoplasmic tibrillar activity involve an energy-releasing mechanism comparable to the ATPase system of myotibrils. The present findings that the ATPase of nerve fibers appears to be located in tibrillar structures, possibly including the neurofibrils, lend support to the hypothesis that ATI’ase activity is associated with nerve conduction (13, 1). On the basis of present evidence it cannot be determined to what extent the microsomal fraction of neural tissue originated from the neurofibrils. Readlike hbrils observed by DeRobertis and Schmitt (2) in the electron Experimentnl
Cell Research 8
L. G. Abood and L. Romanchek
Fig. 3. Single nerve fiber stained for ATPase. Magnification 1000 x .
microscope were described as axonal constituents, but later believed to be from conneclive tissue sheath (17). In nerve fibers fixed in osmic acid it has been possible to observe within the axoplasm contorted beadlike filaments 100-200 A wide, which are believed to be neurohbrils (17). The finding of Libet (10) that the ATPase of the squid nerve is largely in the sheath would tend to vitiate the hypothesis that the ATPase containing microsomes are of axoplasmic origin. Undoubtedly the microsomes isolated from rat nerve originated from the sheath as well as axoplasm, although their enzymatic and chemical homogeneity remains a question. The presence of high K/Na ratios within the microsomes, particularly those of neural or muscular origin suggested that the artificial tlbers might manifest a surface potential. With the ultramicroelectrode potentials were measured exceeding 30 millivolts in freshly prepared threads immersed in 0.25 M sucrose containing 0.001 M magnesium chloride. Potentials of threads Experimental
Cell Research
8
Properties of microsomes
465
prepared from liver microsomes measured less than 10 millivolts. This difference in potential is to be expected because of the greater electrolyte content of conductive tissue. SUMMARY
Microsomes from a number of rat tissues have been examined wifh respect to certain chemical and physical properties. In addition to a Mg-activated ATP-ase, the microsomes contain adenylic kinase and adenylic deaminase. By extrusion of the microsomal gel into 1O-3 magnesium chloride, artificial fibers are obtained with internal structures resembling cytoplasmic fibrils. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19.
ABOOD, L. G., and GERARD, R. W., J. Cellular Comp. Physiol., 43, 379 (1954). DE ROBERTIS, E., and SCHMITT, F. O., ibid., 31, 1 (1948). DUBOIS, K. P., and POTTER, V. R., J. Biol. Chem., 150, 185 (1943). FREY-WYSSLING, A., Submicroscopic Morphology. Elsevier Publ. Co., New York, 1953. GLASSTONE, S., Textbook of Physical Chemistry. 2nd ed. Van Nostrand, New York, 1946. GOMORI. G., Proc. Sot. Exptl. Biol. Med., 42, 23 (1939). HOGEBOOM, G. H., SCHNEIDER, W. C., and PALADE, G. E., J. Biot. Chem., 172, 619 (1948). KIELLEY, W. W., and MEYERHOF, O., ibid., 183, 391 (1950). KORNBERG, A., ibid., 182, 779 (1950). LIBET, B., Federation Proc., 7, 72 (1948). MDMMAERTS, W. F. H. M., Muscular Contraction. Interscience Publ., New York, 1950. MONNB, L., Experienlia, 2, 153 (1946). __ Aduances in Enzymol., 8, 1 (1948). -Arch. exptl. Zellforsch. GeLvebeziicht, 23, 157 (1949). POTTER, V., Adoances in Enzymol., 4, 201 (1944). RUNNSTRGM, J., ibid., 9, 241 (1949). SCHMITT, F. O., in Genetic Neurology. Ed. P. Weiss, Ii. of Chicago, 1950. SCHNEIDER, W. C., J. Biol. Chem., 161, 293 (1945). SZENT-GYGRGYI, A., Chemistry of Muscular Contraction. Acad. Press, New York, 1947.
