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Inhibition by ergotamine and ergobasinine of Ca2+ uptake from the sarcotubular system
513
SHORT COMMUNICATIONS
filter-paper discs with 5 ml scintillator is probably mainly accounted for by the use of glass-fibre discs which unlike cellulose filter discs become transparent in the scintillator and which cling to the bottom of the bottle, and to a lesser extent by the use of small volumes of scintillator (cf. 58 % counting efficiency obtained using W h a t m a n No. 3 filter discs with 5 ml of scintillator, Fig. IC). Indeed m a n y investigators have failed to appreciate that there is no advantage in the use of volumes of scintillator in excess of I ml for the liquid scintillation counting of beta emitters on filter discs, a fact implicit from the earlier work of BROWN .aND BADMAN7 on scintillation counting in non-aqueous systems. Moreover, the use of small volumes of scintillator is more economical. When using labelled amino acids, nucleotides and proteins all radioactivity remains on the discs resulting in negligible radioactive contamination of counting bottles, as previously noted by NANS AND NOVELLI~, thus eliminating the necessity for rigorous washing procedures. Furthermore, since all radioactivity remains on the discs they can be kept for recounting for future reference. Scintillation counting of samples on discs is simple, rapid, and particularly when using glass-fibre filter discs is highly efficient and reproducible, and possesses a wide applicability to a range of biochemical radioisotope investigations. In this laboratory the uptake of tritiated amino acids and nucleotides by isolated plant protoplasts has been readily detected by pipetting suitable aliquots of suspended protoplasts onto glass-fibre discs. The glass-fibre disc method has also been routinely used in studies both i~, vivo and in vitro of the incorporation of 14C-labelled amino acids and bicarbonate into proteins by plant tissues. One of us (J. W. D.) acknowledges the award of a D.S.I.R. Research Studentship.
Department of Botany, University of Nottingham, Nottingham (Great Britain) I 2 3 4 5 6 7
J . W . DAVIES E . C . COCKING
E. A. DAVlDSON, Packard Technical Bulletin, 4 (1962). M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. F. CRAMER, H. Kf)NTZEL AND J. H. MATTHAEI, Angew. Chem., 76 (1964) 716. T. C. HALL AND E. C. COCKING, Biochem. J., 96 (1965) 626. \v. F. BOUSEQUET AND J. F. CHRISTIAN, Anal. Chem., 32 (196o) 722. t{. J. MANS AND G. D. NOVELLI, Arch. Biochem. Biophys., 94 (1961) 48. "~V. O. BROWN AND ]-I. G. BADMAN, Bioehem. J., 78 (1961) 571.
Received September 2oth, 1965 Biochim. Biophys. Acta, 115 (1966) 511-513
BBA
23195
Inhibition by ergotamine and ergobasinine of Ca2+ uptake from the sarcotubular system Relaxation of the muscle fibre is accomplished through the activity of the sarcotubular system 1-3. The relaxation mechanism seems to involve an energydependent Ca 2+ uptake in the sarcotubules 4. So far, no specific inhibitor of this reaction has been found. Ergotamine is known to cause contraction of smooth muscles. Inhibitory effects of ergotamine and ergobasinine on the relaxation of myofibrils and Ca ~+ uptake are reported below. Biochim. Biophys. Acta, 115 (1966) 513-515
514
SHORT COMMUNICATIONS
