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Purification and properties of cytochromes c from trypanosomatids
225
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35911 P U R I F I C A T I O N AND P R O P E R T I E S OF CYTOCHROMES c FROM TRYPANOSOMATIDS
G E O R G E C. H I L L , W I N S T O N E. G U T T E R I D G E AND N A N C Y W. M A T H E W S O N
Squibb Institute for Medical Research, Department of Microbiology, New Brunswick, N.J., 089o3 (U.S.A.) and Division of Parasitology, National Institute for Medical Research, Mill Hill, London N . W . 7 (Great Britain) (Received May ioth, 1971 )
SUMMARY
Cytochromes c have been purified from four insect trypanosomatids: Trypanosoma cruzi, T. rhodesiense, Crithidia fasciculata and Leptomonas sp. All four cytochromes c have similar spectral properties, with the a-peak of the reduced form between 555 558 nm. Their electrophoretic and molecular weight properties are similar. The cytochromes c are less basic than most mammalian-type cytochromes c. The amino acid compositions of the four cytochromes c are similar but not identical. All have e-N-trimethyllysine present. The properties of trypanosomatid cytochromes c are significantly different from other basic mammalian-type cytochromes c but similar to yeast cytochromes c.
Recent studies have demonstrated the presence of cytochrome c in Kinetoplastidael, 2. A mammalian-type cytochrome c, cytochrome c~5, has been purified and characterized from Crithidia fasciculata, an insect trypanosomatid. Its properties are similar to other basic cytochromes c although it has an isoelectric point of pH 8.8 and an unusual amino acid present, e-N-trimethyllysineL Since C. fasciculata is a monoxenous insect trypanosomatid, we have extended our studies to identifying the cytochromes c that are present in trypanosomes which are pathogenic to man and spend part of their life cycle in both vertebrate and invertebrate hosts. Two culture forms of blood trypanosomes were studied: Trypanosoma cruzi and T. rhodesiense. The objective of this study was to compare the properties of cytochromes c purified from culture forms of T. cruzi, T. rhodesiense, Leptomonas sp. and C. fasciculata. C. fasciculata and Leptomonas sp. were grown in an undefined medium 3. Cells were harvested by centrifugation and frozen until used for cytochrome c extraction. T. cruzi and T. rhodesiense were obtained in the acetone powder form from the Microbiological Research Establishment, Porton, Wilts, England. T. cruzi was grown Biochim. Biophys. ,qcta, 243 (I97 I) 225-229
22()
G. ('. HILl. {'/' a/.
ill a m e d i u m described b y (;I!TTERII)GE el al. 4 and T. rhodesicnse in a modification of P i t t a m ' s m e d i u m *~. C y t o c h r o m e c was e x t r a c t e d from all cells b y procedures similar to MARCOLIASH AND WALESEK6 a n d described in HILL et al. 7 for C. fasciculata. A s s u m i n g an extinction coefficient of 28.0 raM, approx. 40.0/~g of c y t o c h r o m e c were recovered from 1.o g (wet weight) of C. fasciculata and L@lomonas sp. cells. F r o m IO.O g (dry weight) of T. cruzi a n d T. rhodesiense, I . I a n d 4.o mg, respectively, of c y t o c h r o m e c were obtained. The purification of these c y t o c h r o m e s c was done e x a c t l y as described for purification of Ascaris suum c y t o c h r o m e c in our l a b o r a t o r i e s 8. The purified protein was eluted from a S e p h a d e x G-75 column as a single species which d i d not combine with CO. All four c y t o c h r o m e s c y i e l d e d a single b a n d when electrophoresed on cellulose-acetate strips in o . o i 5'I T r i s - c a c o d y l a t e buffer (pH 8.0) for 20 min.
L
A
B
C
D
lqg. I. Electropherogranl of cytochromes c. A, Crithidia fasciculata cytochronle cs55; B, horse heart c y t o c h r o m e c ; C, Trypanosoma rhodesiense c y t o c h r o m e c550 ; D, Candida krusei c y t o c h r o m e c. The a r r o w represents the point of origin of the proteins.
