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Localization of mitochondrial phosvitin kinase
SHORT COMMUNICATIONS The present investigation shows that at least the human lactase has some specificity also for the aglycon part of its substrate. This investigation has been supported by grants from the Swedish Medical Research Council (project No. I3X-I57) and from the Mead Hohnson Company, Evansville, Ind. (U.S.A.). The skillful technical assistance of Mrs B. JOHANNESSON, Mrs K. THORNQVIST, Miss B. OHLIN and Mr O. H. MA is gratefully acknowledged.
Department of Physiological Chemistry, University of Lund, Lund (Sweden) and Department of Pediatrics, Yale University School of Medicine, New Haven, Conn. (U.S.A.)
ARNE
JOYCE
DAHLQVIST
D.
GRYBOSKI
I E. M. MONTGOMERY, in R. L. WHISTLER AND M. L. WOLFROM, Methods in Carbohydrate Chemistry, Vol. I, Academic, New York, 1962, p. 325. 2 ]. D. GRYBOSKI, J. ZILIS AND O. H. MA, Gastroenterology, 47 (1964) 26. 3 F. PETUELY, DtJ1$t. Med. Wochschr., 82 (1957) 1957· 4 K. HOFFMANN, D. A. A. MOSSEL, W. KORUS AND J. H. VAN DE KAMER, Klin, Wochschr, 4 2 (1964) I26, 5 S. AUR1CCHIO, A, DAHLQVIST AND G. SEMENZA, Biocbim, Biopbys. Acta, 73 (19 63) 5 82. 6 A. DAHLQVIST, Anal. Biochem., 7 (1964) 18. 7 C.-H. DE VERDIER AND M. HJELM. CUn. Chim. Acta, 7 (19 6 2) 74 2. 8 A. DAHLQVIST AND B. BORGSTROM, Acta Chem, Scand., 13 (1959) 16 59. 9 Z. D1SCHE AND E. BORENFREUND, j. Bioi, Chem., 192 (195 1) 5 83. 10 A. DAHLQV1ST, Biochim. Biophys, Acta, 50 (19 6 1) 55· II R. HEYWORTH AND A. DAHLQV1ST, Bioehim. Biopbys. Acta, 64 (1962) 182.
Received August 9th, Ig65 Biochim, Biopnys. A eta. I I 0 (1965) 635-636
sc 63Iz8 Localization of mitochondrial phosvitin kinase The phosphorylation of phosphoproteins, such as casein", phosvitins-" and cytoplasmic phosphoproteins- by incubation with rat-liver mitochondria in the presence of 32Pt, has previously been demonstrated. Moreover, in a preceding paper from this laboratory-, evidence was provided that the phosphorylation of such exogenous phosphoproteins (phosvitin) by rat-liver mitochondria is strictly dependent on the synthesis of mitochondrial ATP and the release of ATP into the external medium. It was shown that atractyloside, while not affecting the synthesis of intramitochondrial ATP with a-ketoglutarate as substrate, completely blocks the transit of intramitochondrial ATP across the mitochondrial membrane into the external medium. so preventing the phosphorylation of phosvitin", However, the localization and the behaviour of phosvitin kinase (EC 2.7.1.3.7) were unknown. In particular, the presence of phosvitin kinase activity in the extra-mitochondrial medium after incubation suggested that the enzyme. as well as ATp6, has to pass outside the mitochondrion into the external medium in order to catalyze the phosphorylation of exogenous phosphoproteins. In the present study, two forms of Biochim. Biophys. A eta. I IO (1965) 636-639
SHORT COMMUNICATIONS
TABLE I RELEASE OF SOLUBLE PHOSVITIN KINASE ACTIVITY FROM RAT-LIVER MITOCIl:ONDRIA Rat-liver mitochondria were prepared according to SCHNEIDER AND HOGEBOOM', Mitochondria (equivalent to about 20 mg protein) were gently suspended in 3 ml of a solution of o. I M sucrose, 30 mM Tris-HCI buffer (pH 7.4) and 40 mM KCI for 40 sec at 2°, and then centrifuged at 25000 X g. The same mitochondria were resuspended in the same volume of the above medium and centrifuged under the same conditions, 3 times; the successive separate supernatants so obtained ("extracts") were centrifuged at 105 000 X g, in order to precipitate possible mitochondrial fragments. Original mitochondria, and those collected after the 4th "extraction". were incubated for 15 min at 30° in a medium containing: I mg phosvitin, 100 flmoles Tris-HCI buffer (pH 7.4), I2,umoles MgCl s' I20,umoles KCI, 300,umoles sucrose, 60,umoles a-ketoglutarate (potassium salt), 1.5 ,umoles potassium orthophosphate containing IO,uC of .sp! and 0.25 ,umoles ADP, in a final vol. of 3 ml. The mitochondrial "extracts" (2 ml) were incubated for 15 min at 30° in the presence of I mg phosvitin, 100 ftffioles Tris-HCl buffer (pH 7.4), 12 pmoles MgCl s, 120 pmoles KCI, 300 ,umoles sucrose and I ,umole of [IBP)ATP (prepared according to GLYNN AND CHAPPEL'). containing 30,uC as terminal phosphorus; final vol.: 3 rnl. At the end of incubation. mitochondria, when present, were removed by centrifugation and phosvitin was precipitated with 3 % perchloric acid. ATP was determined in the neutralized supernatant as previously described". ssp! incorporation into phosvitin was estimated by measuring the specific radioactivity of alkalilabile phosphate", The phosvitin kinase activity is always referred to 20 mg of mitochondrial protein.