Rabbit myofibrils were prepared according to PERRY AND GREY5. Fragments of the sarcotubular system were obtained by differential centrifugation between 12ooo : g and 3oooo "~: g of rabbit skeletal-muscle homogenates in o.25 M sucrose2 mM oxalate. Relaxation of rabbit myofibrils was followed by determining simultaneously the changes of absorbance 6 and the Pi liberate&. Ca 2+ uptake was determined by incubating the sarcotubules in the presence of 45CaC12. The suspension was filtered through a Millipore Filter Type HA o25ooo and the number of counts on the filter was measured. Ergotamine-D-tartrate (C3aH~sNsOs)2C4HGOG, mol. wt. 1313.46, and ergobasinine C19H2aN302, mol. wt. 325.42, were purchased from Fluka, Buchs, Switzerland. As we have reported previously 6, addition of ATP to a suspension of myofibrils caused an increase of absorbance which was concluded to be equivalent to the contraction of myofibrils. When sarcotubular fragments were added to the incubation medium, addition of ATP resulted in a decrease of the absorbance. Parallel to the decrease of absorbance the ATPase (EC 3.6.1.3) activity of the myofibrils became inhibited. Both the decrease of absorbance and the inhibition of ATPase activity of the myofibrils caused by the presence of the sarcotubules were abolished by addition of increasing amounts of ergotamine. Complete inhibition was obtained at about 5o ffM ergotamine (Fig. IA). The Ca~+ uptake by the sarcotubular fragments was inhibited by ergotamine at concentrations equal to those required to inhibit the relaxing effect. Concentrations 100
A
100~ B
80
8C
60
.~ N4c
•
4C
20
~9
0
O
2C
20 40 Concn. er'gotamine (HM)
'
60
I
0
0 20 40 Concn. ergobasinine (pM)
I
60
Fig. I. T h e i n c u b a t i o n m e d i u m for d e t e r m i n a t i o n of the r e l a x i n g a c t i v i t y c o n t a i n e d : 2.5 naM MgCI2, 5 mM ATP, 8 mM s o d i u m o x a l a t e , 5 ° mM Tris buffer (pH 7.4), i mM KC1, o. 4 mM b o r a t e buffer ( pH 7.1). The a m o u n t of m y o f i b r i l l a r p r o t e i n was a b o u t i m g a n d t h a t of s a r c o t u b u l a r p r o t e i n w a s b e t w e e n o.1 and o.2 rag. F i n a l volume, 3 ml. The c h a n g e s of a b s o r b a n c e were foilowed s pe c t rop h o t o m e t r i c a l l y as d e s c r i b e d p r e v i o u s l y 7. After 3 m i n of i n c u b a t i o n t he r e a c t i o n was s t o p p e d w i t h I N p e r c h l o r i c acid a n d t h e Pl l i b e r a t e d was d e t e r m i n e d s. The i n c u b a t i o n m e d i u m for t h e d e t e r m i n a t i o n of t h e Ca 2+ u p t a k e c o n t a i n e d : 2.5 mM MgCI~, 5 mM ATP, 5 ° mM Tris buffer (pH 7.4), 5 ° - l ° ° # M 45CAC12. The a m o u n t of s a r c o t u b u l a r p r o t e i n was o.3-o.6 mg. F i n a l v o l u m e , 2 ml; t i m e of i n c u b a t i o n , 5 min. • • , Ca 2+ u p t a k e ; O O, m y o f i b r i l s r e l a x a t i o n .
Biochi~n. 13iophys. Acta, I15 (1966) 513-515
SHORT COMMUNICATIONS
515
of ergotamine which completely inhibited the Ca ~+ uptake did not interfere either with the ATPase activity of the sarcotubular fragments or with the ATPase activity of the myofibrils. Results similar to those obtained with ergotamine were obtained with ergobasinine (Fig. IB). Ergotamine and ergobasinine had no inhibitory effects on the Ca 2+ uptake, whether supported by respiration or by ATP of liver mitochondria or of skeletal muscle mitochondria. Ergotamine and ergobasinine would thus appear to be specific inhibitors of the Ca 2+ uptake of the sarcotubular system. However, clarification of the mechanism of relaxation of smooth muscles will be required in order to establish whether the inhibitory effect of ergotamine on the relaxing factor is related to its pharmacological effect. This investigation was aided by a Grant from the Muscular Dystrophy Association of America, Inc.