Fig. I is an e l e c t r o p h e r o g r a m of C. fasciculata c y t o c h r o m e c55,5, T. rhodesiense c y t o c h r o m e c5~6, horse h e a r t cytochrorne c a n d Candida krusei c y t o c h r o m e c. The isoelectric p o i n t of C. fasciculata c y t o c h r o m e c~ 5 has been d e t e r m i n e d at p H 8.87. The o t h e r t r y p a n o s o m a t i d c y t o c h r o m e s c all m i g r a t e d at a r a t e identical to cytochrome c~55 b u t less t h a n C. krusei or horse h e a r t c y t o c h r o m e s c, b o t h of which have higher isoelectric p o i n t s t h a n c y t o c h r o m e c5~5 (ref. 9,io). The a b s o r p t i o n p e a k s of t h e r e d u c e d form of the four c y t o c h r o m e s are given in Table I. All are similar to C. fasciculata c y t o c h r o m e c555 in h a v i n g t h e a - p e a k of the r e d u c e d form b e t w e e n 555 a n d 558 n m in c o n t r a s t to most m a m m a l i a n - t y p e c y t o c h r o m e s c which are in the v i c i n i t y of 55o nm. The a - p e a k of the r e d u c e d form of t r y p a n o s o m a t i d c y t o c h r o m e s c in p y r i d i n e was at 553 nm in comparison to 55o nm for o t h e r m a m m a l i a n - t y p e c y t o c h r o m e s c. The molecular weight of t h e t r y p a n o s o m a t i d c y t o c h r o m e s c was i d e n t i c a l to t h a t for C. fasciculata as j u d g e d b y S e p h a d e x gel filtration. The m o l e c u l a r weight of Biochim. Biophys. Acta, 243 (1971) 225-229
227
TRYPANOSOMATID CYTOCHROMES C TABLE
I
SPECTRAL
PROPERTIES
OF TRYPANOSOMATID
CYTOCHROMES
C
Source
Peaks in reduced f o r m ( n m )
Soret peak in oxidized f o r m ( n m )
C. fasciculata
555 556 556 558
414 414 412 414
L e p t o m o n a s sp.
7", rhodesiense T. cruzi
TABLE
II
AMINO
ACID
COMPOSITION
525 524 524 525
OF SELECTED
421 421 420.5 421
CYTOCHROMES
C
R e s u l t s f o r t r y p a n o s o m a t i d c y t o c h r o m e s c a r e g i v e n a s / , m o l e s o f a m i n o a c i d / / , m o l e o f Fe. T h e t i m e o f h y d r o l y s i s w a s 24 h a n d no c o r r e c t i o n s h a v e b e e n m a d e f o r h y d r o l y t i c losses. T h e a m i n o a c i d c o m p o s i t i o n s o f C a n d i d a krusei c y t o c h r o m e c, a s r e p o r t e d b y NARITA A N D T I T A N 1 1 4 , o f S a c c h a r o m y c e s ( b a k e r ' s y e a s t ) c y t o c h r o m e c, a s r e p o r t e d b y NARITA et al. 1~, a n d o f Neurospora crassa c y t o c h r o m e c, a s r e p o r t e d b y t - I E L L E R A N D SMITH 16, a r e i n c l u d e d f o r c o m p a r i s o n .
Amino acid
C. fasciculata
Asp Thr Ser Glu Pro Gly Ala Val Cys** Met lle Leu Tyr Phe L y s (CHa)a § Lys His Trp Arg
IO.I * 5 .2 5.3 io.i 7.9 I6. I 14.2 6.8 2.1 2.1 2.2 8-9 4.2 2.9 1.9 lO.2 1.8
Total a-peak of lhe reduced fomr
Leptomonas sp. 8.2" 5 .2 7 .2 i2.2 7 .1 19.8 15.2 5.8 1. 7 1.8 2.2 .5. I 2.2 3. I 2.2 I.I 11. 7
T. rhodesiense
T. cruzi
lO.3" 7 -1 4.9 11.1 8.2 12.8 12.9 6.1 2.1 1. 7 3.1 8.2 2.8 4. I I.I I3.1 2.1
IO. I * 6-4 5. i i2.2 IO.t 19.2 13.8 5.I 1. 7 1.8 2.9 5.8 1. 7 3. i
C. krusei
8"* 7 (~ io 7 12 12 3 2 3 3 6 5 4
2.2
1
8.8 2.t
It 4
S. eerevisiae I I ** 8 4 9 4 12 7 3 3 2 4 8 5 4
I 15 4
Neurospora crassa I3"" 9 3 8 3 15 9 I 2 2 5 7 4 6
1 13 2
i . O §§
1.O §§
I . O §§
I . O §§
1
I
I
5.3
4 .8
5 -1
4 .2
4
3
3
lO9
Io8
lO 7
55 °
55 °
55 °
555
556
556
558
* # m o l e s o f a m i n o a c i d p e r /*mole o f Fe. ** A m i n o a c i d r e s i d u e s p e r m o l e c u l e o f p r o t e i n . § e-N-Trimethyllysine. §§ A s s u m e d v a l u e .