Experimental
Cell Research 8
459
Cell Research, 8, 459-465 (1955)
SOME CHEMICAL AND PHYSICAL OF MICROSOMES’ L. G. AROOD and LORRAIIiE Deportment
PROPERTIES
ROMANCHEK
of Psychiatry and Depnrtment ofBiochemistry, Unitzrsity College of Medicine, Chicago, Ill., U.S.A.
of Illinois,
Received August 12, 1054
TIIP: ground suhstanre of most cells is beIievcd to consist of an elaborate framework of fihrils comprised of submicroscopic particles arranged in headed chains (13, 4). This reticulate structure is estremely plastic, usually assuming the: orientation and configuration intrinsic in the pattern and physical forcrs within the cell (13, 4). In addition to providing a structural framework for the: cell, the c’ytoplasmic fihrils may comprisr units of functional activity such as motility, contractility, and conduction. 1.lusrle contraction is due to the folding of the l)olypcptide chains of m?ofibrils (19), \\hilc the ncurofihrils have long been suspected of hcing involved in nerve conduction (11). hlonn6 is of the opinion that rncrgy-yielding processes kvithin the sublnicroscopic particles arc essential for maintaining the dynamic state of thv fihrils. l’hc prcsencc of aclenosincttriphosl)hatase (A’l’Pasc) in the microsomcs of li\or (15), nerve (l), and niusclc (8) has been cstahlished; and hIonn6 c.oncluded that .4’1’I’asc I\as present in the cytoplasmic fibrils in general since they arc contractile and eahihit hirefringencc of flow (12). ‘I’hc present communication deals \vith an attempt to compare and charactcrizc thr isolated microsomcs from dilfcrtbnt tissues and to rcconstitutc them into fihriliar structures rcsemhling the c\-toplasmic fibrils in uivo. \\‘hen microsomcs from rat liver, kidney, musrlc, brain, and nerve wcrc prcparcd in 0.25 hl suvrosr by diflc~rcntial ccntrifugation, they \vere found to he sphcrcs 20 1.50 111~ in diamctcr (1) and to cshihit hirefringence of Ilo\\-. ‘I’hc scdimrnted microsomcs formed a sol \vhich hehavrd like a thisotropic gel \yhen rxtruded into lvatcr, in mut+h the same manner as attoniy0sin. METHODS Isolation of the microsome fraction of rat tissues was done after the method of Hogcboom cl al. (7). I Iomogcnatcs of the various tissues in 0.25 hl sucrnse were first sedimcntetl al 15,000 x g to rernovc mitochontlria, nuclei, ant1 stroma an11 then at 100,000 i( g to isolate the microsorncs. Centrifugation was (lone in the Spinco Xtoclel I ‘I‘his work ~&IS supported University of Illinois.
by a contract
twtwec~n the Office
of Saval
I~rsrwch
Experimertltrl
and the
Cell Research 8
460
L. G. Abood and L. Romanchek
L ultracentrifuge at -5 degrees. The hydrophilic microsomal sol was drawn into a 0.5 ml hypodermic syringe whose tip was drawn out to a 0.1 mm bore, and when the sol was carefully extruded into 1O-8 M magnesium chloride (pH 6.0-7.0), fine gelatinous threads resembling actomyosin were formed. The histochemical technique for ATPase was that of Gomori (6). Rat sciatic nerves were placed in Maximow depression slides and then dissected into single fibers or bundles of usually less then 5 fibers. A medium containing 0.02 M ATP, 0.001 M magnesium chloride, 0.02 M calcium chloride, and histidine buffer, pH 7.5 was then added to the slide and the system incubated for 20 minutes at 37 degrees. Preliminary fixation of the nerves in ice-cold acetone or 70 per cent alcohol did not in any way improve the histochemical technique. ATPase activity was determined on the microsome fraction according to the method of DuBois and Potter (3), except that histidine buffer replaced Verona1 and 0.001 M magnesium chloride was substituted for calcium chloride. Adenylic kinase was determined by the method of Kornberg (9), and adenylic deaminase was measured by following the disappearance of adenylic acid at 260 rnp in the Beckman DU Spectrophotometer. Separation of the “acid-insoluble” fractions was done following the method of Schneider (18). Sodium and potassium were removed from the microsomes by boiling in water for 10 minutes and then determined on the filtrate, using the Beckman flame photometer. Silica glassware, which was low in sodium and potassium, was used whenever analysis for the alkali metals was made. RESULTS
When
AND
DISCUSSION
particles exhibiting double refraction of flow are forced through a tube they align themselves parallel to the direction of flow (5). With
narrow
TABLE Acid
insoluble
components
I
and electrolytes
Wmg % dry dry wgt. I wgt. I
in microsomes
Micromoles/micromoles
of various
of total
tissues.