The Unit "G. Vernoni" for the Study of Physiopathology, Institute of General Pathology, University of Padua, Padua (Italy)
GIOVANNI FELICE AZZONE
GIOVANNI MILIC GIOVANNA MARCER ALBERTO OTTOLENGHI
I T. NAGAI, M. MAKINOSE AND W. ]:{ASSELBACH,Biochim. Biophys. Acta, 43 (196o) 223. 2 S. EBASHI AND F. LIPMANN, J. Cell Biol., 14 (1962) 389. 3 U. MUSCATELLO, E. ANDERSSON-CEDERGREN AND G. F. AZZONE, Bioehim. Biophys. Acta, 63 (1962) 554 W. HASSELBACH, Progr. Biophys. Biophys. Chem., 14 (1964) 164. 5 S . V . PERRY AND T. C. GREY, Biochem. J., 64 (1956) 184. 6 G. F. AzzoNE AND G. DOBRILLA, Exptl. Cell Res., 36 (1964) 215. 7 0 . LINDBERG AND L. ERNSTER, Methods Biochem. Analy., 3 (1955) I.
Received October Ist, 1965 Biochim. Biophys. Acta, 115 (I966) 513-515
BBA
23 189
Small scale preparation of [|-t4C]fluoroacetic acid During a study of the metabolism of organic fluorine compounds it became necessary to make radioactive fluoroacetic acid. In order that the compound should have high specific activity and yet be produced at reasonable cost a small scale preparation was required. The most suitable method available was that of BERGMANN AND BLANK1 in which a chloroacetic ester is allowed to react with potassium fluoride dissolved in molten acetamide. This preparation had been adapted by GAL, DREWES AND TAYLOR2 but the scale was insufficiently small and the yield of fluoroacetic acid only 35 %. Consequently we examined the method critically, and finally adopted the following procedure. ~I-l~CJchloroacetic acid (6. 9 rag, 0. 5 mC) (The Radiochemical Centre, Amersham, U.K.) was washed from its container with ether (i ml) into a 5-ml tube containing methyl chloroacetate (12o rag). Diazomethane from a micro-generator 3 was passed through the ethereal solution until a yellow colour persisted. In the flask of the reaction apparatus (Fig. I) finely powdered potassium fluoride dried overnight at I2o ° (2 g) and freshly redistilled acetamide (I g) were melted b y heating in an Biochim. Biophys. Acta, 115 (1966) 515-517
SHORT COMMUNICATIONS
filter-paper discs with 5 ml scintillator is probably mainly accounted for by the use of glass-fibre discs which unlike cellulose filter discs become transparent in the scintillator and which cling to the bottom of the bottle, and to a lesser extent by the use of small volumes of scintillator (cf. 58 % counting efficiency obtained using W h a t m a n No. 3 filter discs with 5 ml of scintillator, Fig. IC). Indeed m a n y investigators have failed to appreciate that there is no advantage in the use of volumes of scintillator in excess of I ml for the liquid scintillation counting of beta emitters on filter discs, a fact implicit from the earlier work of BROWN .aND BADMAN7 on scintillation counting in non-aqueous systems. Moreover, the use of small volumes of scintillator is more economical. When using labelled amino acids, nucleotides and proteins all radioactivity remains on the discs resulting in negligible radioactive contamination of counting bottles, as previously noted by NANS AND NOVELLI~, thus eliminating the necessity for rigorous washing procedures. Furthermore, since all radioactivity remains on the discs they can be kept for recounting for future reference. Scintillation counting of samples on discs is simple, rapid, and particularly when using glass-fibre filter discs is highly efficient and reproducible, and possesses a wide applicability to a range of biochemical radioisotope investigations. In this laboratory the uptake of tritiated amino acids and nucleotides by isolated plant protoplasts has been readily detected by pipetting suitable aliquots of suspended protoplasts onto glass-fibre discs. The glass-fibre disc method has also been routinely used in studies both i~, vivo and in vitro of the incorporation of 14C-labelled amino acids and bicarbonate into proteins by plant tissues. One of us (J. W. D.) acknowledges the award of a D.S.I.R. Research Studentship.