Biochim. Biophys. Acta, 243 ( r 9 7 I) 2 2 5 - 2 2 9
22~
(;. ('. HILL C/, a[.
C. fasciculata c y t o c h r o m e c~-,,~,is 13 070 , based on tile # m o l e s of amino acid//~mole of Ire (ref. 7). All t r y p a n o s o m a t i d c y t o c h r o m e s c were h y d r o l y z e d in 6 M HC1 a n d their a m i n o acid composition d e t e r m i n e d as previously described for C. fasciculala cvtochrome c (ref. 7)- The Technicon amino acid a n a l y z e r e m p l o y i n g Chromobeads, T y p e C2 microspherical sulfonic acid t y p e cation exchange resin (7 ° m m in length), was used according to procedures essentially analogous to those described b y PIEZ AND MORRIS11. A m i n o acid s t a n d a r d s were run prior to each series of analyses. The results of three analyses for each t r y p a n o s o m a t i d c y t o c h r o m e c are given in Table II. The iron c o n t e n t was e s t i m a t e d according to ADLER AND GEORGE 12 a n d CAMERONla. This amino acid composition d a t a have been c o m p a r e d with previously published i n f o r m a t i o n on various y e a s t c y t o c h r o m e s c (refs. 14 16). The presence of eN - t r i m e t h y t l y s i n e in y e a s t c y t o c h r o m e s c has been established b y DELANGE et al.lVO~. Some of the similarities which exist between t r y p a n o s o m a t i d a n d y e a s t c y t o c h r o m e c include: (a) both have e - N - t r i m e t h y l l y s i n e p r e s e n t ; (b) b o t h have isoelectric points less t h a n p H IO.O; (c) t h e i r amino acid compositions are similar. Several points seem e v i d e n t from these studies: (a) insect t r y p a n o s o m a t i d c y t o c h r o m e s c are quite similar in t h e i r p r i m a r y s t r u c t u r e ; (b) the presence of e-Nt r i m e t h y l l y s i n e in t r y p a n o s o m a t i d c y t o c h r o m e s c seems to be c h a r a c t e r i s t i c for this group; (c) t h e u n u s u a l spectral p r o p e r t i e s of t h e r e d u c e d form of t h e c y t o c h r o m e s c, with t h e a - p e a k between 555 a n d 558 nm, seem to be characteristic of t r y p a n o s o m a t i d e y t o c h r o m e s c. I t is justifiable to suppose t h a t all cyanide-sensitive blood t r y p a n o s o m e s have a c y t o c h r o m e s y s t e m localized in m i t o c h o n d r i a similar to t h a t of C. fasciculata: c y t o c h r o m e s a + aa, b, c a n d c y t o c h r o m e oxidase o. W h e t h e r the i n h i b i t i o n of the f o r m a t i o n of this m i t o c h o n d r i a l s y s t e m will prove helpful in t r y p a n o s o m i a s i s chemot h e r a p y r e m a i n s to be d e t e r m i n e d .
ACKNOWLEDGMENTS
W e t h a n k Dr. H. G a d e b u s c h for his cooperation a n d Mr. F. Russo-Alesi a n d Mr. W. Lesko for t h e a m i n o acid d e t e r m i n a t i o n s . W e are grateful to Dr. F. W a l l a c e of the U n i v e r s i t y of Minnesota for p r o v i d i n g t h e culture of Leptomonas sp. used ill this s t u d y , Mr. F. Belton of the M.R.E. at P o r t o n for growing T. cruzi a n d T. rhodesiense. The assistance of Miss S. Mezours a n d Mrs. J. W i l b e r - M u r p h y in the p r e p a r a t i o n of this m a n u s c r i p t is a p p r e c i a t e d . REFERENCES I 2 3 4 5 6 7 8
G. C. MILL AND V~;. ANDERSON, Exp. Parasitol., 28 (197o) 356. J. p. KUSEL, J. R. SURIANO AND M. WEBER, Arch. Biochem. Biophys., 133 (1969) 293. G. C. HILL AND W. ANDERSON, J. Cell Biol., 41 (1969) 547. W. E. GUTTERIDGE, J. KNO'~VLERAND J. D. COOMBES, dr. Protozool., 16 (1969) 521. H. DIXON AND J. WILLIAMSON,Comp. Biochem. Physiol., 33 (197o) III. E. MARGOLIASHAND O. WALASEK,Methods Enzymol., IO (1968) 349. G. C. HILL, S. K. CHAN AND L. SMITH, Biochim. Biophys. Acta, in the press. G. C. HILL, C. A. PERKOWSKI AND N. W. MATHEX~'SON, Biochim. Biophys. Acta, 229 (I971) 242.