Nitrogen
. . . .
5.7
57
60.0
1.08
16.0
47
14
3.4
Peripheral nerve . . .
5.5
59
50.1
1.00
3.5
40
12
3.4
Muscle
. _ .
6.5
61
40.1
1.20
4.0
44
11
4.0
. . . .
7.0
60
33.3
1.25
4.7
28
9
3.1
. . .
7.5
63
41.5
1.30
5.1
21
7
3.0
Brain
Liver Kidney
Average Experimental
of 4 values
agreeing
Cell Research 8
within
6 per cent.
461
Properties of microsomes
Fig. 1. Unfixed, unstained fiber prepared artificially from nerve fiber microsomes. Magnification 200 x .
the correct salt concentration they would tend to coalesce and form a stable gel if properly oriented. The microsomes of all tissues studied possessed this property of flow birefringence, and as long as the microsomal salt concentration was at least 3 m M/ml, a stable gel formed. The microsomes aligned themselves into numerous parallel fibrils longitudinal to the fiber axis (Figs. 1 and 2). In a thread 0.5 mm in diameter as many as 50 of these tllaments could be observed at magnifications of 200 x . In the absence of magnesium, in 0.25 M sucrose, or in a slightly alkaline solution the threads lacked the lllar structure and would more readily disintegrate. Unlike actomyosin, the threads would not contract upon the addition of ATP. About 60 per cent of the total dry weight of the microsomes from all tissues was comprised of lipid. The phospholipid content of microsomes from nervous tissues was somewhat greater than in the case of the other tissues where the values were quite similar. In general, the nucleic acid Experimenlal
Cell Research 8
462
L. G. Abood and L. Romanchek
Fig. 2. Fibers prepared by extrusion of nerve fiber microsomes into 5 per cent formalin and stained with 0.01 per cent methylene blue. Magnification 200 x .
content of the various tissues was also similar, although slightly lovver in neural tissue. \\‘ith the exception of an extremely high content in brain, the “phosphoprotein” content of the microsomes of various tissues was in close agreement. The potassium and sodium concentration of the microsomes of neural and muscular tissue were similar, being considerably greater than in liver and kidney. A considerable amount of ATPase vvas found to be present in the microsomcs of all tissues (Table II). The activity of brain and kidney was less than half that of muscle and over twice as great as the ATPase activity of liver and peripheral nerve. Adenylic kinase and adenylic deaminase activity of the microsomes seemed to vary from tissue to tissue in the same proportion as ATPase with the exception of kidney. Cytoplasmic fibrils have long been suspected of being comprised of submicroscopic particles, although considerable controversy exists as to the Experimental
Cell Research
8
Properties of microsomes
463
exact nature of the particles involved. MonnE (13) and Runnstrijm (16) are of the opinion that they are equivalent to the microsomes, while FreyWyssling (4) considers it unlikely that morphogenesis and metabolism are performed by the same elements. The cytoplasmic tlbrils, however, are not mere structural elements but appear to be in a state of continual reorganization and reconstitution as determined by the variable morphogenetic and TABLE Enzymes
of adenylate
system
II
in microsomes -
ATP-ase
of various
Adenylate
tissues.
T
kinase
-
Tissue
-
Adenylic deaminase
I-
PM ATP
split/l5’/pM
N
-
I*M ATP formed/lO’/pM
b
r- IFM AA/lO’/pM
Muscle . . . . . .
1.07
0.30
0.0350
Brain
. . . . . .
0.43
0.10
0.0090
. . . . .
0.45
0.053
0.0046
. . . . . .
0.21
0.035
Kidney Liver
Peripheral Average
nerve
.