"Extract" No.
Phosvitin kinase
actioitv (%) I
z 3 4
roo 30 . 0 11.6 4. 1
Mitochondria
Phosvitin kinase activity (%)
Before extraction 100 After 4 extractions 59.6
phosvitin kinase are recognized: (a) a soluble enzyme, which probably represents a cytoplasmic contamination of mitochondrial preparations, and (b) an insoluble phosvitin kinase, tightly bound to the mitochondrial structure or surface. In Table I phosvitin kinase activities of the supernatants ("extracts"), obtained by resuspending mitochondria in an isosmotic medium of sucrose-Tris bufferReI for 40 sec at 2°, are reported. The release of phosvitin kinase from mitochondrial preparations is practically independent of the temperature and time of extraction, since the results obtained with extractions at 30° and for longer periods of time were roughly the same. It can be seen that after the resuspension of mitochondria has been repeated 3 or 4 times. no more soluble phosvitin kinase is released from mitochondria (even when a further extraction at 30° is carried out) *, However, mitochondria, depleted at this stage of any soluble phosvitin kinase, are still able to catalyze the incorporation of 82Pi into phosvitin (Table I), though at a lower level. On the other hand. it must be emphasized that the synthesis of ATP is also decreased. The above results are consistent with the view that the mitochondrial phosphorylation of exogenous phosphoproteins is not necessarily dependent on the • At this point, incubation of mitochondria with Naja Naja phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4), although solubiliaing about 50% of mitochondrial proteins, does not enhance the release of soluble phosvitin kinase.
Biochim. Biophys, Acta,
IIO
(1965) 636-639
SHORT COMMUNICATIONS
presence of soluble phosvitin kinase outside the mitochondria, but points to the existence of an insoluble phosvitin kinase tightly bound to the mitochondrial structure. The soluble enzyme which is released from mitochondrial preparations (also into hypertonic media such as 0.5 M sucrose) may represent contamination by a cytoplasmic enzyme. Indeed, cytoplasm tested under the same conditions has been found to have a phosvitin kinase activity much higher than the first "extract" from mitochondria (Table II). TABLE II PHOSVITIN KINASE ACTIVITY OF CYTOPLASM AND OF MITOCHONDRIAL "EXTR ACT"
Incubation medium and general conditions were as described in Table I for mitochondrial " ex tracts" . Cytoplasmic fraction was obtained by centrifuging the 25000 X g supernatant of mitochondria preparation at 105 000 X g in order to precipitate microsomes and other fragments. Cytoplasmic fraction and mitochondria.l "extract" both equivalent to 4.5 mg protein.