Department of Botany, University of Nottingham, Nottingham (Great Britain) I 2 3 4 5 6 7
J . W . DAVIES E . C . COCKING
E. A. DAVlDSON, Packard Technical Bulletin, 4 (1962). M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. F. CRAMER, H. Kf)NTZEL AND J. H. MATTHAEI, Angew. Chem., 76 (1964) 716. T. C. HALL AND E. C. COCKING, Biochem. J., 96 (1965) 626. \v. F. BOUSEQUET AND J. F. CHRISTIAN, Anal. Chem., 32 (196o) 722. t{. J. MANS AND G. D. NOVELLI, Arch. Biochem. Biophys., 94 (1961) 48. "~V. O. BROWN AND ]-I. G. BADMAN, Bioehem. J., 78 (1961) 571.
Received September 2oth, 1965 Biochim. Biophys. Acta, 115 (1966) 511-513
BBA
23195
Inhibition by ergotamine and ergobasinine of Ca2+ uptake from the sarcotubular system Relaxation of the muscle fibre is accomplished through the activity of the sarcotubular system 1-3. The relaxation mechanism seems to involve an energydependent Ca 2+ uptake in the sarcotubules 4. So far, no specific inhibitor of this reaction has been found. Ergotamine is known to cause contraction of smooth muscles. Inhibitory effects of ergotamine and ergobasinine on the relaxation of myofibrils and Ca ~+ uptake are reported below. Biochim. Biophys. Acta, 115 (1966) 513-515
514
SHORT COMMUNICATIONS
Rabbit myofibrils were prepared according to PERRY AND GREY5. Fragments of the sarcotubular system were obtained by differential centrifugation between 12ooo : g and 3oooo "~: g of rabbit skeletal-muscle homogenates in o.25 M sucrose2 mM oxalate. Relaxation of rabbit myofibrils was followed by determining simultaneously the changes of absorbance 6 and the Pi liberate&. Ca 2+ uptake was determined by incubating the sarcotubules in the presence of 45CaC12. The suspension was filtered through a Millipore Filter Type HA o25ooo and the number of counts on the filter was measured. Ergotamine-D-tartrate (C3aH~sNsOs)2C4HGOG, mol. wt. 1313.46, and ergobasinine C19H2aN302, mol. wt. 325.42, were purchased from Fluka, Buchs, Switzerland. As we have reported previously 6, addition of ATP to a suspension of myofibrils caused an increase of absorbance which was concluded to be equivalent to the contraction of myofibrils. When sarcotubular fragments were added to the incubation medium, addition of ATP resulted in a decrease of the absorbance. Parallel to the decrease of absorbance the ATPase (EC 3.6.1.3) activity of the myofibrils became inhibited. Both the decrease of absorbance and the inhibition of ATPase activity of the myofibrils caused by the presence of the sarcotubules were abolished by addition of increasing amounts of ergotamine. Complete inhibition was obtained at about 5o ffM ergotamine (Fig. IA). The Ca~+ uptake by the sarcotubular fragments was inhibited by ergotamine at concentrations equal to those required to inhibit the relaxing effect. Concentrations 100
A
100~ B
80
8C
60
.~ N4c
•
4C
20
~9
0
O
2C
20 40 Concn. er'gotamine (HM)
'
60
I
0
0 20 40 Concn. ergobasinine (pM)
I
60
Fig. I. T h e i n c u b a t i o n m e d i u m for d e t e r m i n a t i o n of the r e l a x i n g a c t i v i t y c o n t a i n e d : 2.5 naM MgCI2, 5 mM ATP, 8 mM s o d i u m o x a l a t e , 5 ° mM Tris buffer (pH 7.4), i mM KC1, o. 4 mM b o r a t e buffer ( pH 7.1). The a m o u n t of m y o f i b r i l l a r p r o t e i n was a b o u t i m g a n d t h a t of s a r c o t u b u l a r p r o t e i n w a s b e t w e e n o.1 and o.2 rag. F i n a l volume, 3 ml. The c h a n g e s of a b s o r b a n c e were foilowed s pe c t rop h o t o m e t r i c a l l y as d e s c r i b e d p r e v i o u s l y 7. After 3 m i n of i n c u b a t i o n t he r e a c t i o n was s t o p p e d w i t h I N p e r c h l o r i c acid a n d t h e Pl l i b e r a t e d was d e t e r m i n e d s. The i n c u b a t i o n m e d i u m for t h e d e t e r m i n a t i o n of t h e Ca 2+ u p t a k e c o n t a i n e d : 2.5 mM MgCI~, 5 mM ATP, 5 ° mM Tris buffer (pH 7.4), 5 ° - l ° ° # M 45CAC12. The a m o u n t of s a r c o t u b u l a r p r o t e i n was o.3-o.6 mg. F i n a l v o l u m e , 2 ml; t i m e of i n c u b a t i o n , 5 min. • • , Ca 2+ u p t a k e ; O O, m y o f i b r i l s r e l a x a t i o n .