Biochim. Biophys. Acta, 243 (1971) 225-229
TRYPANOSOMATID CYTOCHROMES C
22 9
SHIRASAKA, N. NAKAYAMA, A. ENDO, T. HANEISHI, AND H. OKAZAKI J. Biochem, Tokyo, (1968) 417 . BARLOW AND E. MARGOLIASH, J. Biol. Chem., 241 (1966) 1473. A. PIEZ AND L. A. MORRIS, Anal. Biochem., i (196o) 187 . D. ADLER AND P. GEORGE, Anal. Biochem., I I (196o) 159. F. CAMERON, Anal. Biochem., I I (1965) 164. NARITA AND I4. TITANI, Proc. Jap. Acad., 41 (1965) 831. NARITA, K. TITANI, Y. YAOI AND H. MURAKANI, Biochim. Biophys. Acta, 77 (1963) 688. 16 j . HELLER AND E. L. SMITH, J. Biol. Chem., 241 (1966) 3158 . 17 R. J. DELANGE, A. N. GLAZER AND E. L. SMITH, J. Biol. Chem., 244 (1969) 1385. 18 R. J. I)ELANGE, A. N. GLAZER AND E. L. SMITH, J. Biol. Chem., 245 (197 o) 3325.
9M. 63 IO G. i i K. 12 A. 13 B. 14 N . 15 K.
Biochim. Biophys. Acta, 243 (1971i 225 229
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35911 P U R I F I C A T I O N AND P R O P E R T I E S OF CYTOCHROMES c FROM TRYPANOSOMATIDS
G E O R G E C. H I L L , W I N S T O N E. G U T T E R I D G E AND N A N C Y W. M A T H E W S O N
Squibb Institute for Medical Research, Department of Microbiology, New Brunswick, N.J., 089o3 (U.S.A.) and Division of Parasitology, National Institute for Medical Research, Mill Hill, London N . W . 7 (Great Britain) (Received May ioth, 1971 )
SUMMARY
Cytochromes c have been purified from four insect trypanosomatids: Trypanosoma cruzi, T. rhodesiense, Crithidia fasciculata and Leptomonas sp. All four cytochromes c have similar spectral properties, with the a-peak of the reduced form between 555 558 nm. Their electrophoretic and molecular weight properties are similar. The cytochromes c are less basic than most mammalian-type cytochromes c. The amino acid compositions of the four cytochromes c are similar but not identical. All have e-N-trimethyllysine present. The properties of trypanosomatid cytochromes c are significantly different from other basic mammalian-type cytochromes c but similar to yeast cytochromes c.
Recent studies have demonstrated the presence of cytochrome c in Kinetoplastidael, 2. A mammalian-type cytochrome c, cytochrome c~5, has been purified and characterized from Crithidia fasciculata, an insect trypanosomatid. Its properties are similar to other basic cytochromes c although it has an isoelectric point of pH 8.8 and an unusual amino acid present, e-N-trimethyllysineL Since C. fasciculata is a monoxenous insect trypanosomatid, we have extended our studies to identifying the cytochromes c that are present in trypanosomes which are pathogenic to man and spend part of their life cycle in both vertebrate and invertebrate hosts. Two culture forms of blood trypanosomes were studied: Trypanosoma cruzi and T. rhodesiense. The objective of this study was to compare the properties of cytochromes c purified from culture forms of T. cruzi, T. rhodesiense, Leptomonas sp. and C. fasciculata. C. fasciculata and Leptomonas sp. were grown in an undefined medium 3. Cells were harvested by centrifugation and frozen until used for cytochrome c extraction. T. cruzi and T. rhodesiense were obtained in the acetone powder form from the Microbiological Research Establishment, Porton, Wilts, England. T. cruzi was grown Biochim. Biophys. ,qcta, 243 (I97 I) 225-229
22()