Specifi c radioactivity ·i tt [sap[pboso itir:
(coun ts/min per um ole phosphorus) 1St mitochondrial "extract" Cytoplasm
589
2071
The existence of an insoluble mitochondrial phosvitin kinase is also strongly indicated by the ability of sonic mitochondrial sub-particles to phosphorylate phosvitin in the presence of [32PJATP. The results shown in Table III clearly demonstrate that the most of phosvitin kinase activity is present in the sub-particles. Unfortunately, a direct comparison between the phosvitin kinase activity of subparticles and of the soluble fraction (mitoplasm) is impossible, owing to the ATPase (ATP phosphohydrolase, EC 3.6.1.4) activity which is much higher in sub-particles than in mitoplasm, in spite of added oligomycin". Data shown in Table III also demonstrate that different times of sonic treatment (Expt . r ) do not produce any significant change in the phosvitin kina se activity of either sub-particles and soluble fraction. Moreover, if sonic sub-particles are submitted to further sonic treatment, no further release of phosvitin kinase can be observed during the second sonication, and accordingly no phosvitin kinase activity is lost by sub-particles (Expt. 2). It seems clear, therefore, that soluble and particulate forms of phosvitin kinase are present in the usual mitochondrial preparations, the former probably being a cytoplasmic contamination, the latter representing an enzyme tightly bound to the mitochondrial structure. Since it is accessible to phosvitin, such an insoluble • In preliminary experiments it was observed that oligomycin was practica.lly without effect on A'I'Pase activity of sonic sub-particles in the absence of phosphate or in the presence of high MgH concentrations. It was therefore found convenient to use an incubation medium containing 0.5 mM phosphate and 2 roM MgH, although these conditions are far from optimal for the phosvitin kinase reaction.
Biochim , BioPhys . Acta , no (1965) 636--639
SHO RT COMMUNICATIONS
TABLE III PHOSPHORYLATION OF PHOSVITIN BY SONIC SUB-PARTICLES AND BY RESPECTIVE SOLUBLE FRACTIONS FROM RAT-LIVER MITOCHONDRIA The incubation conditions were the same as used for mitochondrial " extracts " (see Table I), except that oligom ycin (5 flg/mg protein) and phosphate (0.5 roM) were also present, and MgCl~ was on ly 2 mM. Sonic sub-particles and "mitoplasm" were b oth equivalent to 7 mg protein. I n cubat ion was stopped b y the ad dition of r' ml of 8 mM Versen e, After removal of particles, if presen t , b y centrifugation at 105 0 0 0 X g, ph osvitin was reco v ered and it s radioactivity determined as described in Table 1. Sonic subparticles were prepared a ccording to McMuRRAY et aUo from rat liver mitochondria subj ected to soni c d isruption in water at 20 kilocycles at 0 0 • "Mitoplasm" , i ,e. the soluble mitochondrial fraction obtain ed by son ication (equi valent to about 70% of mitoch ondria l proteins). was passed through a Sephadex G-25 col umn (3 ern! X 7 em) before incu ba t ion. E.rpt. No.
Sonic treatment
r ao-sec sonication :l50-sec son ication ~
r zo-sec son ication r zo-sec resonication
of sonic particles
Specific radioactivity in [82P1ph osvitin (counts/min p er umol« phosphorus) Sub-particles sedimenied at IOS 000 X g
Soluble f racti on
1259 1239
5°2 53 0
784
33 8
837
8
phosvitin kinase is very probably present on the mitochondrial surface. This point of view is supported by the finding that only th e ext ramit och ondrial ATP is able to phosphorylate the phosvitin 6 . Some of these results have been reported at the Symposium on the regulation of metabolic processes in mitochondria, Bali (Italy), April 1965. We wish t o thank Miss C. MUNARI for valuable technical assistance. Institute oj Biological Chemistry and Enzymological Un it oj Consiglio Nazionale delle Ricerche, University oj Padooa, Padova ( I taly) 1 '2 3 4 5 6 7 8 9 10
M. LORINI L. A. PINNA
V.
MORET
N. SILIPRANDI
G. BURNETT AND E. P . KENNEDY, ]. Biol, Chem., 211 (1954 ) 969 . S. P. R. ROSE AND P. ]. HEALD, Biochem, 1-, 81 (1961) 339 · V. MORET, L. A. PINN A, S. S PERTl AND M. LORINI, Boll . S oc. Ital, Bioi. Sper., 38 (1962 ) 18 76. M. B . LIVANOVA, Vop r. M ed, Khim., 8 (1962) 429 . V. MORET, L. A. PI NNA , S. SPERTl, M. LORINI AND N . SILIPRANDI, Biocbim. Biophys. A cta, 82 (1964) 603 . W. C. SCHNEIDER AND G. H . HOGEBOOM, j. B iol. Chem., 183 (1950) 123. I. M. GLYNN AND ] . B . CHAPPEL, B io chem , j., 90 (1964) 147 · V. MORET, L. A. PI NNA, S. SPE RT1 , M. LORINI AND N . SILIPRANDl , Bioohim, Bi opbys, A cta, 78 (19 6 3) 547· M. R ABINOWITZ AND F . L1PMANN, J. B iot. Chem., 235 (1960) 1043 . W. C. McMURRAY, G . F ELDOTI MALEY AND H . A. L ARDY, ] . Biol, Chem. , 23 0 (19 58) 21g .