Biochi~n. 13iophys. Acta, I15 (1966) 513-515
SHORT COMMUNICATIONS
515
of ergotamine which completely inhibited the Ca ~+ uptake did not interfere either with the ATPase activity of the sarcotubular fragments or with the ATPase activity of the myofibrils. Results similar to those obtained with ergotamine were obtained with ergobasinine (Fig. IB). Ergotamine and ergobasinine had no inhibitory effects on the Ca 2+ uptake, whether supported by respiration or by ATP of liver mitochondria or of skeletal muscle mitochondria. Ergotamine and ergobasinine would thus appear to be specific inhibitors of the Ca 2+ uptake of the sarcotubular system. However, clarification of the mechanism of relaxation of smooth muscles will be required in order to establish whether the inhibitory effect of ergotamine on the relaxing factor is related to its pharmacological effect. This investigation was aided by a Grant from the Muscular Dystrophy Association of America, Inc.
The Unit "G. Vernoni" for the Study of Physiopathology, Institute of General Pathology, University of Padua, Padua (Italy)
GIOVANNI FELICE AZZONE
GIOVANNI MILIC GIOVANNA MARCER ALBERTO OTTOLENGHI
I T. NAGAI, M. MAKINOSE AND W. ]:{ASSELBACH,Biochim. Biophys. Acta, 43 (196o) 223. 2 S. EBASHI AND F. LIPMANN, J. Cell Biol., 14 (1962) 389. 3 U. MUSCATELLO, E. ANDERSSON-CEDERGREN AND G. F. AZZONE, Bioehim. Biophys. Acta, 63 (1962) 554 W. HASSELBACH, Progr. Biophys. Biophys. Chem., 14 (1964) 164. 5 S . V . PERRY AND T. C. GREY, Biochem. J., 64 (1956) 184. 6 G. F. AzzoNE AND G. DOBRILLA, Exptl. Cell Res., 36 (1964) 215. 7 0 . LINDBERG AND L. ERNSTER, Methods Biochem. Analy., 3 (1955) I.
Received October Ist, 1965 Biochim. Biophys. Acta, 115 (I966) 513-515
BBA
23 189
Small scale preparation of [|-t4C]fluoroacetic acid During a study of the metabolism of organic fluorine compounds it became necessary to make radioactive fluoroacetic acid. In order that the compound should have high specific activity and yet be produced at reasonable cost a small scale preparation was required. The most suitable method available was that of BERGMANN AND BLANK1 in which a chloroacetic ester is allowed to react with potassium fluoride dissolved in molten acetamide. This preparation had been adapted by GAL, DREWES AND TAYLOR2 but the scale was insufficiently small and the yield of fluoroacetic acid only 35 %. Consequently we examined the method critically, and finally adopted the following procedure. ~I-l~CJchloroacetic acid (6. 9 rag, 0. 5 mC) (The Radiochemical Centre, Amersham, U.K.) was washed from its container with ether (i ml) into a 5-ml tube containing methyl chloroacetate (12o rag). Diazomethane from a micro-generator 3 was passed through the ethereal solution until a yellow colour persisted. In the flask of the reaction apparatus (Fig. I) finely powdered potassium fluoride dried overnight at I2o ° (2 g) and freshly redistilled acetamide (I g) were melted b y heating in an Biochim. Biophys. Acta, 115 (1966) 515-517