G. ('. HILl. {'/' a/.
ill a m e d i u m described b y (;I!TTERII)GE el al. 4 and T. rhodesicnse in a modification of P i t t a m ' s m e d i u m *~. C y t o c h r o m e c was e x t r a c t e d from all cells b y procedures similar to MARCOLIASH AND WALESEK6 a n d described in HILL et al. 7 for C. fasciculata. A s s u m i n g an extinction coefficient of 28.0 raM, approx. 40.0/~g of c y t o c h r o m e c were recovered from 1.o g (wet weight) of C. fasciculata and L@lomonas sp. cells. F r o m IO.O g (dry weight) of T. cruzi a n d T. rhodesiense, I . I a n d 4.o mg, respectively, of c y t o c h r o m e c were obtained. The purification of these c y t o c h r o m e s c was done e x a c t l y as described for purification of Ascaris suum c y t o c h r o m e c in our l a b o r a t o r i e s 8. The purified protein was eluted from a S e p h a d e x G-75 column as a single species which d i d not combine with CO. All four c y t o c h r o m e s c y i e l d e d a single b a n d when electrophoresed on cellulose-acetate strips in o . o i 5'I T r i s - c a c o d y l a t e buffer (pH 8.0) for 20 min.
L
A
B
C
D
lqg. I. Electropherogranl of cytochromes c. A, Crithidia fasciculata cytochronle cs55; B, horse heart c y t o c h r o m e c ; C, Trypanosoma rhodesiense c y t o c h r o m e c550 ; D, Candida krusei c y t o c h r o m e c. The a r r o w represents the point of origin of the proteins.
Fig. I is an e l e c t r o p h e r o g r a m of C. fasciculata c y t o c h r o m e c55,5, T. rhodesiense c y t o c h r o m e c5~6, horse h e a r t cytochrorne c a n d Candida krusei c y t o c h r o m e c. The isoelectric p o i n t of C. fasciculata c y t o c h r o m e c~ 5 has been d e t e r m i n e d at p H 8.87. The o t h e r t r y p a n o s o m a t i d c y t o c h r o m e s c all m i g r a t e d at a r a t e identical to cytochrome c~55 b u t less t h a n C. krusei or horse h e a r t c y t o c h r o m e s c, b o t h of which have higher isoelectric p o i n t s t h a n c y t o c h r o m e c5~5 (ref. 9,io). The a b s o r p t i o n p e a k s of t h e r e d u c e d form of the four c y t o c h r o m e s are given in Table I. All are similar to C. fasciculata c y t o c h r o m e c555 in h a v i n g t h e a - p e a k of the r e d u c e d form b e t w e e n 555 a n d 558 n m in c o n t r a s t to most m a m m a l i a n - t y p e c y t o c h r o m e s c which are in the v i c i n i t y of 55o nm. The a - p e a k of the r e d u c e d form of t r y p a n o s o m a t i d c y t o c h r o m e s c in p y r i d i n e was at 553 nm in comparison to 55o nm for o t h e r m a m m a l i a n - t y p e c y t o c h r o m e s c. The molecular weight of t h e t r y p a n o s o m a t i d c y t o c h r o m e s c was i d e n t i c a l to t h a t for C. fasciculata as j u d g e d b y S e p h a d e x gel filtration. The m o l e c u l a r weight of Biochim. Biophys. Acta, 243 (1971) 225-229
227
TRYPANOSOMATID CYTOCHROMES C TABLE
I
SPECTRAL
PROPERTIES
OF TRYPANOSOMATID
CYTOCHROMES
C
Source
Peaks in reduced f o r m ( n m )
Soret peak in oxidized f o r m ( n m )
C. fasciculata
555 556 556 558
414 414 412 414
L e p t o m o n a s sp.