Received August rjth , 1965 B iochim . B iophys, A cia, 110 (19 65\ 636-639
Department of Physiological Chemistry, University of Lund, Lund (Sweden) and Department of Pediatrics, Yale University School of Medicine, New Haven, Conn. (U.S.A.)
ARNE
JOYCE
DAHLQVIST
D.
GRYBOSKI
I E. M. MONTGOMERY, in R. L. WHISTLER AND M. L. WOLFROM, Methods in Carbohydrate Chemistry, Vol. I, Academic, New York, 1962, p. 325. 2 ]. D. GRYBOSKI, J. ZILIS AND O. H. MA, Gastroenterology, 47 (1964) 26. 3 F. PETUELY, DtJ1$t. Med. Wochschr., 82 (1957) 1957· 4 K. HOFFMANN, D. A. A. MOSSEL, W. KORUS AND J. H. VAN DE KAMER, Klin, Wochschr, 4 2 (1964) I26, 5 S. AUR1CCHIO, A, DAHLQVIST AND G. SEMENZA, Biocbim, Biopbys. Acta, 73 (19 63) 5 82. 6 A. DAHLQVIST, Anal. Biochem., 7 (1964) 18. 7 C.-H. DE VERDIER AND M. HJELM. CUn. Chim. Acta, 7 (19 6 2) 74 2. 8 A. DAHLQVIST AND B. BORGSTROM, Acta Chem, Scand., 13 (1959) 16 59. 9 Z. D1SCHE AND E. BORENFREUND, j. Bioi, Chem., 192 (195 1) 5 83. 10 A. DAHLQV1ST, Biochim. Biophys, Acta, 50 (19 6 1) 55· II R. HEYWORTH AND A. DAHLQV1ST, Bioehim. Biopbys. Acta, 64 (1962) 182.
Received August 9th, Ig65 Biochim, Biopnys. A eta. I I 0 (1965) 635-636
sc 63Iz8 Localization of mitochondrial phosvitin kinase The phosphorylation of phosphoproteins, such as casein", phosvitins-" and cytoplasmic phosphoproteins- by incubation with rat-liver mitochondria in the presence of 32Pt, has previously been demonstrated. Moreover, in a preceding paper from this laboratory-, evidence was provided that the phosphorylation of such exogenous phosphoproteins (phosvitin) by rat-liver mitochondria is strictly dependent on the synthesis of mitochondrial ATP and the release of ATP into the external medium. It was shown that atractyloside, while not affecting the synthesis of intramitochondrial ATP with a-ketoglutarate as substrate, completely blocks the transit of intramitochondrial ATP across the mitochondrial membrane into the external medium. so preventing the phosphorylation of phosvitin", However, the localization and the behaviour of phosvitin kinase (EC 2.7.1.3.7) were unknown. In particular, the presence of phosvitin kinase activity in the extra-mitochondrial medium after incubation suggested that the enzyme. as well as ATp6, has to pass outside the mitochondrion into the external medium in order to catalyze the phosphorylation of exogenous phosphoproteins. In the present study, two forms of Biochim. Biophys. A eta. I IO (1965) 636-639
SHORT COMMUNICATIONS
TABLE I RELEASE OF SOLUBLE PHOSVITIN KINASE ACTIVITY FROM RAT-LIVER MITOCIl:ONDRIA Rat-liver mitochondria were prepared according to SCHNEIDER AND HOGEBOOM', Mitochondria (equivalent to about 20 mg protein) were gently suspended in 3 ml of a solution of o. I M sucrose, 30 mM Tris-HCI buffer (pH 7.4) and 40 mM KCI for 40 sec at 2°, and then centrifuged at 25000 X g. The same mitochondria were resuspended in the same volume of the above medium and centrifuged under the same conditions, 3 times; the successive separate supernatants so obtained ("extracts") were centrifuged at 105 000 X g, in order to precipitate possible mitochondrial fragments. Original mitochondria, and those collected after the 4th "extraction". were incubated for 15 min at 30° in a medium containing: I mg phosvitin, 100 flmoles Tris-HCI buffer (pH 7.4), I2,umoles MgCl s' I20,umoles KCI, 300,umoles sucrose, 60,umoles a-ketoglutarate (potassium salt), 1.5 ,umoles potassium orthophosphate containing IO,uC of .sp! and 0.25 ,umoles ADP, in a final vol. of 3 ml. The mitochondrial "extracts" (2 ml) were incubated for 15 min at 30° in the presence of I mg phosvitin, 100 ftffioles Tris-HCl buffer (pH 7.4), 12 pmoles MgCl s, 120 pmoles KCI, 300 ,umoles sucrose and I ,umole of [IBP)ATP (prepared according to GLYNN AND CHAPPEL'). containing 30,uC as terminal phosphorus; final vol.: 3 rnl. At the end of incubation. mitochondria, when present, were removed by centrifugation and phosvitin was precipitated with 3 % perchloric acid. ATP was determined in the neutralized supernatant as previously described". ssp! incorporation into phosvitin was estimated by measuring the specific radioactivity of alkalilabile phosphate", The phosvitin kinase activity is always referred to 20 mg of mitochondrial protein.