0.22
-
of 4 values
0.028 -
agreeing
within
N
0.0020
! I
0.0015
8 per cent.
functional requirements of the cell. Among the numerous types of tibrils, known to respond during functional activity are the myofibrils, neurotibrils, epithelial tibrils, and cilia. This activity usually consists of a visible contraction or motility involving rearrangements in polypeptide chains. Polymerization of actomyosin occurs upon the addition of ATP (1 l), and dissociation of the protein complex follows the dephosphorylation of ATP. It is believed that the polypeptide rearrangements associated with most cytoplasmic tibrillar activity involve an energy-releasing mechanism comparable to the ATPase system of myotibrils. The present findings that the ATPase of nerve fibers appears to be located in tibrillar structures, possibly including the neurofibrils, lend support to the hypothesis that ATI’ase activity is associated with nerve conduction (13, 1). On the basis of present evidence it cannot be determined to what extent the microsomal fraction of neural tissue originated from the neurofibrils. Readlike hbrils observed by DeRobertis and Schmitt (2) in the electron Experimentnl
Cell Research 8
L. G. Abood and L. Romanchek
Fig. 3. Single nerve fiber stained for ATPase. Magnification 1000 x .
microscope were described as axonal constituents, but later believed to be from conneclive tissue sheath (17). In nerve fibers fixed in osmic acid it has been possible to observe within the axoplasm contorted beadlike filaments 100-200 A wide, which are believed to be neurohbrils (17). The finding of Libet (10) that the ATPase of the squid nerve is largely in the sheath would tend to vitiate the hypothesis that the ATPase containing microsomes are of axoplasmic origin. Undoubtedly the microsomes isolated from rat nerve originated from the sheath as well as axoplasm, although their enzymatic and chemical homogeneity remains a question. The presence of high K/Na ratios within the microsomes, particularly those of neural or muscular origin suggested that the artificial tlbers might manifest a surface potential. With the ultramicroelectrode potentials were measured exceeding 30 millivolts in freshly prepared threads immersed in 0.25 M sucrose containing 0.001 M magnesium chloride. Potentials of threads Experimental
Cell Research
8
Properties of microsomes
465
prepared from liver microsomes measured less than 10 millivolts. This difference in potential is to be expected because of the greater electrolyte content of conductive tissue. SUMMARY
Microsomes from a number of rat tissues have been examined wifh respect to certain chemical and physical properties. In addition to a Mg-activated ATP-ase, the microsomes contain adenylic kinase and adenylic deaminase. By extrusion of the microsomal gel into 1O-3 magnesium chloride, artificial fibers are obtained with internal structures resembling cytoplasmic fibrils. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19.
ABOOD, L. G., and GERARD, R. W., J. Cellular Comp. Physiol., 43, 379 (1954). DE ROBERTIS, E., and SCHMITT, F. O., ibid., 31, 1 (1948). DUBOIS, K. P., and POTTER, V. R., J. Biol. Chem., 150, 185 (1943). FREY-WYSSLING, A., Submicroscopic Morphology. Elsevier Publ. Co., New York, 1953. GLASSTONE, S., Textbook of Physical Chemistry. 2nd ed. Van Nostrand, New York, 1946. GOMORI. G., Proc. Sot. Exptl. Biol. Med., 42, 23 (1939). HOGEBOOM, G. H., SCHNEIDER, W. C., and PALADE, G. E., J. Biot. Chem., 172, 619 (1948). KIELLEY, W. W., and MEYERHOF, O., ibid., 183, 391 (1950). KORNBERG, A., ibid., 182, 779 (1950). LIBET, B., Federation Proc., 7, 72 (1948). MDMMAERTS, W. F. H. M., Muscular Contraction. Interscience Publ., New York, 1950. MONNB, L., Experienlia, 2, 153 (1946). __ Aduances in Enzymol., 8, 1 (1948). -Arch. exptl. Zellforsch. GeLvebeziicht, 23, 157 (1949). POTTER, V., Adoances in Enzymol., 4, 201 (1944). RUNNSTRGM, J., ibid., 9, 241 (1949). SCHMITT, F. O., in Genetic Neurology. Ed. P. Weiss, Ii. of Chicago, 1950. SCHNEIDER, W. C., J. Biol. Chem., 161, 293 (1945). SZENT-GYGRGYI, A., Chemistry of Muscular Contraction. Acad. Press, New York, 1947.
Experimental
Cell Research 8