7", rhodesiense T. cruzi
TABLE
II
AMINO
ACID
COMPOSITION
525 524 524 525
OF SELECTED
421 421 420.5 421
CYTOCHROMES
C
R e s u l t s f o r t r y p a n o s o m a t i d c y t o c h r o m e s c a r e g i v e n a s / , m o l e s o f a m i n o a c i d / / , m o l e o f Fe. T h e t i m e o f h y d r o l y s i s w a s 24 h a n d no c o r r e c t i o n s h a v e b e e n m a d e f o r h y d r o l y t i c losses. T h e a m i n o a c i d c o m p o s i t i o n s o f C a n d i d a krusei c y t o c h r o m e c, a s r e p o r t e d b y NARITA A N D T I T A N 1 1 4 , o f S a c c h a r o m y c e s ( b a k e r ' s y e a s t ) c y t o c h r o m e c, a s r e p o r t e d b y NARITA et al. 1~, a n d o f Neurospora crassa c y t o c h r o m e c, a s r e p o r t e d b y t - I E L L E R A N D SMITH 16, a r e i n c l u d e d f o r c o m p a r i s o n .
Amino acid
C. fasciculata
Asp Thr Ser Glu Pro Gly Ala Val Cys** Met lle Leu Tyr Phe L y s (CHa)a § Lys His Trp Arg
IO.I * 5 .2 5.3 io.i 7.9 I6. I 14.2 6.8 2.1 2.1 2.2 8-9 4.2 2.9 1.9 lO.2 1.8
Total a-peak of lhe reduced fomr
Leptomonas sp. 8.2" 5 .2 7 .2 i2.2 7 .1 19.8 15.2 5.8 1. 7 1.8 2.2 .5. I 2.2 3. I 2.2 I.I 11. 7
T. rhodesiense
T. cruzi
lO.3" 7 -1 4.9 11.1 8.2 12.8 12.9 6.1 2.1 1. 7 3.1 8.2 2.8 4. I I.I I3.1 2.1
IO. I * 6-4 5. i i2.2 IO.t 19.2 13.8 5.I 1. 7 1.8 2.9 5.8 1. 7 3. i
C. krusei
8"* 7 (~ io 7 12 12 3 2 3 3 6 5 4
2.2
1
8.8 2.t
It 4
S. eerevisiae I I ** 8 4 9 4 12 7 3 3 2 4 8 5 4
I 15 4
Neurospora crassa I3"" 9 3 8 3 15 9 I 2 2 5 7 4 6
1 13 2
i . O §§
1.O §§
I . O §§
I . O §§
1
I
I
5.3
4 .8
5 -1
4 .2
4
3
3
lO9
Io8
lO 7
55 °
55 °
55 °
555
556
556
558
* # m o l e s o f a m i n o a c i d p e r /*mole o f Fe. ** A m i n o a c i d r e s i d u e s p e r m o l e c u l e o f p r o t e i n . § e-N-Trimethyllysine. §§ A s s u m e d v a l u e .
Biochim. Biophys. Acta, 243 ( r 9 7 I) 2 2 5 - 2 2 9
22~
(;. ('. HILL C/, a[.
C. fasciculata c y t o c h r o m e c~-,,~,is 13 070 , based on tile # m o l e s of amino acid//~mole of Ire (ref. 7). All t r y p a n o s o m a t i d c y t o c h r o m e s c were h y d r o l y z e d in 6 M HC1 a n d their a m i n o acid composition d e t e r m i n e d as previously described for C. fasciculala cvtochrome c (ref. 7)- The Technicon amino acid a n a l y z e r e m p l o y i n g Chromobeads, T y p e C2 microspherical sulfonic acid t y p e cation exchange resin (7 ° m m in length), was used according to procedures essentially analogous to those described b y PIEZ AND MORRIS11. A m i n o acid s t a n d a r d s were run prior to each series of analyses. The results of three analyses for each t r y p a n o s o m a t i d c y t o c h r o m e c are given in Table II. The iron c o n t e n t was e s t i m a t e d according to ADLER AND GEORGE 12 a n d CAMERONla. This amino acid composition d a t a have been c o m p a r e d with previously published i n f o r m a t i o n on various y e a s t c y t o c h r o m e s c (refs. 14 16). The presence of eN - t r i m e t h y t l y s i n e in y e a s t c y t o c h r o m e s c has been established b y DELANGE et al.lVO~. Some of the similarities which exist between t r y p a n o s o m a t i d a n d y e a s t c y t o c h r o m e c include: (a) both have e - N - t r i m e t h y l l y s i n e p r e s e n t ; (b) b o t h have isoelectric points less t h a n p H IO.O; (c) t h e i r amino acid compositions are similar. Several points seem e v i d e n t from these studies: (a) insect t r y p a n o s o m a t i d c y t o c h r o m e s c are quite similar in t h e i r p r i m a r y s t r u c t u r e ; (b) the presence of e-Nt r i m e t h y l l y s i n e in t r y p a n o s o m a t i d c y t o c h r o m e s c seems to be c h a r a c t e r i s t i c for this group; (c) t h e u n u s u a l spectral p r o p e r t i e s of t h e r e d u c e d form of t h e c y t o c h r o m e s c, with t h e a - p e a k between 555 a n d 558 nm, seem to be characteristic of t r y p a n o s o m a t i d e y t o c h r o m e s c. I t is justifiable to suppose t h a t all cyanide-sensitive blood t r y p a n o s o m e s have a c y t o c h r o m e s y s t e m localized in m i t o c h o n d r i a similar to t h a t of C. fasciculata: c y t o c h r o m e s a + aa, b, c a n d c y t o c h r o m e oxidase o. W h e t h e r the i n h i b i t i o n of the f o r m a t i o n of this m i t o c h o n d r i a l s y s t e m will prove helpful in t r y p a n o s o m i a s i s chemot h e r a p y r e m a i n s to be d e t e r m i n e d .