"Extract" No.
Phosvitin kinase
actioitv (%) I
z 3 4
roo 30 . 0 11.6 4. 1
Mitochondria
Phosvitin kinase activity (%)
Before extraction 100 After 4 extractions 59.6
phosvitin kinase are recognized: (a) a soluble enzyme, which probably represents a cytoplasmic contamination of mitochondrial preparations, and (b) an insoluble phosvitin kinase, tightly bound to the mitochondrial structure or surface. In Table I phosvitin kinase activities of the supernatants ("extracts"), obtained by resuspending mitochondria in an isosmotic medium of sucrose-Tris bufferReI for 40 sec at 2°, are reported. The release of phosvitin kinase from mitochondrial preparations is practically independent of the temperature and time of extraction, since the results obtained with extractions at 30° and for longer periods of time were roughly the same. It can be seen that after the resuspension of mitochondria has been repeated 3 or 4 times. no more soluble phosvitin kinase is released from mitochondria (even when a further extraction at 30° is carried out) *, However, mitochondria, depleted at this stage of any soluble phosvitin kinase, are still able to catalyze the incorporation of 82Pi into phosvitin (Table I), though at a lower level. On the other hand. it must be emphasized that the synthesis of ATP is also decreased. The above results are consistent with the view that the mitochondrial phosphorylation of exogenous phosphoproteins is not necessarily dependent on the • At this point, incubation of mitochondria with Naja Naja phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4), although solubiliaing about 50% of mitochondrial proteins, does not enhance the release of soluble phosvitin kinase.
Biochim. Biophys, Acta,
IIO
(1965) 636-639
SHORT COMMUNICATIONS
presence of soluble phosvitin kinase outside the mitochondria, but points to the existence of an insoluble phosvitin kinase tightly bound to the mitochondrial structure. The soluble enzyme which is released from mitochondrial preparations (also into hypertonic media such as 0.5 M sucrose) may represent contamination by a cytoplasmic enzyme. Indeed, cytoplasm tested under the same conditions has been found to have a phosvitin kinase activity much higher than the first "extract" from mitochondria (Table II). TABLE II PHOSVITIN KINASE ACTIVITY OF CYTOPLASM AND OF MITOCHONDRIAL "EXTR ACT"
Incubation medium and general conditions were as described in Table I for mitochondrial " ex tracts" . Cytoplasmic fraction was obtained by centrifuging the 25000 X g supernatant of mitochondria preparation at 105 000 X g in order to precipitate microsomes and other fragments. Cytoplasmic fraction and mitochondria.l "extract" both equivalent to 4.5 mg protein.
Specifi c radioactivity ·i tt [sap[pboso itir:
(coun ts/min per um ole phosphorus) 1St mitochondrial "extract" Cytoplasm
589
2071
The existence of an insoluble mitochondrial phosvitin kinase is also strongly indicated by the ability of sonic mitochondrial sub-particles to phosphorylate phosvitin in the presence of [32PJATP. The results shown in Table III clearly demonstrate that the most of phosvitin kinase activity is present in the sub-particles. Unfortunately, a direct comparison between the phosvitin kinase activity of subparticles and of the soluble fraction (mitoplasm) is impossible, owing to the ATPase (ATP phosphohydrolase, EC 3.6.1.4) activity which is much higher in sub-particles than in mitoplasm, in spite of added oligomycin". Data shown in Table III also demonstrate that different times of sonic treatment (Expt . r ) do not produce any significant change in the phosvitin kina se activity of either sub-particles and soluble fraction. Moreover, if sonic sub-particles are submitted to further sonic treatment, no further release of phosvitin kinase can be observed during the second sonication, and accordingly no phosvitin kinase activity is lost by sub-particles (Expt. 2). It seems clear, therefore, that soluble and particulate forms of phosvitin kinase are present in the usual mitochondrial preparations, the former probably being a cytoplasmic contamination, the latter representing an enzyme tightly bound to the mitochondrial structure. Since it is accessible to phosvitin, such an insoluble • In preliminary experiments it was observed that oligomycin was practica.lly without effect on A'I'Pase activity of sonic sub-particles in the absence of phosphate or in the presence of high MgH concentrations. It was therefore found convenient to use an incubation medium containing 0.5 mM phosphate and 2 roM MgH, although these conditions are far from optimal for the phosvitin kinase reaction.
Biochim , BioPhys . Acta , no (1965) 636--639
SHO RT COMMUNICATIONS
TABLE III PHOSPHORYLATION OF PHOSVITIN BY SONIC SUB-PARTICLES AND BY RESPECTIVE SOLUBLE FRACTIONS FROM RAT-LIVER MITOCHONDRIA The incubation conditions were the same as used for mitochondrial " extracts " (see Table I), except that oligom ycin (5 flg/mg protein) and phosphate (0.5 roM) were also present, and MgCl~ was on ly 2 mM. Sonic sub-particles and "mitoplasm" were b oth equivalent to 7 mg protein. I n cubat ion was stopped b y the ad dition of r' ml of 8 mM Versen e, After removal of particles, if presen t , b y centrifugation at 105 0 0 0 X g, ph osvitin was reco v ered and it s radioactivity determined as described in Table 1. Sonic subparticles were prepared a ccording to McMuRRAY et aUo from rat liver mitochondria subj ected to soni c d isruption in water at 20 kilocycles at 0 0 • "Mitoplasm" , i ,e. the soluble mitochondrial fraction obtain ed by son ication (equi valent to about 70% of mitoch ondria l proteins). was passed through a Sephadex G-25 col umn (3 ern! X 7 em) before incu ba t ion. E.rpt. No.
Sonic treatment
r ao-sec sonication :l50-sec son ication ~
r zo-sec son ication r zo-sec resonication
of sonic particles
Specific radioactivity in [82P1ph osvitin (counts/min p er umol« phosphorus) Sub-particles sedimenied at IOS 000 X g
Soluble f racti on
1259 1239
5°2 53 0
784
33 8
837
8
phosvitin kinase is very probably present on the mitochondrial surface. This point of view is supported by the finding that only th e ext ramit och ondrial ATP is able to phosphorylate the phosvitin 6 . Some of these results have been reported at the Symposium on the regulation of metabolic processes in mitochondria, Bali (Italy), April 1965. We wish t o thank Miss C. MUNARI for valuable technical assistance. Institute oj Biological Chemistry and Enzymological Un it oj Consiglio Nazionale delle Ricerche, University oj Padooa, Padova ( I taly) 1 '2 3 4 5 6 7 8 9 10
M. LORINI L. A. PINNA
V.
MORET
N. SILIPRANDI
G. BURNETT AND E. P . KENNEDY, ]. Biol, Chem., 211 (1954 ) 969 . S. P. R. ROSE AND P. ]. HEALD, Biochem, 1-, 81 (1961) 339 · V. MORET, L. A. PINN A, S. S PERTl AND M. LORINI, Boll . S oc. Ital, Bioi. Sper., 38 (1962 ) 18 76. M. B . LIVANOVA, Vop r. M ed, Khim., 8 (1962) 429 . V. MORET, L. A. PI NNA , S. SPERTl, M. LORINI AND N . SILIPRANDI, Biocbim. Biophys. A cta, 82 (1964) 603 . W. C. SCHNEIDER AND G. H . HOGEBOOM, j. B iol. Chem., 183 (1950) 123. I. M. GLYNN AND ] . B . CHAPPEL, B io chem , j., 90 (1964) 147 · V. MORET, L. A. PI NNA, S. SPE RT1 , M. LORINI AND N . SILIPRANDl , Bioohim, Bi opbys, A cta, 78 (19 6 3) 547· M. R ABINOWITZ AND F . L1PMANN, J. B iot. Chem., 235 (1960) 1043 . W. C. McMURRAY, G . F ELDOTI MALEY AND H . A. L ARDY, ] . Biol, Chem. , 23 0 (19 58) 21g .
Received August rjth , 1965 B iochim . B iophys, A cia, 110 (19 65\ 636-639