ACKNOWLEDGMENTS
W e t h a n k Dr. H. G a d e b u s c h for his cooperation a n d Mr. F. Russo-Alesi a n d Mr. W. Lesko for t h e a m i n o acid d e t e r m i n a t i o n s . W e are grateful to Dr. F. W a l l a c e of the U n i v e r s i t y of Minnesota for p r o v i d i n g t h e culture of Leptomonas sp. used ill this s t u d y , Mr. F. Belton of the M.R.E. at P o r t o n for growing T. cruzi a n d T. rhodesiense. The assistance of Miss S. Mezours a n d Mrs. J. W i l b e r - M u r p h y in the p r e p a r a t i o n of this m a n u s c r i p t is a p p r e c i a t e d . REFERENCES I 2 3 4 5 6 7 8
G. C. MILL AND V~;. ANDERSON, Exp. Parasitol., 28 (197o) 356. J. p. KUSEL, J. R. SURIANO AND M. WEBER, Arch. Biochem. Biophys., 133 (1969) 293. G. C. HILL AND W. ANDERSON, J. Cell Biol., 41 (1969) 547. W. E. GUTTERIDGE, J. KNO'~VLERAND J. D. COOMBES, dr. Protozool., 16 (1969) 521. H. DIXON AND J. WILLIAMSON,Comp. Biochem. Physiol., 33 (197o) III. E. MARGOLIASHAND O. WALASEK,Methods Enzymol., IO (1968) 349. G. C. HILL, S. K. CHAN AND L. SMITH, Biochim. Biophys. Acta, in the press. G. C. HILL, C. A. PERKOWSKI AND N. W. MATHEX~'SON, Biochim. Biophys. Acta, 229 (I971) 242.
Biochim. Biophys. Acta, 243 (1971) 225-229
TRYPANOSOMATID CYTOCHROMES C
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SHIRASAKA, N. NAKAYAMA, A. ENDO, T. HANEISHI, AND H. OKAZAKI J. Biochem, Tokyo, (1968) 417 . BARLOW AND E. MARGOLIASH, J. Biol. Chem., 241 (1966) 1473. A. PIEZ AND L. A. MORRIS, Anal. Biochem., i (196o) 187 . D. ADLER AND P. GEORGE, Anal. Biochem., I I (196o) 159. F. CAMERON, Anal. Biochem., I I (1965) 164. NARITA AND I4. TITANI, Proc. Jap. Acad., 41 (1965) 831. NARITA, K. TITANI, Y. YAOI AND H. MURAKANI, Biochim. Biophys. Acta, 77 (1963) 688. 16 j . HELLER AND E. L. SMITH, J. Biol. Chem., 241 (1966) 3158 . 17 R. J. DELANGE, A. N. GLAZER AND E. L. SMITH, J. Biol. Chem., 244 (1969) 1385. 18 R. J. I)ELANGE, A. N. GLAZER AND E. L. SMITH, J. Biol. Chem., 245 (197 o) 3325.
9M. 63 IO G. i i K. 12 A. 13 B. 14 N . 15 K.
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